Fluorescent sensor for detecting kanamycin and aflatoxin B1 in feed based on two-color fluorescent silver nanoclusters
By using a fluorescence sensor based on dual-color fluorescent silver nanoclusters and utilizing G-rich sequences to enhance the fluorescence emission of AgNCs to form a sandwich structure, the problems of complexity and high cost of traditional detection methods were solved, and rapid, simple, and highly sensitive detection of KAN and AFB1 was achieved.
Patent Information
- Application Number
- CN202510994781.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-18
- Publication Date
- 2025-10-14
AI Technical Summary
In the existing technology, the traditional methods for detecting kanamycin (KAN) and aflatoxin B1 (AFB1) are complex to operate and costly, and cannot meet the needs of rapid and large-scale testing.
A fluorescence sensor based on dual-color fluorescent silver nanoclusters was used, and the fluorescence emission of AgNCs was enhanced by the G-rich sequence. The aptamer and the target were specifically bound to form a sandwich structure, thereby achieving high-sensitivity detection of KAN and AFB1.
A rapid, simple, and low-cost detection of KAN and AFB1 was achieved with high sensitivity and selectivity, suitable for stable detection in complex sample matrices.
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Abstract
Description
TECHNICAL FIELD
[0001] The application provides a fluorescence sensor for detecting kanamycin and aflatoxin B1 in feed based on two-color fluorescent silver nanoclusters, and belongs to the field of nanobiosensors under food safety detection technology. BACKGROUND
[0002] Aflatoxin B1 (AFB1) and kanamycin (KAN) are two types of substances that are of great concern in the fields of food safety and animal health. KAN belongs to aminoglycoside antibiotics and is widely used in the treatment of mastitis and various bacterial infections in dairy cows due to its wide antibacterial spectrum and strong antibacterial activity. AFB1 is a secondary metabolite produced by Aspergillus flavus and parasitic Aspergillus, commonly found in agricultural products such as grains and oil crops, and has strong toxicity, carcinogenicity and mutagenicity. However, some illegal breeders will add KAN to feed by feeding to treat, and agricultural products are prone to contamination during storage and transportation, resulting in AFB1 residues. When these residues enter the human body, KAN can cause ototoxicity, nephrotoxicity and neuromuscular blockade, while AFB1 can seriously threaten liver health and increase the risk of liver cancer and other diseases.
[0003] Currently, the traditional detection methods for KAN and AFB1 include liquid chromatography tandem mass spectrometry, enzyme-linked immunosorbent assay, and high-performance liquid chromatography-ultraviolet detection. These traditional methods have high accuracy and sensitivity, but also have obvious disadvantages, such as complex operation process, need for professional technicians, expensive instruments, high detection cost, etc., which makes it difficult to meet the needs of rapid and large-scale detection. Therefore, it is particularly urgent to develop an efficient, simple and low-cost detection method.
[0004] Fluorescent sensor technology provides a new idea and solution for the detection of KAN and AFB1. Compared with other detection strategies and methods, fluorescent sensors have the advantages of high sensitivity, good selectivity, simple operation, low cost, and good biocompatibility. When using fluorescent sensors for detection, the presence of target substances can be quickly responded, and the quantitative detection of KAN and AFB1 can be realized through the change of fluorescence signal. In particular, the fluorescence intensity and emission wavelength of the sensor based on specific fluorescent materials can be adjusted according to actual needs, and it has good optical stability and chemical stability, which is conducive to the construction and practical application of the sensor.
