Silver nanoclusters and gold-palladium nanoparticles to construct a fluorescent sensor for the detection of streptomycin in milk

Abstract

The present invention provides a method for preparing a fluorescent aptamer sensor for detecting streptomycin (STR) residues in milk, and belongs to the field of food safety detection. The present invention includes the synthesis of hairpin DNA-silver nanoclusters (DNA-AgNCs) regulated by guanine (G) bases, the synthesis of core-shell gold palladium nanoparticles (Au@PdNPs) and the construction of a fluorescent sensor. The present invention achieves the red shift of the emission spectrum and the increase of the fluorescence intensity of DNA-AgNCs by adding different numbers of G bases in the hairpin loop. Au@PdNPs are prepared by seed growth method and deposition method. The Au@PdNPs composite material not only solves the problem of weak quenching ability of palladium nanoparticles (PdNPs) in the long wavelength range, but also effectively expands the fluorescence quenching range of gold nanoparticles (AuNPs). The invented sensor has a good response to STR in the range of 50-1250 nM, and the detection limit (LOD) is 18.7 nM. In addition, the invention has been successfully applied to STR detection in milk, indicating that it has good application prospects in STR detection in the field of animal-derived food.

Description

Technical Field

[0001] The invention provides a method for preparing an aptamer sensor for detecting streptomycin residues in milk, belonging to the technical field of food safety detection. Background Art

[0002] Streptomycin (STR) is a common aminoglycoside antibiotic used to treat diseases such as mastitis caused by bacterial infection in dairy cows. Improper use will cause STR residues in milk and other dairy products, which will not only affect the quality of dairy products, but also cause serious harm to the human body, including allergies, damage to intestinal flora and kidney toxicity.

[0003] Therefore, it is crucial to develop sensitive and specific techniques to detect STR in animal-derived foods such as milk. Currently, sensitive detection methods such as capillary electrophoresis, high-performance liquid chromatography and enzyme-linked immunosorbent assay have been developed for the detection of STR in milk residues, but their applications are limited by their long turnaround time, complex steps, expensive equipment and high technical requirements. Summary of the invention

[0004] The purpose of the invention is to establish a rapid, simple and efficient detection technology for detecting STR in milk.

[0005] The technical solution is: aptamers are RNA or ssDNA sequences with high affinity and specificity screened by systematic evolution of ligands by enrichment (SELEX). In recent years, aptamer-based fluorescent sensors have been widely used due to their advantages of being fast, sensitive and easy to operate.

[0006] The preparation method of the fluorescent aptamer sensor for detecting STR residues in milk ( Figure 1 ), characterized in that: a G base is added to the hairpin DNA-silver nanocluster (DNA-AgNCs) ring to achieve the regulation of the fluorescence spectrum of DNA-AgNCs, and the template chain of DNA-AgNCs contains a STR aptamer (STP) sequence. In the absence of STR, the aptamer of DNA-AgNCs is adsorbed on the surface of gold palladium nanoparticles (Au@PdNPs) through coordination. Therefore, energy is transferred from the fluorescent donor DNA-AgNCs to Au@PdNPs, resulting in fluorescence quenching. When STR is added, the specific binding of STR to the aptamer causes the conformation of the aptamer to change, and the interaction between the aptamer and Au@PdNPs is weakened, resulting in the recovery of the fluorescence signal. Based on the change in the fluorescence signal, the detection of STR in milk can be successfully achieved.

[0007] The method for preparing a fluorescent aptamer sensor for detecting STR residues in milk is characterized in that different numbers of G bases are added to the hairpin loop to achieve a red shift in the emission spectrum and an increase in the fluorescence intensity of DNA-AgNCs. In addition, an Au@PdNPs composite material with excellent quenching properties is synthesized, which not only solves the problem of weak quenching ability of palladium nanoparticles (PdNPs) in the long wavelength range, but also effectively expands the fluorescence quenching range of gold nanoparticles (AuNPs).

