An electrochemical biosensor and electrochemical detection method for enrofloxacin

Abstract

This invention discloses an electrochemical biosensor and electrochemical detection method for enrofloxacin. The electrochemical biosensor includes an electrode and G-quadruplex ribozyme nanowires coated on the electrode. The G-quadruplex ribozyme nanowires are obtained by amplification and extension after complementary hybridization of a single-stranded R-DNA probe and an S-DNA probe pre-modified on the electrode surface. The single-stranded R-DNA probe is obtained by releasing a DNA double-stranded probe, which is obtained by partial complementary hybridization of an R-DNA probe with a free 3'-OH end and an enrofloxacin aptamer sequence. This invention establishes an electrochemical detection method for enrofloxacin based on a signal amplification strategy designed by terminal deoxynucleotidyl transferase (TdT) in conjunction with G-quadruplex ribozyme. The linear detection range of this method for enrofloxacin is 0.5–50 ng / mL, with a detection limit as low as 0.043 ng / mL. Furthermore, this label-free electrochemical biosensor is simple, rapid, and low-cost, and has been successfully applied to the analysis and detection of actual food samples, showing good application potential.

Description

Technical Field

[0001] This invention relates to an electrochemical biosensor and an electrochemical detection method, and more particularly to an electrochemical biosensor and an electrochemical detection method for enrofloxacin. Background Technology

[0002] Enrofloxacin (ENR), a synthetically produced quinolone antibacterial drug, is widely used in the treatment of animal and plant diseases due to its effective inhibitory effect on both Gram-positive and Gram-negative bacteria. However, inappropriate use or even abuse of ENR can lead to excessive residues in animal and plant foods, which can then accumulate in the food chain and cause various adverse effects on the human body, including skin allergies, intestinal flora imbalance, and carcinogenic mutations. Therefore, the research and development of ENR detection methods is of great significance for ensuring food safety and human health. Currently, methods used for ENR residue detection mainly include high-performance liquid chromatography (HPLC), immunochromatography, quartz crystal microbalance, chemiluminescence immunoassay, and surface-enhanced Raman scattering (SERS). However, these traditional methods are usually time-consuming, costly, complex in operation, or have low sensitivity, making it difficult to meet the high standards required for actual antibiotic residue detection. Therefore, there is an urgent need to develop new detection methods to achieve efficient detection and analysis of ENR.

[0003] As a novel analytical detection method, electrochemical biosensors have attracted widespread attention due to their advantages such as fast response speed, low cost, high sensitivity, simple operation, and ease of miniaturization, showing great potential in the field of antibiotic residue detection. Tabanico et al. constructed a portable ENR electrochemical biosensor using molecularly imprinted polymers as recognition elements; Zhang et al. designed a novel ENR residue electrochemical aptamer sensor based on DNA ribozymes. However, most existing ENR electrochemical biosensors have low sensitivity or involve complex DNA sequence designs, limiting their application in practical food sample detection. Therefore, it is necessary to develop novel electrochemical biosensors to achieve simple, rapid, highly sensitive, and highly specific detection of ENR. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a simple, rapid, highly sensitive and specific electrochemical biosensor for enrofloxacin; another purpose of this invention is to provide an electrochemical detection method for enrofloxacin.

[0005] Technical Solution: The present invention provides an electrochemical biosensor for enrofloxacin, comprising an electrode and G-quadruplex ribozyme nanowires coated on the electrode; the G-quadruplex ribozyme nanowires are obtained by amplification and extension of a single-stranded R-DNA probe after complementary hybridization with an S-DNA probe pre-modified on the electrode surface; the single-stranded R-DNA probe is obtained by the release of a DNA double-stranded probe, which is obtained by partial complementary hybridization of an R-DNA probe with a free 3'-OH end and an enrofloxacin aptamer sequence; when enrofloxacin is present, the DNA double-stranded probe separates, releasing the single-stranded R-DNA probe, thereby inducing an amplification reaction and forming a G-quadruplex ribozyme nanowire structure.

[0006] As a further improvement to the above scheme, the amplification reaction is a reaction in which terminal deoxynucleotidyl transferase amplifies and extends the amplification at the 3'-OH end.

[0007] As a further improvement to the above scheme, the S-DNA sequence is: 5'-TTA GTG CCC TG-SH-3';

[0008] The R-DNA sequence is: 5'-CAG GGC ACT AAG GTA-3';

[0009] The enrofloxacin aptamer sequence is: 5'-GCT GTG TGA CTC CTG CAA GTC CGA CAT ACC TTA GTGCCC TGA TAT AAT GTA ACA CTA TTG AGC AGC TGT ATC TTG TCT CC-3'.

