Kanamycin fluorescence-electrochemical dual-mode biosensor based on three-dimensional DNAzyme walker and application of kanamycin fluorescence-electrochemical dual-mode biosensor
By using a fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker, the complexity and high cost of detecting kanamycin in existing technologies have been solved, achieving high accuracy and high specificity in detection, and expanding its application to the detection of other antibiotics.
Patent Information
- Application Number
- CN202511320166.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-16
- Publication Date
- 2026-01-09
AI Technical Summary
Existing technologies for detecting kanamycin suffer from problems such as cumbersome instrument operation, high cost, and complex sample processing, making it difficult to achieve sensitive and accurate detection.
A dual-mode fluorescence-electrochemical biosensor for kanamycin based on a three-dimensional DNAzyme walker was designed. The DNAzyme walker, which is self-protected by a ring domain, is separated from the target binding domain. The self-protected secondary structure formed by the target binding domain is used to combine the specific recognition of aptamers and kanamycin to achieve dual-mode fluorescence-electrochemical detection.
It achieves high accuracy and specificity in the detection of kanamycin, is suitable for trace analysis in complex environments, and has the potential for sensitive detection of other antibiotics, making it suitable for food safety and environmental monitoring.
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Figure CN121294622A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biosensor technology, and in particular to a kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker and its applications. Background Technology
[0002] Kanamycin, an important aminoglycoside antibiotic, is widely used in livestock and aquaculture for the prevention and treatment of bacterial infections due to its significant antibacterial effects, while also promoting animal growth and improving feed conversion rates. However, its broad-spectrum antibacterial activity and low cost have led to frequent and even abusive use. With increasing production and usage, more and more antibiotics are entering the natural environment and accumulating, resulting in antibiotic resistance in bacteria. The spread of drug-resistant bacteria poses serious risks to environmental quality, ecological security, and even human health. Therefore, achieving sensitive and accurate detection of kanamycin is of paramount importance.
[0003] Kanamycin is currently detected using various methods, including high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and enzyme-linked immunosorbent assay (ELISA). However, these analytical techniques still suffer from drawbacks such as cumbersome instrument operation, high cost, and complex sample processing. Therefore, this invention proposes a convenient and accurate fluorescence-electrochemical dual-mode detection method for kanamycin analysis based on a three-dimensional DNAzyme walker. Summary of the Invention
[0004] To address the above technical problems, this invention provides a kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker and its applications. The self-protected ring-domain DNAzyme walker designed in this invention separates the catalytic core from the target-binding domain to reduce spontaneous reaction background. Simultaneously, the self-protective secondary structure formed by the target-binding domain significantly improves the stability of the DNAzyme walker, thereby enhancing the sensor's practicality and reliability in complex environments.
[0005] The first objective of this invention is to provide a kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker, comprising a gold electrode AuE modified with a trap strand CP, and a three-dimensional DNAzyme walker and a kanamycin target recognition probe Apt / R; The three-dimensional DNAzyme walker includes a G-quadruplex substrate strand embedded with MB, a DNAzyme strand with a ring domain self-protection, and gold nanoparticles (AuNPs). The target recognition probe Apt / R is obtained by hybridization of the aptamer chain Apt and the aptamer complementary chain R.
[0006] In some embodiments of the present invention, the sequence of the capture chain CP is 5'-HS-TTTTTTATAGTGCATCTGCTGA-3' (as shown in SEQ ID NO. 1); The sequence of the tetra-stranded substrate strand is 5'-GGGTAGGGCGGGTTGGGCAGCAGATGCACTATrAGGAAGAGACACGTAGTTTTTTTTTT-HS-3` (as shown in SEQ ID NO. 2); (where Tr represents RNA).
[0007] The DNAzyme strand sequence is 5'-CTACGTGTCTCTTCTCCGAGCGGCTAAGCGCCAACCCAGGGTTGACGGTCGAAATAGTGCAT-3' (as shown in SEQ ID NO. 3); The sequence of the aptamer chain Apt is 5`-TGGGGGTTGAGGCTAAGCCGA-3` (as shown in SEQ ID NO. 4); The sequence of the aptamer complementary strand R is 5`-GGCGCTTAGCCTCAACCCTGGG-3` (as shown in SEQ ID NO. 5).
