Fluorescent sensor for detecting kanamycin and tobramycin in milk based on two-color fluorescent silver nanoclusters

Abstract

The invention relates to a fluorescence sensor for detecting kanamycin and tobramycin in milk based on two-color fluorescent silver nanoclusters, and belongs to the field of food safety detection. The preparation method comprises the following steps: linking an aptamer and a silver nano-cluster template fragment by using a carbon 12 spacer arm, and synthesizing the double-color fluorescent silver nano-cluster. The synthesized silver nanocluster can generate strong red fluorescence and yellow fluorescence which do not interfere with each other at the wavelength of 570 nm and the wavelength of 677 nm, synthesis of the silver nanocluster is promoted through introduction of a C12 spacer group, the fluorescence intensity is enhanced, and cross reaction in the quenching process is reduced. After the aptamer fragment is combined with kanamycin and tobramycin, the structure and conformation of the aptamer fragment are changed, so that the fluorescence of the corresponding silver nano-cluster is quenched. The fluorescence sensor has good sensitivity, the detection limits of kanamycin and tobramycin are 180 pM and 860 pM respectively, the recovery rates of labeled milk are 93.6%-110.1% and 91.4%-98.6% respectively, and meanwhile, the specificity is good. It is proved that the method can be applied to detection of kanamycin and tobramycin in a milk sample and has important application prospects in simultaneous detection and analysis of various antibiotics in food safety application.

Description

Technical Field

[0001] The invention provides a fluorescent sensor for detecting kanamycin and tobramycin in milk based on dual-color fluorescent silver nanoclusters, belonging to the field of food safety detection. Background Art

[0002] Kanamycin KAN and tobramycin TOB are both aminoglycoside antibiotics, which are widely used to treat mastitis and various bacterial infections in dairy cows due to their broad antibacterial spectrum and strong antibacterial activity. However, improper use of these drugs can cause residues in milk, which can cause ototoxicity, nephrotoxicity, and neuromuscular blockade to humans.

[0003] The existing traditional detection methods for KAN and TOB include: liquid chromatography tandem mass spectrometry, enzyme-linked immunosorbent assay, and liquid chromatography-evaporative light scattering detection, etc. These methods have the disadvantages of being complex to operate, requiring professional personnel to operate, expensive instruments, and high costs. In view of these shortcomings, it is necessary to develop an efficient, simple, and inexpensive detection method for KAN and TOB.

[0004] Compared with other strategies and methods, fluorescence sensors have high sensitivity, good selectivity, simple operation, low cost and good biocompatibility. The method of fluorescence detection through AgNCs has adjustable fluorescence intensity and position, good fluorescence lifetime, good photostability and chemical stability, all of which are conducive to the construction of sensors and the sensing detection of more targets.

[0005] C12 spacer is a commonly used modification in oligonucleotide synthesis. It consists of 12 carbon atoms and is used to introduce a certain distance between the oligonucleotide and the modification group. This distance helps to reduce steric hindrance, avoid adverse interactions between the modification group and the oligonucleotide, and increase the flexibility of the molecule. In this way, the DNA chain structure can reduce adverse folding rotations. And it can minimize the cross-reaction and influence between the two clusters during quenching. Summary of the invention

[0006] The purpose of the invention is to provide an efficient, convenient, economical and sensitive detection method for detecting KAN and TOB in milk.

[0007] The technical solution is as follows: Aptamers have the advantages of high sensitivity and strong specificity, and are often used and constructed as sensing elements of biosensors. Compared with other sensors, the fluorescent aptamer sensor method has the advantages of high sensitivity and good selectivity. Nanometal clusters have the advantages of good signal intensity, simple preparation method, and fast response speed. However, the fluorescence signals of various metal clusters are prone to signal overlap and interference with each other, and cross-reactions are prone to occur when the signals change. Therefore, the silver nanocluster AgNCs template is screened and modified with C12 spacer to avoid signal overlap and cross-reactions between multiple fluorescences.

