Preparation method of a ratio fluorescent aptamer sensor for detecting streptomycin

Abstract

The application provides a preparation method of a ratio fluorescent aptamer sensor for detecting streptomycin in milk, and belongs to the field of food safety detection. The application comprises screening of streptomycin (STR) specific aptamer and construction of a ratio fluorescent sensor. The STR aptamer (Apt) is obtained by using a magnetic graphene oxide screening technology for the first time, the binding site is analyzed according to the secondary structure and molecular docking analysis, and the Apt52 with the best affinity to the target is obtained by truncation. The carbon quantum dots (CQDs) modified by the Apt52 are used as a reference signal, the AgNCs-SMP@ZIF-8 is used as a response signal to construct a fluorescent sensing method for judging whether the streptomycin is contained in the sample to be detected. The specific binding of the aptamer to the target causes the AgNCs-SMP@ZIF-8 to expose and quickly adsorb a large amount of Cu 2+ The fluorescence signal of the AgNCs-SMP@ZIF-8 is quenched, and the fluorescence signal of the CQDs always remains stable, and the streptomycin residue is detected by the fluorescence signal ratio change of the AgNCs-SMP@ZIF-8 and the CQDs. The detection limit of the sensor for the STR is 0.978 nM, and the sensor has satisfactory specificity and sensitivity.

Description

Technical Field

[0001] This invention provides a method for preparing a ratiometric fluorescent aptamer sensor for detecting streptomycin, belonging to the field of food safety detection technology. Background Technology

[0002] Streptomycin (STR) is an important aminoglycoside antibiotic used to treat diseases caused by Gram-negative bacilli. However, the misuse of STR can leave residues in animal-derived foods, spreading to humans through the food chain and causing serious side effects such as ototoxicity and nephrotoxicity, and even leading to bacterial mutations and antibiotic resistance. Therefore, establishing rapid, sensitive, and accurate methods for detecting STR residues is crucial. Currently, sensitive detection methods such as high-performance liquid chromatography, microbial detection, enzyme-linked immunosorbent assay (ELISA), and colorimetric sensors are used to detect STR residues in milk, but their application is limited by drawbacks such as long detection times, cumbersome procedures, expensive equipment, and high technical requirements. Summary of the Invention

[0003] The purpose of this invention is to provide a rapid, accurate, convenient, and sensitive detection technology for detecting STRs in milk.

[0004] The technical solution involves obtaining single-stranded DNA or RNA fragments with high affinity for target substances from random libraries using the Systematic Evolutionary Ligand Expansion (SELEX) technique. Due to its advantages such as ease of in vitro synthesis, low immunogenicity, and high stability, it is widely used in food safety analysis, environmental monitoring, and drug target screening.

[0005] The aforementioned method for screening aptamers for detecting STR in milk ( Figure 1 The key feature is the use of magnetic graphene oxide (MGO)-SELEX screening to select STR-specific aptamers (Apt). Based on high-throughput sequencing results, secondary structure, and molecular docking simulation, the aptamers were rationally truncated, retaining the long stem portion that binds to the STR. The binding affinity of the aptamers to the STR before and after truncation was compared using fluorescent labeling, based on the dissociation constant (K). d The aptamer with the strongest affinity for STR is selected as the recognition element of the sensor.

[0006] The method for preparing a ratiometric fluorescent aptamer sensor for detecting STRs in milk (as described above) Figure 2The key feature is that the reference signal is aptamer-functionalized carbon quantum dots (CQDs) that emit green fluorescence. The response signal is AgNCs-SMP@ZIF-8 encapsulated in a skim milk powder (SMP) protein and zeolite imidazole framework-8 (ZIF-8). ZIF-8 can both utilize the rigid framework of metal-organic materials to spatially separate AgNCs in a certain direction, improving the luminescence performance and stability of AgNCs; and its porous structure can adsorb accumulated quencher copper ions (Cu). 2+ It rapidly penetrates the pores of ZIF-8, forming larger aggregates with AgNCs. Simultaneously, it binds to the AgNCs template glutathione, quenching fluorescence and ensuring the red fluorescence signal of the nanocomposite material is consistent with Cu. 2+ The response is faster. In summary, we have established a ratio fluorescence detection method for detecting STRs.