[0005] The G-rich sequence refers to a nucleic acid sequence rich in guanine (G). The G-rich sequence is a DNA fragment composed of multiple guanine (G), and its core function is to form a G-quadruplex structure. This structure connects four G bases through Hoogsteen hydrogen bonds to form a planar tetramer and stack into a stable three-dimensional structure. This property plays a key role in the fluorescence regulation of silver nanoclusters (AgNCs). When the G-rich sequence is close to AgNCs, the high electron density of the G base enhances the fluorescence emission efficiency of AgNCs through π-π stacking or charge transfer. The sandwich structure is a commonly used detection structure model, which is usually composed of a capture probe, a target molecule and a signal probe. In the detection of KAN and AFB1, the capture probe can be fixed on the sensor surface to specifically capture the target molecule, and then the signal probe binds to the target molecule bound to the capture probe to produce a detectable fluorescent signal through the fluorescent label carried on the signal probe. This sandwich structure can improve the sensitivity and specificity of detection, and enhance the accuracy and reliability of the detection results. SUMMARY
[0006] The purpose of the application is to demand a rapid, sensitive, efficient and economic detection method for detecting KAN and AFB1 in feed.
[0007] The technical scheme is that the aptamer and the split aptamer have good specificity and affinity, are commonly used in the construction of biosensors as core response elements, can tightly bind to lay the foundation for the reaction under the condition that the target exists. The fluorescence method has good sensitivity and responsiveness, and the signal intensity is high. The fluorescence of AgNCs synthesized by the template reduction method has the advantages of convenient regulation, good biocompatibility, etc., which can solve the problems of signal cross and interference reaction of biosensors for multi-detection, and the "lighting" of the G-rich sequence to AgNCs plays a role in signal amplification, and the tight binding of "aptamer-target-aptamer" provides an important guarantee for the stability of the signal response.
[0008] The fluorescence sensor for detecting KAN and AFB1 in milk based on double-color fluorescence silver nanoclusters, wherein the DNA template for synthesizing AgNCs is obtained from literature and coupled with the aptamer to form the final fluorescence probe. The AgNCs prepared by the template reduction method can generate two kinds of fluorescent nanoclusters with low initial fluorescence intensity. Figure 1As shown, green fluorescent AgNCs are best excited at 430 nm with an emission wavelength of 530 nm; red fluorescent AgNCs are best excited at 554 nm with an emission wavelength of 630 nm. In the presence of KAN or AFB1, the aptamer fragments on the capture probe and fluorescent probe specifically recognize and bind to the target molecules. This binding brings the probes close to each other, promoting the formation of hydrogen bonds between the ATA and TAT bases on the probes, thereby enhancing the hybridization efficiency. Therefore, the G-rich sequence with strong reducing properties activates the otherwise dark AgNCs, significantly enhancing their fluorescence intensity, and achieving detection of KAN and AFB1.
[0009] The two-color fluorescent silver nanocluster-based fluorescence sensor for detecting KAN and AFB1 in feed is characterized in that the DNA template of AgNCs is screened, two kinds of fluorescent AgNCs and two aptamer fragments are synthesized on one DNA strand, and the overlap of the two signals is avoided. The selected template can be activated by a G-rich sequence to enhance the fluorescence intensity, providing a basis for the fluorescence response; meanwhile, the sandwich structure formed by the aptamer and the target provides a guarantee for the stability of the fluorescence response.
[0010] The preparation principle is as follows: the synthesis of DNA-AgNCs is based on the previously established scheme and is partially improved. The DNA template is prepared at a concentration of 100 μM in 1×TE buffer (pH 6.5), and the sequence amount of each tube of solution is 2 OD. During the synthesis of silver nanoclusters, first, 30 μL of TE buffer is added to dissolve the single-tube DNA sequence to a concentration of 100 μM, and then 234 μL of TE buffer is used for dilution at room temperature. Then 21 μL of 2 mM silver nitrate solution is added to the mixture, vortexed for 1 minute, sealed, and incubated in an ice water bath for 30 minutes in the dark. Then 21 μL of freshly prepared 2 mM sodium borohydride solution is added, and the final concentration ratio of DNA:silver nitrate:sodium borohydride is maintained at 1:14:14, and vortexed for 1 minute again. After the resulting solution is incubated at room temperature for 5 hours, it is stored at -4 ℃ in the dark for standby use.