[0010] On the other hand, the present invention provides an electrochemical detection method for enrofloxacin, the electrochemical detection method comprising the following steps:

[0011] (1) The gold electrode is pretreated to obtain the pretreated gold electrode;

[0012] (2) Modify S-DNA on the surface of the pretreated gold electrode;

[0013] (3) Mix the ENR aptamer sequence and R-DNA probe evenly and react for a period of time; then add enrofloxacin solution and incubate for a period of time to obtain a mixed solution;

[0014] (4) Change the concentration of enrofloxacin solution and repeat steps (1) to (3) several times to obtain several mixed solutions containing gradient concentrations of enrofloxacin; immerse several gold electrodes modified with S-DNA in the above mixed solutions; then immerse the gold electrodes in terminal deoxynucleotidyl transferase reaction buffer to amplify continuous G-quadruplex sequences; finally introduce Hemin onto the surface of the gold electrodes and record the electrochemical signals; obtain the standard curve of enrofloxacin concentration and corresponding electrochemical signals.

[0015] (4) Replace the enrofloxacin solution in step (3) with the test sample, and repeat steps (1) to (3) to obtain a mixed solution containing the test sample; immerse the S-DNA modified gold electrode in the above mixed solution; then immerse the gold electrode in terminal deoxynucleotidyl transferase reaction buffer to amplify a continuous G-quadruplex sequence; finally introduce Hemin onto the surface of the gold electrode and record the electrochemical signal; compare the electrochemical signal with the standard curve in step (3) to calculate the enrofloxacin concentration in the test sample.

[0016] As a further improvement to the above scheme, in step (1), the preprocessing includes:

[0017] The gold electrode was polished and then soaked in a piranha solution for a period of time to obtain the first gold electrode. The first gold electrode was polished to obtain the second gold electrode with a mirror-like surface. The second gold electrode was then subjected to ultrasonic cleaning and piranha solution soaking to obtain the third gold electrode. The third gold electrode was then subjected to electrochemical scanning cleaning and then the electrode surface was thoroughly rinsed with ultrapure water to obtain the pretreated gold electrode.

[0018] Preferably, the polishing process uses an alumina slurry solution for polishing.

[0019] Preferably, in step (2), the incubation time of enrofloxacin is 30 min to 50 min; more preferably, the incubation time of enrofloxacin is 40 min.

[0020] Preferably, in steps (4) and (5), the reaction time for immersing the gold electrode in the terminal deoxynucleotidyl transferase reaction buffer is 45 min to 75 min; more preferably, the reaction time is 60 min.

[0021] Preferably, in steps (4) and (5), the concentration of Hemin is 0.4 mM to 0.8 mM, and more preferably, the concentration of Hemin is 0.6 mM.

[0022] Preferably, in steps (4) and (5), an electrochemical analysis method is used to measure the electrochemical signal, wherein the electrochemical analysis method is chronoamperometry, electrochemical impedance spectroscopy, or cyclic voltammetry.

[0023] Terminal deoxynucleotidyl transferase (TdT) is a template-free DNA polymerase that catalyzes the addition of oligonucleotide repeats to the 3'-OH end of single-stranded or double-stranded DNA, and the elongation product sequence is highly dependent on the composition of the oligonucleotide pool. When the oligonucleotide pool contains only a single deoxynucleoside triphosphate (dNTP), TdT catalyzes the addition of dNTP repeats to the substrate chain terminus, producing a single polynucleotide chain. When the oligonucleotide pool contains 60% deoxyguanosine triphosphate (dGTP) and 40% deoxyadenosine triphosphate (dATP), TdT catalyzes the production of a continuous G-quadruplex sequence. The G-quadruplex is a special secondary structure with diverse structures and conformations, formed by hydrogen bonds from guanine-rich single-stranded nucleic acids, and possesses multi-functional binding activity; it is often used as a stabilizer for hemin. G-quadruplex ribozymes, formed by the combination of G-quadruplexes and Hemin, possess horseradish peroxidase activity and can catalyze redox reactions of substrates such as 3,3',5,5'-tetramethylbenzimidazole (TMB), 2,2'-azobis(diammonium) salt (ABTS), and luminol. Compared to traditional enzymes, G-quadruplex ribozymes are often used to construct biosensors for the analysis and detection of various targets due to their advantages such as high thermal stability and ease of synthesis and modification.