[0007] 2+ Unquenchable, the sensor exhibits dual-emission fluorescence signals. In the presence of STR, competition between STR and ZIF-8 weakens π-π stacking, leading to the dissociation of CQDs-Apt and AgNCs-SMP@ZIF-8, Cu... 2+ AgNCs-SMP@ZIF-8 forms aggregates and binds to glutathione to quench the fluorescence signal of AgNCs-SMP@ZIF-8. The change in the ratio of fluorescence signals can enable the quantitative detection of STRs.

[0008] To achieve the above objectives, the following technical solution was adopted: For the preparation of AgNCs-SMP@ZIF-8, firstly, 50 mM glutathione was dissolved in 4.8 mL of ultrapure water, and 125 μL of silver nitrate solution (20 mM) was added under magnetic stirring at room temperature. The pH was adjusted to 9.0 with sodium hydroxide solution. Subsequently, 5 mg of α-lipoic acid was dissolved in 100 mM sodium borohydride solution and stirred vigorously until the solution became clear. This solution was then immediately added to the silver mixture, and the mixture was stirred for 30 min, followed by incubation at room temperature for 2 h. The obtained AgNCs were then incubated with 6 mL of SMP (10 mg / mL) for 1 h, followed by stirring with 10 mL of zinc nitrate hexahydrate solution (35 mM) at room temperature for 5 min. Next, 10 mL of 2-methylimidazole solution (280 mM) was added, and stirring was continued for 24 h. The resulting milky white mixture was centrifuged at 6500 rpm for 10 min and washed three times. Finally, the synthesized AgNCs-SMP@ZIF-8 was stored in the dark at 4°C.

[0009] To achieve the above objectives, the following technical solution was adopted: Before detecting STRs, 1 μM of synthesized CQDs-Apt52 probe solution (based on DNA concentration) was incubated overnight at 4°C with 225 μL of AgNCs-SMP@ZIF-8 (40 μM). Then, a series of STR solutions of different concentrations (1 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, and 500 nM) were added to the AgNCs-SMP@ZIF-8 / CQDs-Apt52 solution, and the mixture was incubated at 37°C for 3 h. Then, 50 μL of Cu was added. 2+ After the solution reacted for 90 seconds, it was transferred to a micro-cubic cell, and the fluorescence emission spectrum in the range of 450~800nm ​​was collected at an excitation wavelength of 425nm and a slit width of 5nm.

[0010] The fabrication process of the aptamer sensor is as follows: Before pretreatment, milk samples were supplemented with STR at concentrations of 20 nM, 100 nM, and 200 nM, respectively. Then, the samples were pretreated using the following method: First, three 4 mL aliquots of milk samples with the target were each added to 16 mL of methanol, frozen at -20°C for 20 min, and then centrifuged at 12000 rpm for 15 min to remove the precipitate. The supernatant was passed through a 0.22 μm filter membrane, the filtrate was dried at 60°C, and the remaining solution was dried with nitrogen. After resuspending in ultrapure water, the solution was passed through a 0.22 μm filter membrane again, and the volume of the resulting solution was set to 4 mL. Attached Figure Description

[0011] Figure 1 The process of aptamer selection.

[0012] Figure 2 The process of constructing an aptamer sensor.

[0013] Figure 3 To screen for recovery rate and gel electrophoresis imaging.

[0014] Figure 4 Structural prediction and truncation strategies for candidate aptamers.

[0015] Figure 5 To determine the affinity between the aptamer and STR.

[0016] Figure 6 Electron microscopy characterization and fluorescence intensity measurement of AgNCs-SMP@ZIF-8.

