Label-free fluorescent aptamer biosensor of kanamycin

Abstract

The invention relates to a label-free fluorescent aptamer biosensor of kanamycin. According to the method, a fluorescence signal generated after a known malachite green aptamer is combined with a fluorescent dye is utilized, a target small molecule kanamycin is introduced, and combination of the aptamer and MG is disturbed under a competitive equilibrium mechanism, so that the fluorescence intensity is changed, and sensitive detection of the kanamycin is realized. The constructed biosensor has good affinity and conformational plasticity, has a linear relationship with the concentration of kanamycin in a range of 0.1-20 [mu] M, has a detection limit as low as 0.40 nM, has strong specificity and good anti-interference capability, and is suitable for detecting kanamycin residues in food, environment and drugs. The invention provides a new thought for constructing a universal micromolecule sensing platform, has the advantages of being rapid in detection, high in sensitivity and simple and convenient to operate, and has a wide practical application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biosensing, and in particular relates to a label-free fluorescent aptamer biosensor for kanamycin. Background Art

[0002] With the widespread use of antibiotics in animal husbandry and medicine, their residues have attracted widespread global attention. Kanamycin is a commonly used aminoglycoside antibiotic, widely used in the treatment of bacterial infections and as an animal feed additive. However, excessive or improper use of kanamycin may leave residues in food or the environment, and long-term ingestion can cause serious damage to the human kidneys and hearing system. Therefore, the development of a sensitive, simple, and highly selective kanamycin detection method is of great practical significance.

[0003] Currently, kanamycin detection methods primarily include high-performance liquid chromatography (HPLC), mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). While these methods offer good accuracy and stability, they are generally subject to complex procedures, high costs, strong equipment dependence, and long detection cycles, limiting their widespread application for rapid on-site testing and in resource-limited areas. In recent years, nucleic acid aptamers (aptamers) have become important recognition elements in the development of novel biosensors due to their high target recognition specificity, excellent chemical synthesis stability, and flexible modification.

[0004] Although aptamers targeting kanamycin have been screened and reported, most detection strategies still rely on the construction of specific aptamer recognition systems, resulting in long development cycles and limited applications. Based on the known high-affinity aptamer malachite green (MG), the present invention proposes a small molecule biosensor strategy for detecting kanamycin using a competitive equilibrium mechanism. By introducing kanamycin to replace MG in binding to the aptamer and regulating the fluorescence changes of the system, a universal and versatile aptamer sensing platform is constructed, addressing the problems of existing detection systems with strong specificity and difficult target switching. This strategy not only achieves highly sensitive detection of kanamycin, but also expands the application spectrum of known aptamers, and has good market prospects and promotion value. Summary of the Invention

[0005] Based on this, the present invention successfully constructed a label-free fluorescent aptamer biosensor for kanamycin.

[0006] In one aspect, the present invention provides a label-free fluorescent aptamer biosensor for kanamycin, wherein the label-free biosensor comprises: (1) a sequence of the biosensor; (2) a signal reporter molecule of the biosensor; (3) a detection principle of the biosensor;

[0007] The sequence of the biosensor is: 5'-CTCGATGTAACCTTGTTAAATCGAG-3', as shown in SEQ ID NO: 1.

[0008] The signal reporter molecule of the above-mentioned biosensor is malachite green fluorescent dye.

[0009] The detection principle of the above-mentioned biosensor is to utilize the fluorescent signal generated by the binding of a known malachite green aptamer to a fluorescent dye. By introducing the target small molecule kanamycin, the binding of the aptamer to MG is disrupted under the competitive equilibrium mechanism, resulting in a change in fluorescence intensity, thereby achieving sensitive detection of kanamycin.

[0010] The buffer solution of the biosensor contains 20 mM Tris-HCl.

[0011] The pH of the buffer solution of the above biosensor is 7.4.

[0012] The above biosensor is selective for kanamycin; other types of antibiotics: vancomycin, chloramphenicol, erythromycin and tetracycline are selected as detection targets, and the biosensor is used to conduct detection experiments to detect the fluorescence value of malachite green at the characteristic wavelength.

[0013] On the other hand, the present invention provides a method for quantitative detection of kanamycin by the above-mentioned biosensor, wherein the standard curve is established:

[0014] Different concentrations of kanamycin and a fixed concentration of malachite green were simultaneously added to a 20 mM Tris-HCl buffer solution containing a 1 μM aptamer solution. After oscillation and mixing at room temperature, the fluorescence intensity of malachite green in the solution system was immediately measured using a fluorescence spectrophotometer with an excitation wavelength of 617 nm and an emission wavelength of 653 nm. A standard curve was plotted based on the fluorescence value and kanamycin concentration.