[0008] In some embodiments of the present invention, the gold electrode AuE modified with the trapping chain CP is prepared by the following method: A chain-capturing CP solution is provided, which is then added to a TCEP solution to react and obtain a mixture. The bare gold electrode AuE was soaked in piranha solution, polished with alumina powder, washed and dried, and then electrochemically activated. A mixture containing CP chains was dropwise added to the surface of an activated gold electrode, incubated at a constant temperature, and then sealed at room temperature to obtain the gold electrode AuE modified with the CP-capturing chain.
[0009] In some embodiments of the present invention, the concentration of the chain-capturing CP solution is 1~5 μM; The concentration of the tri-(2-formylethyl)phosphonic acid hydrochloride TCEP solution is 10-20 mM. This invention is based on the fact that thiol (SH-)-modified DNA sequences are prone to spontaneous oxidation during transport or storage, forming disulfide-bonded dimer DNA structures that cannot be modified onto AuNPs or gold electrode surfaces via Au-S bonds. Therefore, thiol (SH-)-modified DNA sequences need to be reduced using TCEP before use to reduce the stable disulfide bonds to reactive thiol (SH-) groups.
[0010] The constant temperature incubation is divided into two stages: incubation at 4 ℃ for 12~24 h, and constant temperature incubation at 37 ℃ for 2~4 h.
[0011] Room temperature sealing operation: 6-mercapto-1-hexanol (MCH) is added dropwise and sealed at room temperature for more than 1 h; the concentration of the 6-mercapto-1-hexanol MCH solution is 1~5 mM.
[0012] In some embodiments of the present invention, the three-dimensional DNAzyme walker is prepared by the following method: (1) Add TCEP solution to the GS solution containing G-tetrachain substrate chain modified with thiol group at 3' end, mix well, and let stand to activate to obtain a mixed solution; (2) Add the obtained mixed solution to the AuNPs colloidal solution, add the DNAzyme chain W, and incubate at a constant temperature to obtain GS / W-AuNPs, which is the three-dimensional DNAzyme walker.
[0013] In some embodiments of the present invention, in step (1), the concentration of the solution containing the G-quadriplex substrate chain GS is 50~100 μM; The concentration of the TCEP solution is 10~20 mM.
[0014] In some embodiments of the present invention, the concentration of the AuNPs colloidal solution is 2~10 nM; The molar ratio of G-quadruplex substrate chain GS, TCEP, AuNPs and DNAzyme chain W is (100:10000:1:10) to (100:10000:1:30).
[0015] A second objective of this invention is to provide the application of the aforementioned kanamycin fluorescence-electrochemical dual-mode biosensor in the qualitative or quantitative detection of kanamycin.
[0016] In some embodiments of the present invention, the detection of kanamycin includes the following steps: (1) The target recognition probe Apt / R was synthesized by annealing and hybridizing the aptamer chain Apt and the complementary aptamer chain R. The mixture was then mixed with a solution containing kanamycin and incubated at a constant temperature to obtain a mixed solution. (2) Add a three-dimensional DNAzyme walker consisting of a G-quadruplex substrate chain GS and a ring domain-protected DNAzyme chain embedded with methylene blue MB to the mixture in step (1), mix it with PBS solution containing MgCl2 and continue to incubate at a constant temperature to produce a solution containing a large number of G-quadruplex nucleic acid fragments embedded with methylene blue MB. (3) Detect the fluorescence intensity value of the solution obtained in step (2) at 680 nm to achieve qualitative or quantitative detection of the fluorescence pattern of kanamycin; (4) Centrifuge the solution obtained in step (2) to obtain the supernatant, and drop it onto the gold electrode AuE modified with the capture chain CP. Incubate at a constant temperature as the working electrode. Use differential pulse voltammetry (DPV) to measure the change in the electrical signal of methylene blue (MB) to achieve qualitative or quantitative detection of the electrochemical mode of kanamycin.
[0017] In step (2), the three-dimensional DNAzyme walker embedded with methylene blue MB in this invention is prepared by the following method: A certain concentration of methylene blue solution was added to the dispersion of the three-dimensional DNAzyme walker (GS / W-AuNPs). After incubation at 37 °C for 60 min, the solution was centrifuged to remove the supernatant. Then, it was washed three times with PBS buffer (10 mM, pH 7.4) and redispersed in PBS buffer (10 mM, pH 7.4) to obtain the three-dimensional DNAzyme walker embedded with methylene blue MB.
[0018] In some embodiments of the present invention, in step (2), the Mg 2+ The solution is a 10mM PBS solution containing 40-100 mM MgCl2. The specific components of the solution include: potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), and magnesium chloride (MgCl2).