[0008] The fluorescent sensor for detecting kanamycin and tobramycin in milk based on dual-color fluorescent silver nanoclusters ( Figure 1 ), characterized in that: the sensor uses two types of AgNCs with red and yellow fluorescence as output signals for detecting TOB and KAN respectively. After each cluster template fragment is linked to the corresponding aptamer fragment, the two groups are connected by C12 spacer to form the DNA chain used. Two types of AgNCs with yellow and red fluorescence are generated by reduction method, with peaks at wavelengths of 570 nm and 677 nm respectively. When KAN or TOB exists, it will recognize and bind to the aptamer fragment of the DNA sequence, affecting its structure and conformation, and then produce a quenching effect on the AgNCs nearby, and the corresponding fluorescence intensity decreases, thereby realizing the detection of KAN and TOB.

[0009] The fluorescent sensor for detecting kanamycin and tobramycin in milk based on dual-color fluorescent silver nanoclusters is characterized by: synthesizing two fluorescent AgNCs and containing two aptamer fragments on one DNA chain, which is prone to overlap of two signals and cross-reaction of signals in the quenching process. In the process of DNA chain synthesis, the intermediate modifying group is used to modify it at intervals to avoid bending or folding of the DNA chain as much as possible, and the stable chain structure is maintained, which is conducive to the synthesis of AgNCs on the template and has a higher fluorescence intensity. At the same time, the candidate template chain is measured and screened, and the template sequence with good signal and no overlap is selected to synthesize AgNCs, avoiding signal interference.

[0010] The preparation principle is as follows: Using the synthesized DNA sequence, we designed a fluorescent sensor based on AgNCs with two fluorescent colors and the cluster fluorescence quenching scheme of DNA chain structure conformation change to detect KAN and TOB in milk. The aptamer fragments of KAN and TOB are close to the template fragments of yellow and red silver nanoclusters respectively. When KAN or TOB binds to the aptamer fragment, it will cause changes in the structure and conformation of the DNA sequence, resulting in quenching of the corresponding cluster fluorescence and a decrease in the signal.

[0011] To achieve the above objectives, the following technical schemes were adopted: Preparation of DNA-silver nanoclusters DNA-AgNCs, according to the regulations of the synthesis company, first dilute the DNA chain to a concentration of 100 μM with 1×TE buffer (pH 5.0-8.0). Further adjust and dilute with a fixed ratio of DNA to TE buffer of 1:7.8. Add silver nitrate (2 mM) to the diluted DNA solution, and finally the molar concentration ratio of DNA (100 μM): silver nitrate: sodium borohydride is 1:12:12. Subsequently, the solution is thoroughly mixed on a mixer for 1 min to ensure sufficient mixing. The mixed solution is sealed and incubated in ice water for 30 min under light-proof conditions. Subsequently, prepare a new sodium borohydride solution (2 mM): 0.038 g of sodium borohydride is dissolved in 10 mL of ultrapure water to prepare a 100 mM sodium borohydride solution, which is further diluted to a sodium borohydride solution with a concentration of 2 mM. Freshly prepared sodium borohydride solution (2 mM) was added to the mixture and immediately mixed thoroughly for 1 min. The mixture was then incubated at room temperature in the dark for 4 h. Finally, the mixture was stored at 4°C in the dark overnight.

[0012] To achieve the above objectives, the following technical scheme was adopted: appropriate concentrations of KAN and TOB were prepared for detection and calibration. 50 μL of DNA-AgNCs (10 μM) solution was added to 200 μL of ultrapure water. Subsequently, 50 μL of different concentrations of KAN and TOB were added to the solution and reacted at room temperature in the dark for about 2 min to achieve competitive binding. After the reaction was completed, the fluorescence intensity was measured by a fluorescence spectrophotometer. In the KAN detection, the excitation light of 480 nm was used for excitation, and the emission spectrum between 500-655 nm was collected. There was an obvious peak at around 570 nm, corresponding to the AgNCs of the KAN aptamer template. For the detection of TOB, the excitation light of 600 nm was used for excitation, and the emission spectrum between 620-700 nm was collected. There was an obvious peak at around 677 nm, corresponding to the AgNCs of the TOB aptamer template. The difference in fluorescence intensity was compared with the blank control without antibiotics.

