CN115452787A - Method for measuring streptomycin in milk by using fluorescence sensor constructed by silver nanoclusters and gold palladium nanoparticles - Google Patents

Method for measuring streptomycin in milk by using fluorescence sensor constructed by silver nanoclusters and gold palladium nanoparticles Download PDF

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CN115452787A
CN115452787A CN202211154362.7A CN202211154362A CN115452787A CN 115452787 A CN115452787 A CN 115452787A CN 202211154362 A CN202211154362 A CN 202211154362A CN 115452787 A CN115452787 A CN 115452787A
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pdnps
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李发兰
王晓阳
田宇航
孙霞
郭业民
杨青青
张妍妍
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Shandong University of Technology
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Abstract

The invention provides a preparation method of a fluorescence aptamer sensor for detecting Streptomycin (STR) residues in milk, belonging to the field of food safety detection. The invention comprises a method for synthesizing a hairpin type DNA-silver nano cluster (DNA-AgNCs) regulated and controlled by guanine (G) base, synthesizing core-shell gold-palladium nano particles (Au @ PdNPs) and constructing a fluorescent sensor. According to the invention, different numbers of G bases are added into the hairpin loop, so that the red shift of the emission spectrum and the increase of the fluorescence intensity of DNA-AgNCs are realized. The Au @ PdNPs is prepared by a seed growth method and a deposition method, and the Au @ PdNPs composite material not only solves the problem that the quenching capacity of the palladium nanoparticles (PdNPs) is weak in the long wavelength range, but also effectively expands the fluorescence quenching range of the gold nanoparticles (AuNPs). The inventive sensor responded well to STRs in the range of 50-1250 nM with a limit of detection (LOD) of 18.7 nM. In addition, the invention has been successfully applied to STR detection in milk, and shows that the invention has good application prospect in STR detection in the field of animal-derived foods.