[0019] The constant temperature incubation is 90~100 ℃, and the time is 10~20 min.
[0020] This invention provides a method for preparing a kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker, using a double-stranded structure (Apt / R) formed by an aptamer (Apt) and its complementary strand (R) as the target recognition unit. A three-dimensional DNAzyme walker is obtained by modifying the surface of AuNPs with a substrate chain (GS) containing a G-quadruplex embedded with methylene blue (MB) and a DNAzyme chain (W) with a ring domain self-protection. Utilizing the specific binding of the target and the aptamer, a free complementary strand (R) is obtained, which can bind to the ring domain of the DNAzyme, activating the catalytic activity of the DNAzyme walker in Mg... 2+Under the influence of the autologous walking cycle, the substrate chain is cleaved, generating a large number of G-quadruplex nucleic acid fragments embedded with bromine (MB), achieving transcriptional amplification of the target. As the substrate chain is cleaved, the MB-embedded G-quadruplex nucleic acid fragments detach from the AuNP surface, allowing the fluorescence of previously quenched MB to recover, generating a fluorescence response. Simultaneously, the free, MB-embedded G-quadruplex nucleic acid fragments can also be captured by the trapping strand (CP) on the surface of the gold electrode (AuE), and a significantly enhanced electrochemical signal of MB can be obtained using differential pulse voltammetry (DPV). Finally, by detecting the fluorescence intensity of the solution and the DPV electrochemical signal, a fluorescence-electrochemical dual-mode detection of kanamycin is achieved.
[0021] The mechanism of fluorescence detection is as follows: when the target is present, the catalytic activity of the DNAzyme walker is specifically activated, and in Mg 2+ With assistance, autonomous walking cycles cleave the rA site containing the G-quadruplex substrate chain (GS), causing the G-quadruplex nucleic acid fragment embedded with MB to detach from the surface of AuNPs, thereby restoring the fluorescence of MB after quenching. The fluorescence intensity depends on the concentration of the target. Therefore, the quantitative analysis of kanamycin can be achieved by detecting the intensity of the fluorescence signal.
[0022] The mechanism of electrochemical detection is as follows: when the target is present, a large number of G-quadruplex nucleic acid fragments embedded with MB, generated by DNAzyme walker cleavage, are immobilized on the surface of a gold electrode (AuE) modified with capture strand (CP) through base complementarity pairing. The concentration of the target is indicated by the differential pulse voltammetry (DPV) electrochemical signal intensity of MB on the electrode surface, thereby achieving electrochemical detection of kanamycin.
[0023] The technical solution of the present invention has the following advantages compared with the prior art: (1) This invention combines fluorescence and electrochemical methods, and by taking advantage of the specific recognition of kanamycin by aptamers, it achieves the goal of high accuracy and high specificity in the detection of kanamycin.
[0024] (2) This invention utilizes a three-dimensional DNAzyme walker with a self-protected ring domain to achieve efficient and stable amplification of the target, ensuring the sensitivity of trace kanamycin analysis in complex environments.
[0025] (3) The dual-mode sensor prepared by the present invention is not only suitable for the trace detection of kanamycin, but also has the potential to achieve sensitive detection of other antibiotics by simply changing the aptamer. It has great application potential in food safety, environmental monitoring and other fields. Attached Figure Description
[0026] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is a schematic diagram illustrating the working principle of the dual-mode biosensor of the present invention.
[0027] Figure 2 These are transmission electron microscope images of AuNPs and the three-dimensional DNAzyme walker obtained in Embodiment 2 of the present invention.
[0028] Figure 3 These are the spectra of kanamycin at different concentrations and the standard curve of the fluorescence sensor in Example 4 of this invention.
[0029] Figure 4 These are the differential pulse voltammetry curves and standard curves of the electrochemical sensor for different concentrations of kanamycin in Example 5 of this invention.
[0030] Figure 5 This is a specificity diagram of the dual-mode sensor studied in Embodiment 7 of the present invention.
[0031] Figure 6 This is a reproducibility diagram of the dual-mode sensor studied in Embodiment 8 of the present invention. Detailed Implementation
[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0033] Example 1 This invention provides a method for preparing a kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker, as detailed below: (1) Preparation of kanamycin recognition element (Apt / R): 10 μL of 0.2 μM aptamer chain (Apt chain) and 10 μL of 0.22 μM complementary chain (R chain) were mixed in TE buffer solution (10 mM Tris-HCl, 1 mM disodium EDTA, 12.5 mM MgCl2), kept at 90 ℃ for 10 min, and then gradually cooled to room temperature to successfully prepare the kanamycin recognition element (Apt / R).