[0013] The preparation process of the aptasensor is as follows: milk samples were purchased from the local market. Before sample pretreatment, KAN at concentrations of 1, 2, and 3 nM and TOB at concentrations of 2, 5, and 7 nM were added to the milk samples, and then protein and fat were removed. First, 4 mL of each added milk sample was added to 16 mL of ice methanol and stored at -20°C for 20 min. Then centrifuged at 12000 rpm for 15 min to remove the precipitate. The supernatant was filtered with a 0.22 μm membrane, the filtrate was dried at 60°C, and the residual liquid was further dried with nitrogen. The resulting residue was re-dissolved with ultrapure water and filtered again through a 0.22 μm filter membrane. Finally, the volume was made up to 4 mL with ultrapure water. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The construction process of fluorescent aptasensor.

[0015] Figure 2 A uses the emission spectrum of AgNCs. Figure 2 B Effects of multiple intermediate modifiers.

[0016] Figure 3 Characterization diagram of DNA-AgNCs (A transmission electron microscopy of DNA-AgNCs, B diameter distribution of AgNCs, C excitation and emission spectra of AgNCs, D element mapping of DNA-AgNCs).

[0017] Figure 4 XPS characterization of DNA-AgNCs (A full spectrum, B detailed spectrum of Ag element).

[0018] Figure 5 Optimization of experimental conditions (A and B optimize pH, C and D optimize reaction time).

[0019] Figure 6 A. Fluorescence spectra and B. standard curves of KAN and TOB determination.

[0020] Figure 7 A: Circular dichroism (CD) spectrum during the competitive binding process; B: Cross-reaction effect diagram.

[0021] Figure 8 Sensor-specific performance graph. DETAILED DESCRIPTION

[0022] Embodiment 1: Figure 2 In order to ensure that the fluorescence peaks of the synthesized AgNCs do not overlap and interfere with each other, we selected multiple templates (DNA 1 , DNA 2 and DNA3 ) to synthesize AgNCs and then perform fluorescence detection. The fluorescence intensity results of AgNCs, such as Figure 2 As shown in A. Using DNA 1 , DNA 2 and DNA 3 The emission peaks of the synthesized AgNCs are around 550 nm (blue), 575 nm (yellow), and 675 nm (red). By comparison, it was found that the spacing between red and blue or yellow AgNCs was more appropriate, and there was no obvious overlap between the emission peaks. At the same time, the fluorescence intensity of red and yellow AgNCs was high and comparable, while the fluorescence intensity of blue AgNCs was low, which was not conducive to sensing. Therefore, we chose DNA 2 and DNA 3 The template was used as the best template for synthesizing yellow and red AgNCs. In order to further reduce the cross-reaction between yellow and red AgNCs, we introduced a C spacer to connect the two template fragments and explored three C spacers of different lengths. The fluorescence intensity of AgNCs synthesized using DNA sequences with C12 spacer, C6 spacer and spacer 18 is shown in Figure 2. Figure 2 As shown in Figure B, it is clear that the length of C spacer affects the synthesis of AgNCs. The AgNCs synthesized using the C12 spacer to construct the DNA sequence showed a fluorescence intensity significantly higher than that synthesized using the C6 spacer to construct the DNA sequence. The red fluorescence intensity of the AgNCs synthesized using Spacer 18 to construct the DNA sequence did not increase significantly compared to the C12 spacer, but the yellow fluorescence intensity decreased slightly. This indicates that too long a spacer will destroy the DNA chain structure and lead to unnecessary rotation effects. Considering the requirements between fluorescence intensity and effective sensing, C12spacer was finally selected to connect the two DNA templates to obtain the DNA sequence-DNA 5 The choice of C12 spacer balances the cost considerations of DNA sequence synthesis and structural integrity.

[0023] Embodiment 2: Figure 1 The process of constructing the sensor is demonstrated. After the dual fluorescent AgNCs were synthesized using the template method, 50 μL of DNA-AgNCs were mixed with different concentrations of KAN and TOB, and finally ultrapure water was added to make the volume 350 μL. After 2 minutes of competitive reaction, the solution was placed in a fluorescence spectrophotometer, excited with a wavelength of 480 nm or 600 nm, and the fluorescence spectrum was measured to calculate the corresponding target concentration. When KAN or TOB is present, the target will bind to the corresponding aptamer fragment, thereby changing the structural conformation of the DNA chain and quenching its corresponding fluorescence, and the fluorescence intensity will decrease, such as Figure 1As shown in A; when KAN and TOB are not present, no fluorescence quenching occurs, and AgNCs still maintain the corresponding bright fluorescence, thereby completing the construction of the sensor.