Description

Method for measuring streptomycin in milk by using fluorescence sensor constructed by silver nanoclusters and gold palladium nanoparticles
Technical Field
The invention provides a preparation method of an aptamer sensor for detecting streptomycin residue in milk, and belongs to the technical field of food safety detection.
Background
Streptomycin (STR) is a common aminoglycoside antibiotic, is used for treating mastitis and other diseases caused by bacterial infection of cows, and can cause STR residues in dairy products such as milk if unreasonably used, thereby not only affecting the quality of the dairy products, but also causing serious harm to human bodies, including allergy, damage to intestinal flora and toxicity to kidneys.
Therefore, it is crucial to develop sensitive and specific technologies for detecting STR in foods of animal origin, such as milk, and at present, sensitive detection methods such as capillary electrophoresis, high performance liquid chromatography and enzyme-linked immunosorbent assay have been developed for detecting STR in milk residues, but their application is limited due to the disadvantages of long turnaround time, complicated steps, expensive equipment and high technical requirements.
Disclosure of Invention
The invention aims to establish a rapid, simple and efficient detection technology for detecting STR in milk.
The technical scheme is as follows: aptamers are high-affinity and specific RNA or ssDNA sequences screened by a number-enriched ligand systematic evolution technology (SELEX), and in recent years, due to the advantages of rapidness, sensitivity and easiness in operation, fluorescent sensors based on aptamers are widely applied.
The preparation method of the fluorescent aptamer sensor for detecting STR residues in milk (figure 1) is characterized by comprising the following steps: the method is characterized in that a G base group is added into a hairpin type DNA-silver nanocluster (DNA-AgNCs) ring to adjust the fluorescence spectrum of the DNA-AgNCs, a template chain of the DNA-AgNCs contains an STR aptamer (STP) sequence, the aptamer of the DNA-AgNCs is adsorbed on the surface of gold palladium nanoparticles (Au @ PdNPs) through coordination under the condition of no STR, therefore, energy is transferred from the fluorescence donor DNA-AgNCs to the Au @ PdNPs to cause fluorescence quenching, when the STR is added, the conformation of the aptamer is changed due to specific combination of the STR and the aptamer, the interaction between the aptamer and the Au @ PdNPs is weakened, the fluorescence signal is recovered, and the STR in the milk can be successfully detected based on the change of the fluorescence signal.
The preparation method of the fluorescent aptamer sensor for detecting STR residues in milk is characterized by comprising the following steps: in addition, the Au @ PdNPs composite material with excellent quenching property is synthesized, so that the problem of weak quenching capability of palladium nanoparticles (PdNPs) in a long wavelength range is solved, and the fluorescence quenching range of gold nanoparticles (AuNPs) is effectively expanded.
The preparation principle is as follows: on the basis of Fluorescence Resonance Energy Transfer (FRET), a fluorescence sensor utilizing DNA-AgNCs and core-shell gold-palladium nanoparticles (Au @ PdNPs) is designed to detect Streptomycin (STR) in milk, and an aptamer of the DNA-AgNCs is adsorbed on the surface of the Au @ PdNPs through coordination. DNA-AgNCs and Au @ PdNPs generate FRET, energy is transferred from fluorescence donor DNA-AgNCs to Au @ PdNPs, fluorescence quenching is caused, when STR is added, the specific binding of the STR and the aptamer causes the conformation of the aptamer to change, the FRET between the aptamer and the Au @ PdNPs is destroyed, and the recovery of fluorescence signals is caused.
In order to achieve the purpose, the following technical scheme is adopted for realizing the purpose: silver nanoclusters were prepared by first preparing all DNA template solutions (100. Mu.M) with TE buffer and holding in a water bath (95 ℃) for 10 minutes, then cooling the solution to room temperature in an ice-water bath, followed by 500. Mu.L of 100. Mu.M DNA solution and 300. Mu.L of 1 mM silver nitrate (AgNO) 3 ) The solutions were added to 3.9 ml of TE buffer, mixed thoroughly and shaken, all the mixed solutions were incubated in ice bath and in the dark for 30 minutes, and 300. Mu.L of 1 mM freshly prepared and ice-cold sodium borohydride (NaBH) was added to the mixed solutions, respectively 4 ) Solution, after the reaction is finished, fully shaking the mixed solution for 1 minute, and finally obtaining the productStoring the obtained solution at 4 deg.C in dark place, preparing Au @ PdNPs, and mixing 100 ml of 0.01% (w/v) gold chloride trihydrate (HAuCl) 4 ·3H 2 O) solution was heated to boiling, after which 2.7 mL of 1% (w/v) trisodium citrate (Na) were added rapidly 3 C 6 H 5 O 7 ) The 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, followed by mixing 2.0 mL of the gold seed solution and 4 mL of 1% (w/v) HAuCl under vigorous stirring 4 ·3H 2 The O solution was added to 200 mL of ultrapure water, and after stirring well, 800. Mu.L of 1% (w/v) Na was added 3 C 6 H 5 O 7 Solution and 400. Mu.L of 30 mM Hydroquinone (C) 6 H 6 O 2 ) The solution is quickly added into the mixed solution for reduction. The above reducing agent was repeatedly added to the mixed solution 7 times at intervals of 10 minutes, and after the addition was completed, the mixed solution was further stirred for 1 hour, and finally, 1 mM H was synthesized by the following method 2 PdCl 4 Solution, 10.64 mg of palladium chloride (PdCl) 2 ) Is dissolved in 6 mL of 0.02M HCl solution and 54 mL of ultrapure water are added to the mixture in a water bath at 70 ℃ until PdCl 2 Complete dissolution to give 1 mM H 2 PdCl 4 Solutions of Au @ PdNPs obtained by deposition of Palladium, 30 mL of 75 nm AuNPs solution with 1 mL, 4 mL, 7 mL and 10 mL of H, respectively 2 PdCl 4 The solutions (1 mM) were mixed, the mixed solution was cooled in an ice bath, 1 mL, 4 mL, 7 mL and 10 mL ascorbic acid solution and 18 mL, 12 mL, 6 mL and 0 mL ultrapure water were respectively and slowly added to the mixed solution under vigorous stirring, then the mixed solution was further stirred for 30 minutes, after stirring, the resulting solution was centrifuged, washed with water-ethanol solution (1, 2, v/v) several times, and finally, au @ PdNPs were dissolved in C 6 H 5 Na 3 O 7 In solution (0.02%, w/v).
In order to achieve the purpose, the following technical scheme is adopted to realize the purpose: before detecting STR, the concentration of hairpin DNA-AgNCs is diluted to 1 μ M and the excitation wavelength is 586 nm in the whole experiment process, and the detection result of STR is obtained under the optimal experiment condition, 500 μ L of DNA-AgNCs and 500 μ L of Au @ PdNPs solution are mixed for 15 minutes, a series of STR solutions with different concentrations of 500 μ L are respectively added into the mixed solution of DNA-AgNCs and Au @ PdNPs, the reaction lasts for 60 minutes, and then the fluorescence intensity is measured.