[0011] To achieve the above object, the following technical scheme is adopted: KAN and AFB1 of appropriate concentration are prepared for detection and establishment of standard curve. KAN and AFB1 are detected simultaneously as follows: the G-rich sequence is diluted to 10 μM with 1×TE buffer (pH 6.5). 100 μL of the diluted DNA-AgNCs solution and 220 μL of the corresponding G-rich sequence solution are added to the reaction system, and then 100 μL of the target solution is added. The mixed system is incubated for 35 minutes to promote competitive binding. Fluorescence spectrum is detected using an RF-6000 fluorescence spectrophotometer. When detecting AFB1, the excitation wavelength is set to 430 nm, and the spectrum is collected in the range of 450-600 nm, with a peak at 530 nm. When detecting KAN, the spectrum is collected in the range of 570-700 nm, the excitation wavelength is set to 554 nm, and a peak appears at 630 nm. When detecting simultaneously, the excitation wavelength is 395 nm. The fluorescence intensity is compared with the blank control without target.
[0012] Actual sample testing selects grain feed as the actual sample matrix for recovery rate calculation. The feed is purchased from the local market and ground into fine powder. 1 g of the feed sample is dried at 40 °C and thoroughly pulverized, and 5 mL of organic solvent (methanol / water = 80:20, v / v) is added to dissolve the powder. After ultrasonic extraction for 30 minutes, centrifugation is performed at 12,000 rpm for 30 minutes, and the supernatant is filtered through a 0.22 μm sterile syringe filter to obtain the sample to be tested, which is finally diluted to 5 mL. The extract is diluted to prepare different concentrations of KAN (2.5, 20, 40 nM) and AFB1 (24.04, 48.08, 96.15 nM) standard addition solutions, which are stored at -4 °C in the dark for standby use. The developed sensor is used to measure the fluorescence intensity, and the fluorescence spectrum under different target concentrations is collected to evaluate the performance of the sensor in detecting KAN and AFB1 in actual feed samples. BRIEF DESCRIPTION OF DRAWINGS
[0013] Figure 1 Construction process of the fluorescence aptamer sensor.
[0014] Figure 2 Feasibility diagram (A "lighting" effect diagram and logic gate system experimental results, B DNA-AgNCs, AFB1, KAN, and the interaction of Apt1 and Apt2 chains with AFB1, C corresponding signal output result bar chart, D gel electrophoresis spectrum of different combinations; E-F excitation and emission spectra of KAN and AFB1 before and after adding the target).
[0015] Figure 3Characterization diagrams of DNA-AgNCs (A transmission electron microscopy of DNA-AgNCs, B diameter distribution of AgNCs, C excitation and emission spectra of AgNCs, D element distribution of DNA-AgNCs).
[0016] Figure 4 Fluorescence intensity plots of two AgNCs under different conditions (AB: effect of different pH values, CD: effect of different sodium borohydride:silver nitrate ratios, EF: effect of different G-rich sequence ratios, GH: effect of different reaction times).
[0017] Figure 5 A Fluorescence spectra of KAN at different concentrations, B Fluorescence spectra of AFB1 at different concentrations, C Standard curve of KAN, D Standard curve of AFB1, E Specificity performance of KAN, F Specificity performance of AFB1, G Fluorescence spectra of KAN and AFB1 at different concentrations using a single excitation wavelength of 395 nm, H Standard curve of KAN, I Standard curve of AFB1, J Selectivity analysis of KAN detection, K Selectivity analysis of AFB1 detection. Error bars are from three replicate experiments (n = 3).
[0018] Figure 6 Cross-reactivity analysis of targets at different concentrations.