[0024] This invention presents a highly sensitive ENR detection method based on a TdT-co-G-quadruplex ribozyme signal amplification strategy. In the presence of ENR, the aptamer sequence specifically recognizes ENR, triggering dual-probe separation. This results in the generation of a DNA double strand with a 3'-OH terminus on the electrode surface, inducing a TdT amplification reaction and generating G-quadruplex ribozyme nanowires. These nanowires then catalyze TMB signal amplification, with the signal magnitude positively correlated with the ENR concentration. This method achieves highly sensitive and specific detection of ENR.

[0025] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The linear detection range of this method for enrofloxacin is 0.5–50 ng / mL, with a detection limit as low as 0.043 ng / mL. Furthermore, this label-free electrochemical biosensor is simple, rapid, and low-cost, and has been successfully applied to the analysis and detection of actual food samples, demonstrating good application potential. The constructed sensing and detection method is simple and rapid, requires no labeling, and does not involve complex DNA strand design or cumbersome nanomaterial synthesis processes. This method has been successfully applied to the analysis and detection of actual food samples, demonstrating good application potential. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of an ENR electrochemical detection method based on TdT-synergistic G-quadruplex ribozyme signal amplification.

[0027] Figure 2 This is a graph showing the quantitative detection of ENR using chronoamperometry. (A) It graphs for different concentrations of target ENR (a-h: 0.5, 1, 2, 5, 10, 20, 50, 200 ng / mL), (B) Current values ​​as a function of ENR concentration. The inset shows the linear relationship between current values ​​and the logarithm of ENR concentration. Error bars represent the standard deviation of three parallel experiments.

[0028] Figure 3 This is a diagram for the feasibility verification of the electrochemical detection method. (A) EIS characterization of electrode surface state: (a) bare electrode, (b) S-DNA modified gold electrode, (c) modified electrode after incubation with a mixed reaction solution containing ENR, (d) modified electrode after incubation with TdT reaction solution. (B) CV verification of feasibility: (a) without ENR, (b) with 50 ng / mL ENR and no TdT amplification reaction, (c) with 50 ng / mL ENR and TdT amplification reaction. (C) It verification of feasibility: (a) without ENR, (b) with 50 ng / mL ENR.

[0029] Figure 4 The results of the optimized experimental conditions are shown in the figure. (A) Change in current value with ENR incubation time, (B) Change in current value with TdT reaction time, (C) Relationship between current value and Hemin concentration. The error bars represent the standard deviation of three parallel experiments.

[0030] Figure 5 This is a specificity validation graph for the electrochemical detection method. Blank control: 10 mM HEPES (pH 8.0), CM: chloramphenicol, KANA: kanamycin, OFL: ofloxacin, TET: tetracycline, Mixture: a mixture of CM, KANA, OFL, TET, and ENR. All antibiotics were at a concentration of 50 ng / mL. Error bars represent the standard deviation of three parallel experiments. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0032] The materials and reagents involved in the embodiments of this invention are as follows:

[0033] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and mercaptohexanol (MCH) were purchased from Bailingwei Technology Co., Ltd. (Beijing, China); ENR was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; TdT was purchased from New England Biotechnology (Beijing) Co., Ltd.; Hemin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and TMB chromogenic reagent was purchased from Shanghai Beyotime Biotechnology Co., Ltd. All DNA probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The DNA sequences are shown below:

[0034] S-DNA: 5'-TTA GTG CCC TG-SH-3'

[0035] R-DNA: 5'-CAG GGC ACT AAG GTA-3'

[0036] Enrofloxacin Aptamer: 5'-GCT GTG TGA CTC CTG CAA GTC CGA CAT ACC TTAGTG CCC TGA TAT AAT GTA ACA CTA TTG AGC AGC TGT ATC TTG TCT CC-3'

[0037] The embodiments of the present invention acquire electrochemical signals using the following method:

[0038] The electrochemical workstation (CHI 660E) was purchased from Shanghai Chenhua Instrument Co., Ltd., and employed a three-electrode system: a modified gold electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum electrode as the counter electrode. All electrochemical measurements were performed at room temperature. The electrochemical analytical methods used included chronoamperometry (It), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The It curve was measured at -0.4V, and the current value was recorded at 60 seconds after the G-quadruplex ribozyme-catalyzed redox reaction reached steady state. EIS measurements were performed in 5 mM [Fe(CN)6] containing 0.1 M KCl. 3- / 4- The measurements were performed in solution with the following parameters: frequency range of 0.1 Hz to 10 kHz, bias potential of 0.224 V, and amplitude of 5 mV. CV measurements were performed in 10 mM HEPES buffer containing 1 mM H2O2, and the results were recorded at a scan rate of 100 mV / s.