The preparation process of the aptamer sensor comprises the following steps: STRs were added to milk at three concentration levels (300 nM, 600 nM and 900 nM) before pretreatment, and subsequently, the milk sample was pretreated by the following method, 20 mL of a milk-methanol (1, 4, v/v) mixture was kept at-20 ℃ for 20 minutes, then the mixture was centrifuged at 12500 rpm for 25 minutes, 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 ℃, the residue was dried with nitrogen and then reconstituted with ultrapure water, the reconstituted solution was filtered through a membrane filter (0.22 μm), and finally, the volume of the filtrate was set to 4 mL.
Drawings
FIG. 1 shows a process for constructing a fluorescence aptamer sensor.
FIG. 2 excitation and emission wavelengths of DNA-AgNCs synthesized from different template strands.
FIG. 3 fluorescence spectrum and fluorescence intensity of DNA-AgNCs.
FIG. 4 Electron microscopy characterization of nanomaterials.
Fig. 5 optimization of palladium shell thickness.
Figure 6 experimental conditions were optimized.
FIG. 7 fluorescent determination of STR.
FIG. 8 specific assay.
FIG. 9 STR testing in real samples of milk.
Detailed Description
Example 1: FIG. 3A shows fluorescence spectra of DNA-AgNCs of different template strand synthesis, excitation and emission wavelengths are listed in FIG. 2, and fluorescence intensity is shown in FIG. 3B, however, in DNA 2 、DNA 3 And DNA 4 、DNA 5 In (2), 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 0 to 6In time, the fluorescence intensity did not change much, however, when the number of G bases increased to 8, significant enhancement and change occurred due to DNA 4 -AgNCs、DNA 5 -AgNCs、DNA 6 AgNCs and DNA 7 The emission spectrum of AgNCs is close to the near infrared region, which means that it requires large metal nanoparticles, but larger metal nanoparticles may cause scattering, leading to a distortion of the fluorescence spectrum, DNA 3 Fluorescence intensity of AgNCs in DNA 1 -AgNCs、DNA 2 AgNCs and DNA 3 The strongest of the AgNCs, DNA 3 -position ratio DNA of maximum emission peak of AgNCs 2 The AgNCs are short, so the quencher nanoparticles required are smaller in diameter and less susceptible to scattering, and in summary, DNA was selected 3 AgNCs as fluorescent material used in this experiment.
Example 2: DNA Observation by TEM 3 Morphology of AgNCs, DNA synthesized as shown in FIG. 4A 3 AgNCs showed a uniform dispersion, almost circular, with an average size of 2.47 nm, and measured for DNA 3 Stability of AgNCs over 120 hours, results show, DNA 3 AgNCs are stable for 12-24 hours, so the experiment is on DNA 3 Measured DNA, performed within 12-24 hours after AgNCs synthesis 3 Absolute quantum yield of-AgNCs is 10.76%, TEM image of gold seeds is shown in FIG. 4B, from which it can be seen that the gold seeds are spherical particles with diameter of 19.9 nm, FIG. 4E shows large size gold nanoparticles synthesized by seed growth method with diameter of 75 nm, from FIGS. 4F-I, with different amount of H 2 PdCl 4 And ascorbic acid on the surface of AuNPs, it can be seen that the thickness of the palladium shell is about 4 nm, 7 nm, 11 nm and 16 nm, respectively, FIG. 4J-M shows EDX of Au @ PdNPs with diameter of 75+16 nm, it can be seen that palladium is dispersed in the outer layer of the gold core, the weight ratio of gold/palladium is 33.36/66.64, FIG. 4C, D shows SEM of Au @ PdNPs with diameter of 75+16 nm, according to the image, au @ PdNPs are almost spherical particles with diameter of 107.11 nm, the above analysis shows that Au @ PdNPs and DNA are almost spherical particles 3 The synthesis effect of the AgNCs is good.
Example 3: the optimization of the thickness of the palladium shell (FIG. 5), the energy transfer efficiencies (E) of 75+4 nm, 75+7 nm, 75+11 nm and 75+16 nm Au @ PdNPs are respectively 26.7%, 46.6%, 56.7% and 62.8%, it can be found that the energy transfer efficiency is gradually increased along with the increase of the thickness of the palladium shell, the 75+16 nm Au @ PdNPs has very high energy transfer efficiency which is enough to meet the experimental requirements, and meanwhile, the interference of metal nanoparticle scattering is increased by further increasing the particle size, so the 75+16 nm Au @ PdNPs with the highest quenching efficiency is selected as the quenching agent of the experiment.
Example 4: optimization of the conditions in the assay (FIG. 6), (A) optimization of the pH in the DNA-AgNCs synthesis (6.5-9.0), pH optimum 7.5, (B) addition of 0-24.8 pm concentrations of Au @ PdNPs to 1. Mu.M DNA 3 In AgNCs, the optimal concentration of Au @ PdNPs is 17.2 pM, (C) the incubation time for fluorescence quenching (0-30 min), the optimal quenching time is 15min, and (D) the incubation time for fluorescence recovery (0-90 min), and the optimal recovery time is 60min.
Example 5: the performance of the sensor for determining the analyte is evaluated by measuring STRs with different concentrations, as shown in FIG. 7, the fluorescence intensity gradually increases with the increase of the STR concentration within the range of 50 to 2800 nM, the fluorescence intensity and the STR concentration have a positive linear relation within the range of 50 to 1250 nM, and the equation of a linear calibration curve is as follows: i =0.43314C STR +102.94683, correlation coefficient (R) 2 ) 0.99258, with a limit of detection (LOD) of the STR fluorometry of 18.7 nM according to 3 σ/k, where σ is the standard deviation of the measurement of the calibration standard blank (n = 10) and k is the slope of the calibration curve.
Example 6: to evaluate the specificity of the sensor, the specificity of the DNA-AgNCs-based fluorescence aptamer was determined by comparison with other antibiotics such as Telavancin (TER), chloramphenicol (CHL), tetracycline (TET), erythromycin (ERY), chlortetracycline hydrochloride (CHH) and Penicillin (PEN), at a concentration 10 times that of STR, as shown in FIG. 8, I and I 0 Indicating the fluorescence intensity in the presence and absence of different types of antibiotics, respectively, and that STR responds to fluorescence aptamers more strongly than other antibiotics, these results indicate that fluorescence adaptationThe instrument has strong specificity for detecting STR, and shows that the developed fluorescence adapter can be used for detecting STR.
Example 7: to evaluate the feasibility of this invention to detect STR in milk samples, we added three different concentrations of streptomycin to actual milk, and the recovery is shown in fig. 9, and it can be seen that the peak recovery of kanamycin detection is between 97.45% and 105.34%, and the Relative Standard Deviation (RSD) is between 1.05% and 3.04%, and these excellent properties indicate the feasibility of our proposed streptomycin detection method in this application.