[0034] (2) Activation of the three-dimensional DNAzyme walker and autonomous walking response: First, 2 μL of the recognition element (Apt / R) prepared in step (1) was added to a centrifuge tube, followed by 1 μL of kanamycin. The mixture was reacted at 37 °C for 2 h to allow the target compound to fully bind to the aptamer and release the R chain. Then, 7 μL of a three-dimensional DNAzyme walker (GS / W-AuNPs) was added, and the mixture was reacted at 37 °C for 30 min to allow the free R chain to target and bind to the ring domain of the DNAzyme chain (W), activating the DNAzyme catalytic activity. Subsequently, 2 μL of Mg was added. 2+ (60 mM, 10 mM PBS solution containing 60 mM MgCl2), reacted at 37 ℃ for 1.5 h, in Mg 2+ With assistance, the DNAzyme autonomously walks and cycles to cleave the substrate chain, generating a large number of free G-quadruplex nucleic acid fragments, thereby achieving the transcription and amplification of the target signal. See the appendix for details. Figure 1 .
[0035] Example 2 This embodiment provides a method for fabricating a three-dimensional DNAzyme walker, as detailed below: (1) Synthesis of gold nanoparticles (AuNPs): First, 10 mL of 1wt% HAuCl4 solution was added to 290 mL of ultrapure water and boiled. Then, 30 mL of trisodium citrate solution (38.8 mM) was added with vigorous stirring, and stirring was continued for 15 min, during which the solution changed from pale yellow to wine red. Finally, the solution was cooled to room temperature and stored at 4 °C for later use. The concentration of 13 nm AuNPs was determined by measuring its maximum absorbance at 520 nm (ε = 2.7 × 10⁻⁶). 8 L·mol −1 ·cm −1 The concentration of AuNPs prepared was 4 nM.
[0036] (2) Modification with gold nanoparticles (AuNPs): Take 6 μL of 100 μM 3'-terminal thiol-modified G-quadruplex substrate chain (GS) diluted with DEPC (diethyl pyrocarbonate-treated water), add 6 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution, mix thoroughly, and incubate at room temperature for 1 h for activation. Then, add the activated mixture to a 2 mL glass bottle containing 400 μL of 4 nM AuNPs and mix again. Then, place the glass bottle in a microwave oven (microwave input power 1150 W, microwave output power 700 W) and heat on medium-high for 18 min until completely dry. The dried product is redispersed with PBS buffer (10 mM, pH 7.4). To remove free DNA from the solution, centrifuge at 12000 rpm for 20 min, remove the supernatant, and then wash three times with PBS buffer (10 mM, pH 7.4) for purification to obtain GS-AuNPs. Finally, the obtained GS-AuNPs were redispersed in 400 μL of PBS buffer. Then, 7 μL of 10 μM DNAzyme chain (W chain) was added to the GS-Au NPs solution, and the mixture was kept at 90 °C for 10 min to allow the W chain to fully bind with the GS modified on the Au NPs, resulting in a three-dimensional DNAzyme walker (GS / W-AuNPs), which was stored at 4 °C for later use. 0.4 mM methylene blue solution was added to the three-dimensional DNAzyme walker (GS / W-AuNPs) dispersion, and the mixture was incubated at 37 °C for 60 min. After centrifugation to remove the supernatant, the solution was washed three times with PBS buffer (10 mM, pH 7.4) and redispersed in PBS buffer (10 mM, pH 7.4) to obtain a three-dimensional DNAzyme walker embedded with methylene blue MB.
[0037] Transmission electron microscopy characterization was performed on Au NPs and three-dimensional DNAzyme walkers without methylene blue embedding (GS / W-AuNPs). The results are shown in the appendix. Figure 2 With AuNPs ( Figure 2 Compared to A), DNA-modified three-dimensional DNAzyme walkers (GS / W-AuNPs) Figure 2 The dispersion of B) is better.
[0038] Example 3 This embodiment provides a method for preparing a gold electrode (AuE) modified with a trap chain (CP), as detailed below: (1) Soak the bare gold electrode in the newly prepared piranha solution (a mixture of 98wt% H2SO4 and 30wt% H2O2 in a volume ratio of 3:1) for 30 min, and then rinse it with ultrapure water for later use.