[0015] On the other hand, the present invention proposes the use of the above-mentioned biosensor or the above-mentioned method in the development of a kanamycin detection method.

[0016] In another aspect, the present invention provides the use of the above-mentioned biosensor or the above-mentioned method in a kanamycin food safety kit.

[0017] On the other hand, the present invention provides the use of the above-mentioned biosensor or the above-mentioned method in a kanamycin environmental detection kit.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This invention is the first to use malachite green aptamers to identify the non-targeted small molecule kanamycin, and to construct a fluorescence sensing system based on competitive binding between the aptamer and the dye;

[0020] 2. Through structural comparison and molecular docking, this study found that kanamycin and MG have similar binding preferences, revealing the potential structural plasticity and target recognition versatility of MG aptamers, and providing theoretical support for the sequence expansion of "known aptamers to recognize new targets across targets";

[0021] 3. The sensor constructed using the MC-DA-LG sequence in this invention has a detection limit of 0.40 nM for kanamycin, significantly exceeding the current EU maximum residue limit. It has extremely high sensitivity and practical value, and has broad application potential in food safety and environmental monitoring.

[0022] 4. In the context of multiple antibiotics, the sensor maintains a stable response to kanamycin, showing excellent specificity and selectivity, providing a demonstration for anti-interference design in complex sample analysis and has good promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the molecular structure of kanamycin.

[0024] Figure 2 This is the secondary structure of the kanamycin aptamer.

[0025] Figure 3 The secondary structure of the CAG-T-ML-S4 malachite green aptamer.

[0026] Figure 4 Schematic diagram of the biosensor detection mechanism.

[0027] Figure 5 Circular dichroism spectroscopy was used to verify the feasibility of the assay.

[0028] Figure 6 This is the molecular docking result of kanamycin and MC-DA-LG sequence.

[0029] Figure 7 This is the molecular docking result of malachite green and MC-DA-LG sequence.

[0030] Figure 8 This is the detection waveform.

[0031] Figure 9 The fluorescence peaks are those in the presence of different target concentrations.

[0032] Figure 10 To detect the linear relationship diagram.

[0033] Figure 11 For sensor specificity assessment. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0035] Example 1. Biosensing strategy and feasibility assessment

[0036] The present invention plans to use the MG aptamer sequence to construct a sensor for detecting other harmful substances besides malachite green, so as to expand the application scenarios of the sequence. Kanamycin is a broad-spectrum aminoglycoside antibiotic that is widely used to treat infections caused by Gram-positive and Gram-negative bacteria. As a small molecule pollutant, it has objective detection needs in food safety testing, environmental monitoring, clinical drug monitoring and drug production. The chemical structure of kanamycin is as follows Figure 1 As shown. A kanamycin DNA aptamer was screened in 2011 and subsequently tailored to improve affinity. The tailoring process primarily focused on optimizing the size and sequence of the loop and stem. The tailored aptamer structure has a similar loop size and stem structure to the high-affinity aptamer for MG, suggesting that kanamycin may have a binding preference for this type of nucleic acid conformation. Figure 2 and Figure 3 Therefore, we attempted to use the MG aptamer to monitor kanamycin based on the principle of competitive equilibrium.

[0037] The detection mechanism of the competitive balance principle is as follows Figure 4 As shown in the figure, the researchers utilized the fluorescence signal generated by the binding of a known malachite green aptamer to a fluorescent dye. By introducing the target small molecule kanamycin, the binding between the aptamer and MG is disrupted under a competitive equilibrium mechanism, resulting in a change in fluorescence intensity and enabling sensitive detection of kanamycin. Specifically, the aptamer binds to the MG molecule and induces it to produce a fluorescent signal. When kanamycin, a competing target, is introduced into the aptamer-MG complex, the small molecule competes with MG for the same binding site, disrupting the binding between MG and the aptamer and causing a change in fluorescence intensity. By monitoring this change in fluorescence signal, sensitive detection of the target substance, kanamycin, can be achieved.