Claims (4)

1. A preparation method of a fluorescence aptamer sensor for detecting streptomycin in milk is characterized by comprising the following steps: a fluorescence aptamer sensor based on guanine (G) base-regulated hairpin-type DNA silver nanoclusters (DNA-AgNCs) and core-shell gold-palladium nanoparticles (Au @ PdNPs) is constructed and used for detecting Streptomycin (STR).
2. The method for preparing an aptamer sensor for detecting streptomycin in milk according to claim 1, wherein the aptamer sensor comprises: 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 the DNA-AgNCs.
3. The method of claim 1, wherein the aptamer sensor for detecting streptomycin in milk comprises: the excellent quenching material Au @ PdNPs is prepared by a seed growth method and a deposition method, the Au @ PdNPs and the DNA-AgNCs are utilized to carry out fluorescence resonance energy transfer, and the streptomycin is detected by the change of fluorescence signals of the Au @ PdNPs and the DNA-AgNCs under the corresponding excitation wavelength.
4. The method of claim 1, wherein the aptamer sensor for detecting streptomycin in milk comprises: the sensor has better sensing performance, and can detect STR residues in an actual milk sample.
CN202211154362.7A 2022-09-22 2022-09-22 Method for measuring streptomycin in milk by using fluorescence sensor constructed by silver nanoclusters and gold palladium nanoparticles Pending CN115452787A (en)

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