[0024] Example 3: The size and element distribution of DNA-AgNCs were characterized by transmission electron microscopy. Figure 3 As shown in A, the AgNCs are nearly round. The size distribution of the synthesized AgNCs is shown in Figure 3 As shown in B, its diameter is mainly in the range of 2-4 nm, with an average diameter of 2.7 nm ( Figure 3 B). Emission and excitation spectra of two different AgNCs (5 μM) are shown in Figure 2. Figure 3 C. The yellow fluorescence corresponding to the KAN aptamer reaches a peak at 575 nm, with a fluorescence intensity of about 12000; the red fluorescence corresponding to the TOB aptamer reaches a peak at 677 nm, with an intensity of about 18000. At the same time, the synthesized DNA-AgNCs were analyzed by UV spectroscopy. Figure 3 The inset in B compares the appearance of the DNA-AgNCs solution under sunlight and 365 nm excitation light. The solution appears light yellow in sunlight and bright orange under 365 nm excitation light. Figure 3 C shows the excitation spectrum and emission spectrum of the AgNCs used. It can be seen that 480 nm is the optimal excitation wavelength for yellow fluorescence, and its peak appears at 570 nm when excited; 600 nm is the optimal excitation wavelength for red fluorescence, and its peak appears at 677 nm when excited. The emission wavelengths and excitation wavelengths of the two fluorescent clusters do not overlap with each other, the peak wavelength intervals are moderate, and the intensities are close, which is suitable for multi-detection signals. Figure 3 D shows the EDX analysis of DNA-AgNCs, showing the distribution of oxygen, nitrogen, phosphorus, and carbon elements, which correspond to the elements contained in the DNA used in the synthesis process, and the distribution of silver elements is consistent with the circular area in the transmission electron microscope image. XPS characterization of the synthesized DNA-AgNCs was performed to understand the oxidation state of the Ag element, such as Figure 4 As shown. Figure 4 In A, the overall elemental mapping shows seven elements: S, P, C, N, O, Ag and Na, proving its existence in the system. Figure 4 In B, the Ag element has a clear 3D 5 / 2 and 3D 3 / 2 The presence of the tracks proves the existence of AgNCs and confirms the successful synthesis of AgNCs on the DNA template.

[0025] Example 4: Optimization of the pH and reaction time of the buffer used during the experiment ( Figure 5 ). Figure 5A and B are the effects of pH on fluorescence intensity. The fluorescence intensity is highest when pH=6.5, indicating that 6.5 is the optimal pH value. Figure 5 C and D are the optimization of reaction time. When the time is 2 min, quenching no longer continues, indicating that 2 min is the best reaction time.

[0026] Embodiment 5: Figure 6 A and B show the changes in fluorescence spectra after adding 500-4000 pM KAN and 1000-8125 pM TOB, respectively. During the detection process, it can be seen that the fluorescence intensity of yellow fluorescence and red fluorescence decreases with the increase of KAN and TOB concentrations, and finally stabilizes when KAN is 8125 pM and TOB is 4000 pM. A linear curve is fitted based on the quenched fluorescence intensity and a standard curve is drawn as shown in Figure 6 As shown in C and D, it can be seen that KAN and TOB have a good linear relationship with the quenching fluorescence intensity in the range of 500-4000 pM and 1000-8125 pM. The calibration curve is represented by I KAN =1.6080C KAN +1218, with a correlation coefficient as high as R 2 = 0.98, the calculated limit of detection (LOD) is as low as 180 pM (based on 3σ / k); I TOB =2.5303C KAN +4134.1, with a correlation coefficient as high as R 2 = 0.97, and the calculated LOD was as low as 860 pM.