[0039] Example 1

[0040] This embodiment provides an electrochemical biosensor for enrofloxacin, comprising an electrode and G-quadruplex ribozyme nanowires coated on the electrode. The G-quadruplex ribozyme nanowires are obtained by complementary hybridization of a single-stranded R-DNA probe with an S-DNA probe pre-modified on the electrode surface, followed by amplification and extension. The single-stranded R-DNA probe is obtained by the release of a DNA double-stranded probe, which is obtained by partial complementary hybridization of an R-DNA probe with a free 3'-OH terminus with an enrofloxacin aptamer sequence. In the presence of enrofloxacin, the DNA double-stranded probe separates, releasing the single-stranded R-DNA probe, thereby inducing an amplification reaction and forming the G-quadruplex ribozyme nanowire structure. In this invention, the binding of the target analyte enrofloxacin to a specific nucleic acid aptamer triggers the amplification reaction of TdT on the electrode surface, generating the G-quadruplex ribozyme nanowire structure, which then exerts horseradish peroxidase activity to catalyze signal amplification, achieving highly sensitive and specific detection of enrofloxacin.

[0041] like Figure 1 As shown, an R-DNA probe with a free 3'-OH terminus was designed and partially hybridized with an ENR aptamer sequence to form a DNA double-stranded probe. In the presence of ENR, the ENR aptamer specifically recognizes and binds to ENR, causing the DNA double-stranded probe to dissociate and release a single-stranded R-DNA probe. The released R-DNA then hybridizes complementaryly with an S-DNA probe pre-modified on the electrode surface, introducing a free 3'-OH terminus onto the electrode surface. Subsequently, TdT is used to amplify and extend the 3'-OH terminus, generating a continuous G-quadruplex sequence. Finally, Hemin is introduced and hybridizes closely with the G-quadruplex to form a G-quadruplex ribozyme nanowire, which catalyzes signal amplification, generating a significant electrochemical signal positively correlated with ENR concentration, thereby achieving sensitive detection of ENR.

[0042] This embodiment provides a method for preparing an electrochemical biosensor for enrofloxacin, namely an electrochemical biosensor for the detection of enrofloxacin, the steps of which are as follows:

[0043] (1) Pretreatment of gold electrodes

[0044] First, the gold electrode was physically polished on sandpaper. Then, it was immersed in a piranha solution (concentrated sulfuric acid: hydrogen peroxide = 3:1) for 5 minutes to remove organic matter from the electrode surface. Next, it was polished sequentially on 1 μm and 0.3 μm alumina slurry solutions to achieve a mirror-like finish. Subsequently, the gold electrode was ultrasonically cleaned with ethanol and ultrapure water for 10 minutes each to remove the alumina slurry adhering to the electrode surface, followed by immersion in a piranha solution for 5 minutes. Finally, it underwent electrochemical scanning cleaning in 0.5 M sulfuric acid, and then thoroughly rinsed with ultrapure water.

[0045] (2) DNA modification and fixation

[0046] The pretreated gold electrode was immersed in a 1 μM S-DNA solution (containing 1 mM TCEP to prevent disulfide bonds from forming between S-DNA molecules) and left to stand at 37 °C for 1 h. Then the electrode was immersed in a 0.1 mM MCH solution, sealed with a Parafilm membrane, and left to stand in a fume hood for 20 min to form an upright and ordered DNA molecule monolayer on the electrode surface, while preventing non-specific adsorption.

[0047] (3) ENR detection

[0048] 20 μL each of the ENR aptamer sequence and R-DNA probe were mixed in a centrifuge tube and heated at 95 °C for 5 min, followed by cooling to room temperature for 1 h. Then, 20 μL of ENR solutions of different concentrations were added to the centrifuge tubes and incubated at room temperature for 40 min. After incubation, the gold electrode was immersed in the above mixed solution and incubated at room temperature for 30 min to allow the released R-DNA to hybridize with the S-DNA. Subsequently, the gold electrode was immersed in TdT reaction buffer (containing 0.2 U μL...) -1 The reaction mixture (TdT, 0.4 mM dATP, 0.6 mM dGTP) was reacted at 37 °C for 60 min to amplify a continuous G-quadruplex sequence. Finally, 0.6 mM Hemin was introduced onto the gold electrode surface, and the electrochemical signal was recorded.

[0049] The results are as follows Figure 2 As shown in Figure A, the current value gradually increases with the increase of ENR concentration. Figure 2 B shows the change in current value with ENR concentration. As can be seen from the inset, within the concentration range of 0.5–50 ng / mL, the current value increases with increasing logarithmic value of ENR concentration, and the linear regression equation is I(μA) = 0.82 + 1.18lgC(ng / mL), R 2 =0.99. Based on the signal-to-noise ratio criterion of 3:1, the limit of detection (LOD) was calculated to be 0.043 ng / mL.