[0017] Figure 7For the stability of AgNCs-SMP@ZIF-8 / CQDs-Apt52.

[0018] Figure 8 To optimize the experimental conditions.

[0019] Figure 9 For fluorescence determination of STR.

[0020] Figure 10 For specific testing.

[0021] Figure 11 STR testing for actual milk samples. Implementation

[0022] Example 1: The binding rate of the screening process is as follows Figure 3 As shown in Figure A, with the increase of the selection rounds, except for rounds 6 and 10 where antibiotics such as penicillin, chloramphenicol, cephalexin, and tetracycline were added simultaneously for reverse screening, the overall binding rate gradually increased, with a recovery rate of 65% in round 13. Compared with the gel electrophoresis of the first round ( Figure 3 B), the gel electrophoresis in round 13 of the figure ( Figure 3 C) When the target band is displayed in the correct position with clearer and brighter bands and no extraneous bands, the screening ends.

[0023] Example 2: The PCR products from the 13th round were cloned and sequenced. The top 30 highly enriched sequences from the sequencing results were analyzed for homology and secondary structure prediction using DNAMAN 8 software. Figure 4 A shows that it can be roughly divided into three different families, and a highly enriched and stable secondary structure representative sequence is selected from each family for binding affinity analysis. Figure 4 B indicates that all three candidate aptamers (Apt3, Apt5, and Apt6) have strong affinity for STR, with Apt5 showing the highest K... d The smallest value indicates the strongest affinity. According to... Figure 4 Molecular simulations of the three candidate aptamers in C were performed, and non-essential bases were truncated to obtain five truncated aptamers: Apt31, Apt51, Apt52, Apt61, and Apt62. Figure 4 D).

[0024] Example 3: Binding curves of 5 truncated aptamers as shown in the figure. Figure 5 As shown in Figure A, the K of Apt52 can be seen in the figure. d The value was the lowest, at 14.52 nM, indicating that Apt52 has the strongest affinity for STR. Figure 5B shows the circular dichroism (CD) spectra before and after aptamer binding to the target. The CD spectrum of the target in the binding buffer is close to baseline, indicating a very weak CD signal. The CD spectrum of Apt52 has a positive peak at 270 nm and a negative peak at 240 nm. This indicates that Apt52 has a B-type DNA structure. After adding the target to the aptamer, the negative peak at 240 nm is significantly enhanced, which means that the target conformation embedded in the aptamer changes, forming a stable complex.