[0027] Example 6: CD analysis of DNA strands before and after binding with KAN and TOB Figure 7 A. The CD spectrum resembles an atypical B-type structure with an A configuration due to the high content of CG bases in the DNA template. At 260 nm, a positive peak was observed, slightly shifted to the right, but still broad. The region around 220 nm is close to the baseline and there is no obvious negative peak. The binding of DNA to KAN reduces the compactness of base stacking, thereby weakening the negative peak near 230 nm. The positive peak at 265 nm, which is related to the n-π* transition of DNA, is enhanced as some bases are exposed. The increased positive peaks at 225 nm and 240 nm indicate that TOB binding enhances base stacking, resulting in more obvious peaks at these wavelengths. The decrease at 280 nm may indicate that TOB binding weakens base stacking and reduces π-π* transitions, thereby reducing the peak. These observations confirm that DNA undergoes conformational changes after binding to KAN and TOB, resulting in quenching of fluorescence intensity.

[0028] Example 7: In order to evaluate the cross-reactivity during quenching, different concentrations (1 nM, 4 nM) of KAN and TOB were introduced into different systems. The fluorescence intensity curves obtained are shown in Figure 7. Figure 7 B. In the test solution and milk samples, the sensor showed significant responses to different concentrations of KAN and TOB targets. In addition, the quenching effect in the presence of KAN and TOB was very close to the quenching effect of the corresponding specific target, and the quenching effect compared with the other target was significantly different. The results showed that the fluorescence quenching effect of these two targets was consistent with the quenching effect of KAN or TOB alone. The quenched fluorescence intensity in the milk sample was also slightly disturbed, indicating that the residual fat, casein, and Ca in the milk 2+ and K + Ions have a slight effect on the quenching effect. The sensor has good anti-cross-reactivity and can accurately detect KAN and TOB in different concentrations and systems.

[0029] Embodiment 8: Figure 8 A and B are the quenching effects of adding 1.5 nM KAN and TOB to the sensor under optimal conditions, compared with adding 15 nM amoxicillin (AMO), doxycycline (DOX), enrofloxacin (ENR), and oxytetracycline (OXY). The results show that the quenching fluorescence intensity of adding other antibiotics is significantly lower than that of adding KAN and TOB, indicating that this sensor has good specificity.

[0030] Table 1 Detection of KAN and TOB in milk samples

Claims

1. A fluorescent sensor for detecting kanamycin and tobramycin in milk based on dual-color fluorescent silver nanoclusters, characterized in that: The kanamycin and tobramycin aptamer fragments were linked to the dual-color silver nanocluster template sequence using the carbon 12 spacer arm and the silver nanoclusters with dual-color fluorescence properties were synthesized by the template reduction method to construct a fluorescent aptamer sensor that can be used to detect kanamycin and tobramycin in milk.

2. A fluorescent sensor for detecting kanamycin and tobramycin in milk based on dual-color fluorescent silver nanoclusters as claimed in claim 1, characterized in that: The carbon 12 spacer arm is used to link multiple aptamer fragments with multicolor silver nanocluster fragments, which, to a certain extent, avoids the folding rotation caused by the sequence being too long, so that more silver nanoclusters are synthesized and the fluorescence intensity is stronger. And when quenched, the cross analysis between the two fluorescences is smaller, which is conducive to the accurate detection of the two targets.

3. A fluorescent sensor for detecting kanamycin and tobramycin in milk based on dual-color fluorescent silver nanoclusters as claimed in claim 1, characterized in that: The appropriate two-color silver nanocluster template was screened, tested and selected, so that the synthesized silver nanoclusters produced fluorescence at 570 nm and 677 nm, respectively, with no cross-over and overlap between them, providing a basis for achieving multiple detection.

4. A fluorescent sensor for detecting kanamycin and tobramycin in milk based on dual-color fluorescent silver nanoclusters as claimed in claim 1, characterized in that: The changes in DNA sequence structure and conformation caused by the recognition and binding of kanamycin and tobramycin with the corresponding aptamer fragments were used, which resulted in a quenching effect on the corresponding fluorescence and a change in the signal. The calibration curve was established using the corresponding fluorescence intensity change value and the antibiotic concentration used, which can be used to efficiently and easily detect kanamycin and tobramycin in milk samples.

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