[0038] In order to verify the versatility of the sensing strategy and the potential of the aptamer sequence, the MC-DA-LG sequence was used to construct a kanamycin aptamer sensor. The sequence of MC-DA-LG is: 5'-CTCGATGTAACCTTGTTAAATCGAG-3', (SEQ ID NO: 1). After MG binds to the aptamer sequence, it can be excited to emit fluorescence. When the target kanamycin is present in the system, MG will be displaced, causing the fluorescence intensity of the system to decrease. The feasibility of the kanamycin sensor was explored through circular dichroism spectroscopy and molecular docking simulation. Figure 5 The CD spectra shown in the figure shift the peaks of the CD spectra after adding kanamycin or malachite green to the MC-DA-LG sequence, indicating that the sequence can interact with MG or kanamycin molecules. Molecular docking results also show that kanamycin interacts with the same pockets as MG and MC-DA-LG sequences, further suggesting the feasibility of the competitive equilibrium principle ( Figure 6 and Figure 7 ).

[0039] Example 2. Biosensor Performance Evaluation

[0040] MC-DA-LG (SEQ ID NO. 1) was used to detect known concentrations of kanamycin, and a standard curve was generated based on the changes in malachite green fluorescence in the solution. Various concentrations of kanamycin and a fixed concentration of malachite green were simultaneously added to a 1 μM aptamer solution in 20 mM Tris-HCl (pH 7.4) buffer. After oscillation at room temperature, the solution was directly tested for fluorescence. The malachite green fluorescence intensity of the solution at 617 nm was measured using a fluorescence spectrophotometer, and a standard curve was plotted based on the fluorescence values.

[0041] Experimental verification shows that the MC-DA-LG nucleic acid sequence is used for kanamycin biosensing based on the competitive equilibrium detection principle. Figure 8 and Figure 9 As shown in Figure 2, as the concentration of the target increases, the fluorescence spectrum intensity of the system gradually decreases. The biosensor can have a good linear relationship (R 2 =0.9912)( Figure 10 ).

[0042] Other antibiotics, such as vancomycin, chloramphenicol, erythromycin, and tetracycline, were selected to evaluate the specificity of the kanamycin biosensor. Vancomycin, chloramphenicol, erythromycin, and tetracycline were selected as detection targets, and the biosensor was used to detect the fluorescence value of malachite green at the characteristic wavelength.

[0043] like Figure 11As shown, the biosensor exhibits good specificity and is resistant to interference from other antibiotics, demonstrating its good selectivity. Calculations indicate that the proposed biosensor has a detection limit as low as 0.40 nM, significantly exceeding the EU maximum residue limit for kanamycin and meeting current market demand.

Claims

1. A label-free fluorescent aptamer biosensor for kanamycin, characterized in that: The label-free biosensor includes: (1) the sequence of the biosensor; (2) the signal reporter molecule of the biosensor; (3) the detection principle of the biosensor; The biosensor sequence is: 5'-CTCGATGTAACCTTGTTAAATCGAG-3', as shown in SEQ ID NO:

1.

2. The biosensor according to claim 1, wherein The signal reporting molecule of the biosensor is malachite green fluorescent dye.

3. The biosensor according to claim 1, wherein The detection principle of the biosensor is to utilize the fluorescent signal generated by the binding of a known malachite green aptamer to a fluorescent dye. By introducing the target small molecule kanamycin, the binding of the aptamer to MG is disrupted under a competitive equilibrium mechanism, resulting in a change in fluorescence intensity, thereby achieving sensitive detection of kanamycin.

4. The biosensor according to claim 1, wherein The buffer solution of the biosensor contains 20 mM Tris-HCl.

5. The biosensor according to claim 1, wherein The pH of the buffer solution of the biosensor is 7.

4.

6. The biosensor according to claim 1, wherein the selectivity of the biosensor for kanamycin; Other types of antibiotics, including vancomycin, chloramphenicol, erythromycin and tetracycline, were selected as detection targets. The detection experiment was carried out using a biosensor to detect the fluorescence value of malachite green at the characteristic wavelength.

7. The method for quantitative detection of kanamycin by a biosensor according to claim 1, wherein: Establishment of standard curve: Different concentrations of kanamycin and a fixed concentration of malachite green were simultaneously added to a 20 mM Tris-HCl buffer solution containing a 1 μM aptamer solution. After oscillation and mixing at room temperature, the fluorescence intensity of malachite green in the solution system was immediately measured using a fluorescence spectrophotometer with an excitation wavelength of 617 nm and an emission wavelength of 653 nm. A standard curve was plotted based on the fluorescence value and kanamycin concentration.

8. Use of the biosensor according to any one of claims 1 to 6 or the method according to claim 7 in the development of a kanamycin detection method.

9. Use of the biosensor according to any one of claims 1 to 6 or the method according to claim 7 in a kanamycin food safety kit.

10. Use of the biosensor according to any one of claims 1 to 6 or the method according to claim 7 in a kanamycin environmental detection kit.

Images