[0039] (2) The electrodes obtained in step (1) are ground and polished to a mirror finish with 0.3 μm and 0.05 μm alumina powder respectively. Then, the electrodes are ultrasonically treated in the order of ultrapure water, anhydrous ethanol, anhydrous ethanol and ultrapure water respectively, and dried for later use.
[0040] (3) The electrode obtained in step (2) is electrochemically cleaned in 0.5 M sulfuric acid solution at a scan rate of 0.1 V / s until a stable cyclic voltammetry curve is obtained. Then it is rinsed with ultrapure water and dried for later use.
[0041] (4) Dilute the captured chain (CP) with TE buffer solution (10 mM Tris-HCl, 1 mM disodium EDTA, 12.5 mM MgCl2), add 10 mM TCEP solution, react at room temperature for 1 h to break the disulfide bonds in the CP chain, and finally dilute the CP chain concentration to 1 μM for later use.
[0042] (5) Take 10 μL of CP chain solution from step (4) and drop it onto the electrode prepared in step (3), incubate at 4 °C for 12 h, and then incubate at 37 °C for 2 h.
[0043] (6) Finally, 10 μL of MCH (6-mercapto-1-hexanol, 1 mM) solution was added dropwise to the electrode obtained in step (5) and incubated at room temperature for 1 h to obtain the CP-modified gold electrode.
[0044] Example 4 This embodiment provides a method for constructing a fluorescence sensor, as detailed below: (1) Hybridize 10 μL of 0.2 μM kanamycin aptamer chain (Apt chain) and 10 μL of 0.22 μM kanamycin complementary chain (R chain) in PBS buffer to form kanamycin recognition element (Apt / R).
[0045] (2) Take 70 μL of three-dimensional DNAzyme walker (GS / W-AuNPs) and add 10 μL of 0.4 mM methylene blue. Incubate at 37℃ for 40 min. Centrifuge the solution at 12000 rmp for 20 min. Remove the supernatant and wash three times with PBS buffer (10 mM, pH 7.4). Redisperse the solution in 80 μL of PBS buffer for later use.
[0046] (3) Add 10 μL of kanamycin at different concentrations (1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM, respectively) to the solution from step (1), react at 37 °C for 2 h, then add 80 μL of the solution from step (2), react at 37 °C for 30 min, and then add 20 μL of Mg 2+ (10 mM PBS solution containing 60 mM MgCl2), react at 37 °C for 1.5 h.
[0047] (4) Take the final reaction solution from step (3) and record the fluorescence intensity at 680 nm (excitation at 645 nm). Perform a linear regression analysis on the logarithm of the kanamycin concentration and the fluorescence intensity of MB at 680 nm. Using the logarithm of the kanamycin concentration (in nM) as the abscissa and the fluorescence intensity as the ordinate, the linear regression equation for kanamycin fluorescence analysis is obtained as follows: FL =158.799+14.27 lgC Kana (R) 2 =0.9969). The detection range of this fluorescent biosensor is 1×10⁻⁶. -3 nM~1×10 3 The detection limit was 0.327 pM (nM). See attached results. Figure 3 .
[0048] Example 5 This embodiment provides a method for constructing an electrochemical sensor, as detailed below: (1) Hybridize 10 μL of 0.2 μM aptamer chain (Apt chain) and 10 μL of 0.22 μM complementary chain (R chain) in PBS buffer to form kanamycin recognition element (Apt / R).
[0049] (2) Take 70 μL of three-dimensional DNAzyme walker (GS / W-AuNPs) and add 10 μL of 400 μM methylene blue. Incubate at 37℃ for 40 min. Centrifuge the solution at 12000 rmp for 20 min. Remove the supernatant and wash three times with PBS buffer (10 mM, pH 7.4). Redisperse the solution in 80 μL of PBS buffer for later use.
[0050] (3) Take 2 μL of the solution from step (1) and add 1 μL of kanamycin of different concentrations (10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM, respectively). React at 37 °C for 2 h. Then add 7 μL of the solution from step (2) and react at 37 °C for 30 min. Finally, add 2 μL of Mg.2+ (10 mM PBS solution containing 60 mM MgCl2), reacted at 37°C for 1.5 h.
[0051] (4) Centrifuge the mixed solution from step (3) at 12000 rpm for 20 min, take 10 μL of the supernatant and drop it onto the surface of the gold electrode (AuE) modified with the trap chain (CP), and incubate at 37 ℃ for 2 h.