[0019] Figure 7 A. Fluorescence intensity curves of DNA-AgNCs synthesized in four parallel experiments. B. Fluorescence intensity changes of DNA-AgNCs stored at -4°C in the dark for 0-12 days. Error bars are from three parallel experiments (n = 3). DETAILED DESCRIPTION Example 1: Figure 1The sensor construction process is shown. The double fluorescent AgNCs are synthesized by using the template method, 100 μL of diluted DNA-AgNCs solution and 220 μL of corresponding G-rich sequence solution are added to the reaction system, and then 100 μL of target solution is added. The mixed system is incubated for 35 minutes to promote competitive binding. The fluorescence spectrum is detected by using an RF-6000 type fluorescence spectrophotometer. When AFB1 is detected, the excitation wavelength is set to 430 nm, and the spectrum is collected in the range of 450-600 nm, and the peak value is at 530 nm. When KAN is detected, the spectrum is collected in the range of 570-700 nm, the excitation wavelength is set to 554 nm, and the peak value is at 630 nm. When both are detected, the excitation wavelength is set to 395 nm. When KAN or AFB1 exists, the aptamer fragments on the capture probe and the fluorescent probe can specifically recognize and bind to the target molecules. This combination brings the probes close to each other, promotes the formation of hydrogen bonds between the ATA and TAT bases on the probes, thereby increasing the hybridization efficiency. Therefore, the G-rich sequence with strong reducing property activates the originally dark AgNCs, and the fluorescence intensity is significantly improved. When KAN or AFB1 does not exist, the probes do not specifically recognize, and the fluorescence intensity of AgNCs remains unchanged, thereby completing the construction of the sensor.
[0020] Example 2: As shown in Figure 2 The sandwich structure of G-rich sequence formed by the connection of target and aptamer can enhance the fluorescence intensity of AgNCs. Figure A is a schematic diagram showing the principle of the sandwich structure formed by the recognition and combination of G-rich sequence, AgNCs and target, and the feasibility thereof is verified by a series of experiments. Figure B is a schematic diagram of the “lighting” effect, logic gate system experiment and fluorescence intensity analysis. Different combinations of DNA template chain, KAN-Apt2-ATA-G-rich sequence, AFB1-Apt1-ATA-G-rich sequence and target and fluorescent AgNCs are prepared, and the fluorescence intensity change curve is drawn. When only G-rich sequence or target exists, the fluorescence is stable, and when both exist, the fluorescence is significantly enhanced. Figure D is a gel electrophoresis verification. Different sequences show different performances before and after the addition of target. When the target exists, a new band is formed, and when the target does not exist, no band is formed, which confirms that base pairing and fluorescence activation require the participation of target. Figures C, E and F are column charts and spectrum diagrams for analyzing the fluorescence response of aptamer sensor, measuring the excitation and emission wavelengths and the change of fluorescence intensity after adding target. The AFB1 and KAN aptamer connection sequences produce fluorescence at the corresponding wavelengths, and the peak fluorescence intensity is greatly improved after adding target. The results comprehensively show that the aptamer sensor can effectively recognize the target and amplify the fluorescence signal, and realize high sensitivity and selectivity detection of KAN and AFB1.
[0021] Example 3: As shown in Figure 3The oxidation states of each element in DNA-AgNCs were determined by STEM technology, and the morphology and particle size distribution were observed. Silver nanoclusters mainly present an elliptical structure, and the particle size is uniformly distributed in the range of 2-5 nm Figure 3 A). The particle size distribution graph Figure 3 B) shows that most of the silver nanoclusters have a diameter of 1.7-5.1 nm, with an average particle size of 3.3 nm. This size meets the Fermi size characteristics of metal clusters, which is consistent with the unique fluorescence characteristics of AgNCs. Under sunlight, AgNCs show a light yellow color; after excitation by 365 nm ultraviolet light, the fluorescence intensity is significantly enhanced, as shown in the inset of Figure 3 B. The high-resolution spectrum of silver element shows clear characteristic peaks at Ag 3d5 / 2 and 3d3 / 2 orbits Figure 3 C), indicating that silver elements exist in the form of atoms in nanoclusters. Further analysis of the elemental composition of DNA-stabilized AgNCs by energy dispersive X-ray spectroscopy Figure 3 D) shows that it contains elements such as carbon, nitrogen, oxygen, phosphorus, and silver. By detecting the distribution of carbon, nitrogen, oxygen, and phosphorus elements, it is confirmed that there is a DNA sequence in the system. In addition, the detection results of silver element provide strong evidence for the successful synthesis of DNA-AgNCs.