[0008] The preparation principle is as follows: Based on fluorescence resonance energy transfer (FRET), we designed a fluorescent sensor using DNA-AgNCs and core-shell gold palladium nanoparticles (Au@PdNPs) to detect streptomycin (STR) in milk. The aptamer of DNA-AgNCs is adsorbed on the surface of Au@PdNPs through coordination. DNA-AgNCs and Au@PdNPs produce FRET, and energy is transferred from the fluorescent donor DNA-AgNCs to Au@PdNPs, resulting in fluorescence quenching. When STR is added, the specific binding of STR to the aptamer causes the conformation of the aptamer to change, and the FRET between the aptamer and Au@PdNPs is destroyed, resulting in the recovery of the fluorescence signal.

[0009] To achieve the above objectives, the following technical scheme was adopted: for the preparation of silver nanoclusters, all DNA template solutions (100 μM) were first prepared with TE buffer and kept in a water bath (95°C) for 10 minutes, and then the solution was cooled to room temperature in an ice water bath, and then 500 μL of 100 μM DNA solution and 300 μL of 1 mM silver nitrate (AgNO3) solution were added to 3.9 ml TE buffer respectively. After thorough mixing and shaking, all mixed solutions were incubated in an ice bath and dark conditions for 30 minutes, and 300 μL of 1 mM freshly prepared and ice-cold sodium borohydride (NaBH4) solution was added to the mixed solution respectively. After the reaction was completed, the mixed solution was shaken thoroughly for 1 minute. Finally, the obtained solution was stored at 4°C in the dark. For the preparation of Au@PdNPs, 100 mL of 0.01% (w / v) gold chloride trihydrate (HAuCl4·3H2O) solution was heated to boiling under uniform stirring, and then 2.7 mL 1% (w / v) trisodium citrate (Na3C6H5O7) solution was continuously heated and boiled until the color of the gold seed solution did not change, and then the gold seed solution was cooled to room temperature under constant stirring. Subsequently, 2.0 mL of the gold seed solution and 4 mL of 1% (w / v) HAuCl4·3H2O solution were added to 200 mL of ultrapure water under vigorous stirring. After stirring evenly, 800 μL of 1% (w / v) Na3C6H5O7 solution and 400 μL of 30 mM hydroquinone (C6H6O2) solution were quickly added to the mixed solution for reduction. The above reducing agent was repeatedly added to the mixed solution for 7 times with an interval of 10 minutes. After the addition was completed, the mixed solution was stirred for 1 hour. Finally, a 1 mM H2PdCl4 solution was synthesized by the following method. 10.64 mg of palladium chloride (PdCl2) was dissolved in 6 mL of 0.02 M HCl solution. Then 54 mL of ultrapure water was added to the mixture in a 70 °C water bath until PdCl2 was completely dissolved to obtain a 1 mM H2PdCl4 solution. Au@PdNPs were obtained by palladium deposition. 30 mL of 75 nm AuNPs solution was mixed with 1 mL, 4 mL, 7 mL and 10 mL of H2PdCl4 solution (1 mM), respectively. The mixed solution was cooled in an ice bath. Under vigorous stirring, 1 mL, 4 mL, 7 mL and 10 mL of ascorbic acid solution and 18 mL, 12 mL, 6 mL and 0 mL of ultrapure water were slowly added to the mixed solution, and then the mixed solution was stirred for 30 min. After stirring, the obtained solution was centrifuged and washed several times with water-ethanol solution (1:2, v / v). Finally, Au@PdNPs were dissolved in C6H5Na3O7 solution (0.02%, w / v).

[0010] To achieve the above objectives, the following technical scheme was adopted: before detecting STR, the concentration of hairpin DNA-AgNCs was diluted to 1 μM and the excitation wavelength was 586 nm during the whole experiment, while the detection results of STR were obtained under the optimal experimental conditions, 500 μl of DNA-AgNCs and 500 μl of Au@PdNPs solution were mixed for 15 min, a series of 500 μL of STR solutions of different concentrations were added to the mixed solution of DNA-AgNCs and Au@PdNPs, the reaction lasted for 60 min, and then the fluorescence intensity was measured.

[0011] The preparation process of the aptamer sensor is as follows: before pretreatment, STR was added to milk at three concentration levels (300 nM, 600 nM and 900 nM). Subsequently, the milk sample was pretreated by the following method: 20 mL of milk-methanol (1:4, v / v) mixture was stored at -20°C for 20 min, and then the mixture was centrifuged at 12500 rpm for 25 min. The supernatant was filtered through a membrane filter (0.22 μm). After filtration, the supernatant was concentrated and dried in a water bath at 60°C. The residue was dried with nitrogen and then reconstituted with ultrapure water. The reconstituted solution was filtered with a membrane filter (0.22 μm). Finally, the volume of the filtrate was set to 4 mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The construction process of fluorescent aptasensor.