[0052] (5) The gold electrode (AuE) from step (4) was rinsed three times with PBS (10 mM, pH 7.4) buffer solution and used as the working electrode. The calomel electrode was used as the reference electrode and the Pt electrode was used as the counter electrode. The electrochemical signal change of MB was measured in PBS (10 mM, pH 7.4) buffer solution using differential pulse voltammetry (DPV). The potential ranged from 0 to -0.6 V, the pulse width was 0.05 V, and the scan rate was 0.05 s.
[0053] (6) A linear regression equation for the electrochemical analysis of kanamycin was obtained by linearly fitting the logarithm of kanamycin concentration and the DPV peak current signal intensity of MB. 10 -6 A = 6.72 + 0.467 lgC Kana (R) 2 =0.9986). Compared with fluorescence methods, this electrochemical biosensor has a wider detection range (1×10⁻⁶). -5 nM~2.5×10 3 The detection limit is even lower (18.48 aM), and the results are attached. Figure 4 .
[0054] Example 7 This embodiment provides a method for testing the anti-interference performance of a fluorescence-electrochemical dual-mode biosensor. The specific steps are as follows: Interfering antibiotics such as amoxicillin (Amox), ciprofloxacin (CIP), levofloxacin (LEV), metronidazole (MET), and tetracycline (TC) at concentrations of 1 μM were added to the fluorescence and electrochemical testing systems, respectively. A mixed solution of multiple antibiotics containing kanamycin (fluorescence: 10 nM; electrochemical: 10 pM) was also added. The anti-interference performance of the dual-mode sensor was tested by recording the fluorescence intensity and DPV peak current intensity, respectively. The results are shown in the appendix. Figure 5 This fluorescence-electrochemical dual-mode sensor only produces a strong fluorescence signal in the presence of the target kanamycin. Figure 5 (A) and electrochemical signals ( Figure 5(B) When the target kanamycin is absent, even in the presence of other interfering antibiotics, the signal intensity remains comparable to that of the blank sample, maintaining a low level. This indicates that the dual-mode sensor has excellent anti-interference performance and can be used for the accurate detection and analysis of kanamycin in complex samples.
[0055] Example 8 This embodiment provides a reproducibility experiment for a fluorescence-electrochemical dual-mode biosensor, as detailed below: The reproducibility of the dual-mode sensor was tested by preparing six batches of fluorescence and electrochemical sensors. The results are shown in the appendix. Figure 6 Data shows that the relative standard deviation (RSD) of the fluorescence sensor is 1.79%. Figure 6 (A) The RSD of the electrochemical sensor is 2.37% ( Figure 6 The percentages (B in the middle section) are all below 5%, indicating that the constructed sensor has excellent reproducibility.
[0056] Example 9 This embodiment provides the actual sample analysis performance of a fluorescence-electrochemical dual-mode biosensor, as shown below: Three groups of spiked samples of tap water and milk at different concentrations were prepared for actual sample analysis using a dual-mode sensor. Tap water samples required no pretreatment; milk samples required simple pretreatment: 20% acetic acid was added to adjust the sample pH to 4.6, and the samples were incubated at 45 °C for 15 min to precipitate proteins; then, the samples were centrifuged at 10,000 rpm for 30 min to remove coagulated proteins and fats, and the supernatant was filtered through a 0.22 μM membrane and the pH was adjusted to neutral. Actual sample analysis results showed that the fluorescence sensor achieved recoveries of 95.3%–108.83% and 95.67%–103.9% for tap water and milk samples, respectively, with relative standard deviations (RSDs) of 1.63%–3.14% and 2.57%–3.71%, respectively. The electrochemical sensor achieved recoveries of 98.07%–101.02% and 96.23%–103.6% for tap water and milk samples, respectively, with RSDs of 1.74%–5.30% and 1.95%–4.99%, respectively. These results demonstrate that the dual-mode sensor developed in this invention is feasible for detecting kanamycin and can meet the needs of practical sample analysis.
[0057] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker, characterized in that, This includes a gold electrode AuE modified with a trapping strand CP, and a three-dimensional DNAzyme walker and a kanamycin target recognition probe Apt / R; The three-dimensional DNAzyme walker includes a G-quadruplex substrate strand embedded with MB, a DNAzyme strand with a ring domain self-protection, and gold nanoparticles (AuNPs). The target recognition probe Apt / R is obtained by hybridization of the aptamer chain Apt and the aptamer complementary chain R.