[0022] Example 4: As shown in Figure 4 A-B, the influence of two kinds of DNA-AgNCs at different pH values, the fluorescence intensity is the highest at pH = 6.5, indicating that 6.5 is the best pH value. As shown in Figure 4 C-D, the influence of different DNA: sodium borohydride and silver nitrate ratios, the fluorescence intensity is the highest when the ratio is 1:14, indicating that 1:14 is the best DNA: sodium borohydride and silver nitrate ratio. As shown in Figure 4 E-F, the influence of different G-rich sequence ratios, the fluorescence intensity is the highest when the ratio is 1:14, indicating that 1:14 is the best DNA: G-rich sequence ratio. As shown in Figure 4 G-H, the influence of different reaction times, when the time is 35 min, the fluorescence intensity no longer continues to enhance, indicating that 35 min is the best reaction time.
[0023] Example 5: Different concentrations of KAN and AFB1 were added to the sensor, and the fluorescence intensity was measured at 430 nm (AFB1) and 554 nm (KAN) excitation wavelengths. The corresponding fluorescence spectra are shown in Figure 5 A and 5B, in KAN detection, the fluorescence intensity has a linear relationship with the concentration (C KAN ) of KAN (1.25-40 nM range, Figure 5 C). The standard curve is y = 32.202 C KAN+ 5692.87, R² value reached 0.9916, and the detection limit (LOD) was 0.86 nM. In the AFB1 detection, the fluorescence intensity was related to the AFB1 concentration (C AFB1 ) showed a linear relationship in the range of 0.8-104.26 nM ( Figure 5 D). The standard curve is y=4.1381C AFB1 +474.11, R² value was 0.9661, and LOD was 0.71 nM. The selectivity of the sensor was evaluated by using antibiotics or toxins such as doxycycline (DOX), neomycin (NEO), enrofloxacin (ENR), oxytetracycline (OXY), fumonisin B (FB), ochratoxin A (OTA), zearalenone (ZEN), and deoxynivalenol (DON). Figure 5 As shown in E and 5F, the changes in fluorescence intensity of the target alone and its mixture with KAN or AFB1 showed significant characteristics. When KAN or AFB1 was present, the fluorescence intensity was significantly enhanced due to the activation of the G-rich sequence. In contrast, the presence of other antibiotics or toxins had little effect on the fluorescence intensity. The fluorescence enhancement effect exhibited by the mixture containing KAN or AFB1 and non-target substances was similar to the results observed when KAN or AFB1 was used alone. These results indicate that the aptamer can specifically recognize and bind to its corresponding target (KAN or AFB1), while exhibiting specificity for other non-target substances. Taken together, this confirms that the aptamer sensor has the advantages of high sensitivity, wide detection range and good selectivity in detecting KAN and AFB1. As Figure 5 As shown in G, the fluorescence spectrum was recorded using a single excitation wavelength of 395 nm. Since the excitation wavelengths of the two fluorescence signals did not reach the optimal value, the fluorescence intensity was significantly lower than that at the optimized wavelength. A standard curve was established based on the concentrations of KAN and AFB1 as shown in Figure 5 H and 5I. The study found that the aptamer sensor is compatible with C KAN The linear range of the relationship is 2.5-40 nM. The standard curve equation is y = 3.085 C KAN + 5143, R² value reached 0.9579. For AFB1, the fluorescence intensity and C AFB1 A significant linear relationship was observed in the concentration range of 24-96 nM, and the standard curve equation was y = 0.6256 C AFB1 The LODs for KAN and AFB1 were 1.79 nM and 19.29 nM, respectively, significantly lower than the required standards. These results demonstrate that the aptasensor has excellent detection performance.