[0013] Figure 2 Excitation and emission wavelengths of DNA-AgNCs synthesized with different template chains.

[0014] Figure 3 Fluorescence spectra and fluorescence intensity of DNA-AgNCs.

[0015] Figure 4 Electron microscopy characterization of nanomaterials.

[0016] Figure 5 Optimization of palladium shell thickness.

[0017] Figure 6 Optimization of experimental conditions.

[0018] Figure 7 Fluorescence assay of STR.

[0019] Figure 8 Specificity test.

[0020] Fig. 9 STR testing in real samples of milk. DETAILED DESCRIPTION

[0021] Embodiment 1: Figure 3 A shows the fluorescence spectra of DNA-AgNCs synthesized with different template chains. Figure 2 The excitation and emission wavelengths are listed in Figure 3 B shows the fluorescence intensity. However, in DNA2, DNA3, DNA4, and DNA5, there is no regular red shift as the number of G bases increases, and when the number of G bases reaches 10 and 12, the red shift no longer occurs. When the number of bases is between 0 and 6, the fluorescence intensity does not change much. However, when the number of G bases increases to 8, a significant enhancement and change occurs. Since the emission spectra of DNA4-AgNCs, DNA5-AgNCs, DNA6-AgNCs, and DNA7-AgNCs are close to the near-infrared region, this means that This means that it requires large-sized metal nanoparticles, but larger metal nanoparticles may cause scattering, resulting in distortion of the fluorescence spectrum. The fluorescence intensity of DNA3-AgNCs is the strongest among DNA1-AgNCs, DNA2-AgNCs and DNA3-AgNCs. The position of the maximum emission peak of DNA3-AgNCs is shorter than that of DNA2-AgNCs, so the diameter of the required quencher nanoparticles is smaller and the effect of scattering is also weaker. In summary, DNA3-AgNCs was selected as the fluorescent material used in this experiment.

[0022] Example 2: The morphology of DNA3-AgNCs was observed by TEM. Figure 4 As shown in A, the synthesized DNA3-AgNCs showed uniform dispersion and were almost round with an average size of 2.47 nm. The stability of DNA3-AgNCs was measured for 120 h. The results showed that DNA3-AgNCs were stable within 12-24 h. Therefore, the experiment was carried out within 12-24 h after the synthesis of DNA3-AgNCs. The absolute quantum yield of DNA3-AgNCs was measured to be 10.76%. The TEM image of the gold seeds was shown in Figure 4 In Figure B, it can be seen that the gold seeds are spherical particles with a diameter of 19.9 nm. Figure 4 E shows the large-sized gold nanoparticles synthesized by the seed-growth method, with a diameter of 75 nm. Figure 4 FI shows that palladium was deposited on the surface of AuNPs with different amounts of H2PdCl4 and ascorbic acid. It can be seen that the thickness of the palladium shell is about 4 nm, 7 nm, 11 nm, and 16 nm, respectively. Figure 4 JM shows the EDX of Au@PdNPs with a diameter of 75+16 nm. It can be seen that the palladium is dispersed in the outer layer of the gold core, and the weight ratio of gold / palladium is 33.36 / 66.64. Figure 4C, D show the SEM of Au@PdNPs with a diameter of 75+16 nm. According to the images, Au@PdNPs are almost spherical particles with a diameter of 107.11 nm. The above analysis shows that the synthesis of Au@PdNPs and DNA3-AgNCs is good.

[0023] Example 3: Optimization of palladium shell thickness ( Figure 5 ), the energy transfer efficiency (E) of 75+4 nm, 75+7 nm, 75+11 nm and 75+16 nm Au@PdNPs were 26.7%, 46.6%, 56.7% and 62.8%, respectively. It can be found that the energy transfer efficiency gradually increases with the increase of palladium shell thickness. The 75+16 nm Au@PdNPs have a high energy transfer efficiency, which is sufficient to meet the experimental requirements. At the same time, considering that further increasing the particle size will increase the interference of metal nanoparticle scattering, therefore, the 75 + 16 nm Au@PdNPs with the highest quenching efficiency were selected as the quencher for the experiment.