[0025] Example 4: Transmission electron microscopy images of AgNCs and AgNCs-SMP@ZIF-8 are shown below. Figure 6 As shown in Figure A, the results indicate that AgNCs exhibit good and uniform monodispersity. The AgNCs-SMP@ZIF-8 encapsulated with SMP and then encapsulated in ZIF-8 exhibits the ZIF-8 contour structure. This is consistent with the dodecahedral structure shown in the SEM image. Figure 6 B). In addition, we also used EDS to study the elemental characteristics of AgNCs-SMP@ZIF-8. Figure 6 C shows the EDS image of AgNCs-SMP@ZIF-8, indicating that the synthesized material elements include C, N, O, Zn and Ag, confirming that AgNCs were successfully encapsulated in ZIF-8. Figure 6 D shows the FTIR spectra of AgNCs, ZIF-8, and AgNCs-SMP@ZIF-8. At 419 cm⁻¹ −1 The peak value at 692 cm corresponds to the tensile vibration of Zn-N, while at 692 cm... −1 The peak value at 600 cm⁻¹ corresponds to the bending vibration of the imidazole ring. Both ZIF-8 and AgNCs-SMP@ZIF-8 show the imidazole ring bending vibration at 600 cm⁻¹. −1 and 1500 cm −1 Vibrations between [variables]. Typically, the C=N stretching mode is considered to occur at 1457 cm. −1 and 1516 cm −1 A significant peak value was observed at 2928 cm⁻¹. These results indicate that the zinc-containing compound interacts appropriately with the imidazole ligand, confirming the correct synthesis of ZIF-8. Furthermore, the imidazole ring showed a significant peak value at 2928 cm⁻¹. −1 and 3139 cm −1 The NH and CH groups exhibit tensile vibrations at 1660 cm⁻¹. However, in AgNCs-SMP@ZIF-8, C=O and NH groups show tensile vibrations at 1660 cm⁻¹. −1 and 3340 cm −1 The stretching vibrations at that location are not present in ZIF-8, but rather originate from AgNCs. These results indicate that AgNCs are encapsulated within ZIF-8, without affecting the original ZIF-8 FTIR peaks. Figure 6As shown in Figure E, the XRD modes of AgNCs-SMP@ZIF-8 and ZIF-8 are consistent. Characteristic diffraction peaks at 2θ = 7.4˚, 10.5˚, 12.8˚, 14.8˚, 16.5˚, 18.1˚, 24.7˚, and 26.8˚ indicate that the crystal structure of ZIF-8 is not affected by AgNCs encapsulation. To verify the fluorescence enhancement effect of coating and encapsulation, the fluorescence intensity of equal volumes of AgNCs, AgNCs-SMP, and AgNCs-SMP@ZIF-8 were measured. Figure 6 The F-value shows that the fluorescence intensity of AgNCs-SMP@ZIF-8 is 8 times and 2 times higher than that of AgNCs and AgNCs-SMP, respectively. This indicates that AgNCs are indeed encapsulated by the SMP coating and ZIF-8, and the fluorescence intensity is gradually enhanced.

[0026] Example 5: Stability of AgNCs-SMP@ZIF-8 / CQDs-Apt52 over 15 days Figure 7 As shown, the fluorescence signal decreased by approximately 3.56% within 15 days in a closed environment at 4°C in the dark. The decrease in fluorescence intensity may be attributed to the oxidation of AgNCs-SMP@ZIF-8 by oxygen in the air, which may have led to the destruction of some AgNCs, resulting in structural changes.

[0027] Example 6: Optimization of experimental conditions ( Figure 8 ). Figure 8 The results from A showed that the fluorescence intensity of AgNCs varied with pH; an alkaline environment increased the fluorescence intensity of AgNCs, but an excessively alkaline environment decreased it. Therefore, the optimal pH for synthesizing AgNCs was determined to be 9.0. Figure 8 The results in B indicate that the coating solutions bovine serum albumin (BSA), ovalbumin (OVA), and SMP all enhanced the fluorescence intensity of the responsive signaling material. The strongest enhancement from the SMP coating is likely due to its higher amino acid residue content, while the AgNCs-SMP interaction may induce energy transfer from different metal ligand residues from SMP to AgNCs. Therefore, we selected SMP, which showed the best performance, for subsequent experiments. Figure 8 C shows that the fluorescence intensity of AgNCs-SMP@ZIF-8 increases with increasing SMP encapsulation concentration, decreasing until 15 mg / mL, with the highest fluorescence intensity observed at a coating concentration of 10 mg / mL. At concentrations greater than 10 mg / mL, the encapsulation effect of SMP on AgNCs is stronger than its fluorescence enhancement effect. Therefore, we chose 10 mg / mL SMP for coating AgNCs. Excessive probe concentration leads to insignificant quenching response, while insufficient concentration reduces the sensor's detection sensitivity. Figure 8D shows that the detection performance is best when the volume ratio of the detection probe to the response material is 1:3. To determine the optimal quenching concentration, Cu at different concentrations ranging from 1 to 800 ng / mL were used. 2+ The fluorescence intensity after quenching was measured when AgNCs-SMP@ZIF-8 solution was introduced into the solution containing 1 nM and 100 nM STR, respectively. Figure 8 E shows Cu 2+ The fluorescence intensity difference was greatest at a concentration of 100 ng / mL, therefore 100 ng / mL Cu was selected. 2+ As the quenching concentration, the optimal fluorescence quenching time was determined by observing the concentration of 100 ng / mL Cu. 2+ The change in fluorescence intensity ratio when added to 100 nM STR was used to determine this. Figure 8 As shown in F, the fluorescence intensity ratio (I) 685 / I 520 The temperature gradually decreases and stabilizes after 90 s; therefore, 90 s is selected as the optimal quenching time.