[0024] Example 6: To evaluate the cross-reactivity of the adapted sensor, the mixed samples of KAN, AFB1 and two targets were tested at different concentrations (10, 40, 32 and 96 nM) and verified in real feed samples. The fluorescence intensity change bar chart is shown in Figure 6 The sensor can effectively detect KAN and AFB1, and the fluorescence intensity change is completely consistent with the target concentration. When there are mixed targets, the fluorescence intensity is highly consistent with the fluorescence intensity of detecting KAN or AFB1 alone, and is significantly different from the sample containing only a single target. At the same time, the fluorescence enhancement phenomenon in the real feed sample is consistent with the results of other experimental systems, further confirming the stability of the sensor. The experimental results show that the adapted sensor exhibits excellent anti-cross-reactivity under different conditions such as complex sample matrix, and can effectively distinguish KAN and AFB1.
[0025] Example 7: To evaluate the practical application value of the sensor, the repeatability and stability of the developed sensor were systematically tested. Four groups of parallel AgNCs fluorescence probes were prepared under the same conditions, and the fluorescence intensity measurement results (Fig. 6A) show that the emission signals between batches are stable, and the red and green fluorescence intensities are stabilized at about 5700 a.u. and 480 a.u., respectively, indicating good batch-to-batch repeatability. At the same time, the stability of the synthesized AgNCs was evaluated: they were stored in a light-free environment at -4 ℃ for 12 days (Fig. 6B), and the results show that under the above conditions, the fluorescence intensity of AgNCs remains relatively stable, only a slight decrease occurs within 0-12 days of storage. It shows that this sensor not only has excellent repeatability, but also has long-term storage stability, fully meeting the actual needs of sensor applications. Figure 7 Figure 7
[0026] Table 1 Detection of KAN and AFB1 in feed samples
Claims
1. A fluorescent sensor for detecting kanamycin and aflatoxin B1 in feed based on dual-color fluorescent silver nanoclusters, characterized by: A fluorescent aptamer sensor was constructed by linking kanamycin and aflatoxin B1 aptamer fragments to the dual-color silver nanocluster template sequence and using the template reduction method to synthesize silver nanoclusters with dual-color fluorescence properties, which can be used to detect kanamycin and aflatoxin B1 in feed.
2. A fluorescent sensor for detecting kanamycin and aflatoxin B1 in feed based on dual-color fluorescent silver nanoclusters as claimed in claim 1, characterized in that: By regulating the reducibility of the G-rich sequence, the fluorescence emission of dark-state silver nanoclusters is activated, thus illuminating the fluorescent signal. Simultaneously, the target and multi-aptamer recognition function forms an "aptamer-target-aptamer" sandwich structure. This allows the G-rich sequence to successfully act on darker AgNCs, amplifying their fluorescence intensity and providing a basis for multi-signal sensing.
3. A fluorescent sensor for detecting kanamycin and aflatoxin B1 in milk based on dual-color fluorescent silver nanoclusters as claimed in claim 1, characterized in that: Appropriate dual-color silver nanocluster templates were tested and screened, so that the synthesized silver nanoclusters produced fluorescence at 530 nm and 630 nm, respectively, without crossing or overlapping with each other, and could interact with G-rich sequences and be illuminated by them, providing a basis for achieving multiplex detection.
4. A fluorescent sensor for detecting kanamycin and aflatoxin B1 in feed based on dual-color fluorescent silver nanoclusters as claimed in claim 1, characterized in that: The system utilizes a tight "aptamer-target-aptamer" sandwich structure, where kanamycin and aflatoxin B1 recognize and bind to their corresponding multi-aptamer fragments. This closes the distance between the aptamer-linked AgNCs template and the G-rich sequence, enabling interaction and amplifying the corresponding fluorescence signal. Standard curves were established using changes in fluorescence intensity versus kanamycin and aflatoxin B1 concentrations, enabling rapid and portable detection of kanamycin and aflatoxin B1 in feed samples.