[0024] Example 4: Optimizing the conditions in the experiment ( Figure 6 ), (A) Optimization of pH in DNA-AgNCs synthesis (6.5-9.0), the optimal pH is 7.5, (B) Au@PdNPs with a concentration of 0-24.8 pm were added to 1 μM DNA3-AgNCs, the optimal Au@PdNPs concentration was 17.2 pM, (C) Incubation time for fluorescence quenching (0-30min), the optimal quenching time was 15min, (D) Incubation time for fluorescence recovery (0-90min), the optimal recovery time was 60min.

[0025] Example 5: The performance of the sensor in determining analytes was evaluated by measuring different concentrations of STR, such as Figure 7 As shown in the figure, in the range of 50~2800 nM, the fluorescence intensity gradually increases with the increase of STR concentration. In the range of 50~1250 nM, the fluorescence intensity and STR concentration show a positive linear relationship. The equation of the linear calibration curve is as follows: I=0.43314C STR +102.94683, correlation coefficient (R 2 ) was 0.99258, and the limit of detection (LOD) of the STR fluorescence assay was 18.7 nM based on 3σ / k, where σ is the standard deviation of the measurements of the calibration standard blank (n=10) and k is the slope of the calibration curve.

[0026] Example 6: To evaluate the specificity of the sensor, the specificity of the fluorescent aptamer based on DNA-AgNCs was determined by comparing with other antibiotics such as teramicin (TER), chloramphenicol (CHL), tetracycline (TET), erythromycin (ERY), chlortetracycline hydrochloride (CHH) and penicillin (PEN). The concentration of other antibiotics was 10 times that of STR, e.g. Figure 8 As shown, I and I0 represent the fluorescence intensity in the presence and absence of different types of antibiotics, respectively, and the response of STR to the fluorescent adaptor is higher than that to other antibiotics. These results indicate that the fluorescent adaptor has a strong specificity for detecting STR, indicating that the developed fluorescent adaptor can be used to detect STR.

[0027] Example 7: In order to evaluate the feasibility of the invention in detecting STR in milk samples, we added three different concentrations of streptomycin to actual milk, and the recovery rates were shown in Table 7. Fig. 9 ,It can be seen that the peak recovery of kanamycin detection is between 97.45%-105.34%, and the relative standard deviation (RSD) is between 1.05%-3.04%. These excellent performances indicate the feasibility of our proposed streptomycin detection method in this application.

Claims

1. A method for preparing a fluorescent aptamer sensor for detecting streptomycin in milk, characterized in that: A fluorescent aptasensor based on hairpin DNA silver nanoclusters DNA-AgNCs and core-shell gold palladium nanoparticles Au@PdNPs regulated by guanine G base was constructed for the detection of streptomycin STR. Different numbers of G bases were added to the hairpin loop to achieve a red shift in the emission spectrum and an increase in the fluorescence intensity of hairpin DNA-AgNCs; Preparation of silver nanoclusters: 100 μM of all DNA template solutions were prepared using TE buffer and kept in a 95°C water bath for 10 minutes, then the solution was cooled to room temperature in an ice water bath, then 500 μL of 100 μM DNA solution and 300 μL of 1 mM silver nitrate AgNO3 solution were added to 3.9 ml TE buffer respectively, after thorough mixing and shaking, all mixed solutions were incubated in an ice bath and dark conditions for 30 minutes, 300 μL of 1 mM freshly prepared and ice-cold sodium borohydride NaBH4 solution was added to the mixed solution respectively, the mixed solution was shaken thoroughly for 1 minute, and finally, the obtained solution was stored at 4°C in the dark, wherein the number of G bases of the DNA template was 0G, 2G, 4G, 6G, 8G, 10G and 12G.

2. The method for preparing a fluorescent aptamer sensor for detecting streptomycin in milk according to claim 1, characterized in that: Core-shell Au@PdNPs, an excellent quenching material, was prepared by seed growth and deposition methods. Fluorescence resonance energy transfer was carried out using core-shell Au@PdNPs and hairpin DNA-AgNCs. Streptomycin was detected by the changes in the fluorescence signals of core-shell Au@PdNPs and hairpin DNA-AgNCs at the corresponding excitation wavelength.

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