2. The kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker according to claim 1, characterized in that, The sequence of the capture chain CP is 5'-HS-TTTTTTATAGTGCATCTGCTGA-3'; The sequence of the tetrachain substrate chain is 5'-GGGTAGGGCGGGTTGGGCAGCAGATGCACTATrAGGAAGAGACACGTAGTTTTTTTTTT-HS-3`; The DNAzyme chain sequence is 5'-CTACGTGTCTCTTCTCCGAGCGGCTAAGCGCCAACCCAGGGTTGACGGTCGAAATAGTGCAT-3'; The sequence of the aptamer chain Apt is 5`-TGGGGGTTGAGGCTAAGCCGA-3`; The sequence of the aptamer complementary strand R is 5`-GGCGCTTAGCCTCAACCCTGGG-3`.
3. The kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker according to claim 1, characterized in that, The gold electrode AuE modified with the trapping chain CP was prepared by the following method: A chain-capturing CP solution is provided, which is then added to a TCEP solution to react and obtain a mixture. The bare gold electrode AuE was soaked in piranha solution, polished with alumina powder, washed and dried, and then electrochemically activated. A mixture containing CP chains was dropwise added to the surface of an activated gold electrode, incubated at a constant temperature, and then sealed at room temperature to obtain the gold electrode AuE modified with the CP-capturing chain.
4. The kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker according to claim 3, characterized in that, The concentration of the CP capture solution is 1~5 μM; The concentration of tri-(2-formylethyl)phosphine hydrochloride solution is 10~20 mM; The constant temperature incubation is divided into two stages: incubation at 4 ℃ for 12~24 h, and constant temperature incubation at 37 ℃ for 2~4 h.
5. A kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker according to claim 1, characterized in that, The three-dimensional DNAzyme walker was prepared by the following method: (1) Add TCEP solution to the GS solution containing G-tetrachain substrate chain modified with thiol group at 3' end, mix well, and let stand to activate to obtain a mixed solution; (2) Add the obtained mixed solution to the AuNPs colloidal solution, add the DNAzyme chain W, and incubate at a constant temperature to obtain GS / W-AuNPs, which is the three-dimensional DNAzyme walker.
6. A kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker according to claim 5, characterized in that, In step (1), the concentration of the solution containing the G-quadruplex substrate chain GS is 50~100 μM; The concentration of the TCEP solution is 10~20 mM.
7. A kanamycin fluorescence-electrochemical dual-mode biosensor based on a three-dimensional DNAzyme walker according to claim 5, characterized in that, The concentration of AuNPs colloidal solution is 2~10 nM; The molar ratio of G-quadruplex substrate chain GS, TCEP, AuNPs and DNAzyme chain W is (100:10000:1:10) to (100:10000:1:30).
8. The application of the kanamycin fluorescence-electrochemical dual-mode biosensor according to any one of claims 1 to 7 in the qualitative or quantitative detection of kanamycin.
9. The application according to claim 8, characterized in that, The detection of kanamycin includes the following steps: (1) The target recognition probe Apt / R was synthesized by annealing and hybridizing the aptamer chain Apt and the complementary aptamer chain R. The mixture was then mixed with a solution containing kanamycin and incubated at a constant temperature to obtain a mixed solution. (2) Add a three-dimensional DNAzyme walker embedded with methylene blue MB to the mixture in step (1), and mix with Mg 2+ After mixing the solutions, the mixture was incubated at a constant temperature to produce a solution containing a large number of G-quadruplex nucleic acid fragments embedded with methylene blue MB. (3) Detect the fluorescence intensity value of the solution obtained in step (2) at 680 nm to achieve qualitative or quantitative detection of the fluorescence pattern of kanamycin; (4) Centrifuge the solution obtained in step (2) to obtain the supernatant, and drop it onto the gold electrode AuE modified with the capture chain CP. Incubate at a constant temperature as the working electrode. Use differential pulse voltammetry (DPV) to measure the change in the electrical signal of methylene blue (MB) to achieve qualitative or quantitative detection of the electrochemical mode of kanamycin.
10. The application according to claim 5, characterized in that, In step (2), the Mg 2+ The solution was a PBS solution containing 40-100 mM MgCl2; The constant temperature incubation is 90~100 ℃, and the time is 10~20 min.