[0028] Example 7: Recording the standard curve for STR fluorescence measurement under optimal experimental conditions. (e.g.) Figure 9 As shown, fluorescence spectra of STR concentrations ranging from 1 nM to 500 nM were measured. The results clearly demonstrate the ratio of STR concentration to fluorescence intensity (IL). 685 / I 520 There is a direct correlation between fluorescence intensity and STR concentration, with the latter continuously decreasing as the former increases. The fluorescence intensity is linearly negatively correlated with STR concentration. The linear regression equation is as follows: y = -0.00221x + 1.3423, and the correlation coefficient (R²) is... 2 The signal-to-noise ratio (SNR) is 0.999. Based on the three signal-to-noise ratios (S / N=3), the detection limit (LOD) of this method is calculated to be 0.978 nM.

[0029] Example 8: The susceptibility of AgNCs-SMP@ZIF-8 / CQDs-Apt52 to various antibiotics was tested using the same experimental conditions. The concentration of STR was 500 nM, while the concentrations of penicillin (PCN), chloramphenicol (CAP), cephalexin (CHP), tetracycline (TET), azithromycin (AZM), amoxicillin (AMX), cefazolin (ZOX), and neomycin (NEO) were 5000 nM. Results are as follows... Figure 10 As shown, the fluorescence intensity ratio (I) 685 / I 520 The decrease was significant only in the presence of STRs, which means that the developed fluorescent aptamer sensor is more sensitive to STRs than other antibiotics, indicating that the sensor has higher specificity for STRs.

[0030] Example 9: To investigate the applicability of ratiometric fluorescent aptamer sensors in milk, we added three different concentrations of STR to actual milk. The recovery rates are shown in [Figure 9]. Figure 11 As can be seen, the peak recoveries for kanamycin detection ranged from 96.4% to 104.6%, with relative standard deviations (RSDs) ranging from 2.65% to 4.47%. These excellent performance characteristics demonstrate the feasibility of our proposed STR detection method in practical applications.

Claims

1. A method for preparing a ratiometric fluorescent aptamer sensor for detecting streptomycin in milk, characterized in that: A novel ratiometric fluorescent aptamer sensor based on CQDs and AgNCs-SMP@ZIF-8 was constructed using the streptomycin (STR)-specific aptamer Apt52 obtained by magnetic graphene oxide (MGO)-SELEX screening as the recognition element for the detection of STR.

2. The method for preparing a ratiometric fluorescent aptamer sensor for detecting streptomycin in milk as described in claim 1, characterized in that: STR-specific aptamers were obtained by screening and truncation using MGO-SELEX, and the high-affinity aptamer Apt52 was obtained by truncation of the docking binding site based on molecular simulation.

3. The method for preparing a ratiometric fluorescent aptamer sensor for detecting streptomycin in milk as described in claim 1, characterized in that: Using Cu 2+ Electron transfer and aggregation induction between AgNCs-SMP and ZIF-8 avoid quenching the fluorescence signal of CQDs in a relatively short wavelength range. STRs are detected by the change in the fluorescence signal ratio between AgNCs-SMP@ZIF-8 and CQDs under the same excitation wavelength.

4. The method for preparing a ratiometric fluorescent aptamer sensor for detecting streptomycin in milk as described in claim 1, characterized in that: This sensor has good sensing performance and can detect STR residues in actual milk samples.

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