[0050] (4) Detection of ENR in actual food samples

[0051] Milk contains a large amount of fat and protein, which significantly interferes with the detection of the target analyte. Therefore, milk samples were pretreated to remove protein and fat. First, 5 mL of milk was centrifuged at 10000 r / min (10℃) for 10 min to remove the supernatant fat. Then, 150 μL of 17.2% potassium ferrocyanide and 150 μL of 53.5% zinc sulfate were added to precipitate the protein, and the mixture was centrifuged at 10000 r / min (15℃) for 10 min. The supernatant was collected and diluted for later use. Different concentrations of ENR solutions were added to the pretreated milk samples, and the established electrochemical detection method was used to determine the ENR, and the recovery rate was calculated.

[0052] Table 1. Detection results of ENR in milk samples using the electrochemical detection method established in this invention.

[0053]

[0054]

[0055] The practicality of the established electrochemical detection method for detecting ENR in milk samples was verified by examining its analytical performance. Different concentrations of ENR standards were added to pretreated milk samples, and the recovery rate was determined using the constructed electrochemical detection method. As shown in Table 2, within the allowable relative error range, the recovery rate was between 98% and 110%, indicating that the established electrochemical detection method can be successfully applied to the detection of ENR residues in complex food samples.

[0056] Example 2: Principle Verification

[0057] The feasibility of the detection method was investigated using EIS, CV, and IT. EIS is an electrochemical analysis method used to characterize the surface electron transport of electrodes after stepwise modification.

[0058] Electrode surface states at different stages of the enrofloxacin electrochemical biosensor fabrication process were characterized using EIS. In Example 2, the electrochemical biosensor was fabricated using the same method as in Example 1, and the surface states of the bare electrode, the S-DNA-modified gold electrode, the modified electrode after incubation with a mixed reaction solution containing ENR, and the modified electrode after incubation with a TdT reaction solution were characterized using EIS.

[0059] The feasibility of modifying some experimental conditions in Example 1 was verified by CV. The modified experimental conditions are as follows: (a) without ENR, (b) with 50 ng / mL ENR and no TdT amplification reaction, and (c) with 50 ng / mL ENR and TdT amplification reaction.

[0060] The feasibility of the experiment was verified by changing some of the experimental conditions in Example 1. The changed experimental conditions are as follows: (a) in the absence of ENR, and (b) in the presence of 50 ng / mL ENR.

[0061] The results are as follows Figure 3 As shown in Figure A, the EIS spectrum of the bare electrode appears as a straight line with almost no semicircular portion (curve a). After S-DNA modification of the gold electrode surface, the EIS spectrum shows a distinct semicircular arc (curve b), indicating that S-DNA self-assembles on the gold electrode surface, increasing the electron transport resistance. When the modified electrode is treated with a mixed reaction solution containing ENR, the semicircular diameter increases slightly, indicating an increase in electron transport resistance on the electrode surface (curve c). After TdT catalytic extension, the semicircular diameter increases significantly (curve d), indicating the formation of a continuous G-quadruplex structure on the electrode surface, hindering electron transport. Furthermore, CV was used to study the electrochemical response of the sensor to ENR and to verify the role of TdT enzyme in catalyzing the amplification of G-quadruplex ribozymes. Figure 3 As shown in curve B, in the absence of ENR, the current peak is smaller (curve a). This is because without the triggering effect of the target compound ENR, the R-DNA with the 3'-OH terminus cannot hybridize with the S-DNA on the gold electrode surface, and cannot initiate the subsequent TdT-catalyzed amplification reaction to produce G-quadruplexes, resulting in a lower electrochemical response. In the presence of the target compound ENR, it specifically binds to the aptamer, causing the double-stranded DNA probe formed by the aptamer and R-DNA to separate, releasing free R-DNA. Subsequently, the R-DNA probe hybridizes complementaryly with the S-DNA immobilized on the gold electrode surface. When CV is measured without the introduction of the TdT amplification reaction, the current peak remains almost unchanged because no G-quadruplex ribozyme is produced (curve b). After the introduction of TdT catalytic extension to form a large amount of G-quadruplex ribozyme, the current peak increases significantly (curve c), indicating that TdT, in conjunction with the G-quadruplex ribozyme, can effectively amplify the detection signal. Finally, the feasibility of the electrochemical sensor is investigated using It. Figure 3 As shown in Figure C, when ENR is absent, G-quadruplex ribozyme nanowires cannot be generated on the electrode surface, and the current value measured by It is extremely weak. When ENR is present, it triggers the separation of the double-stranded DNA probe, thereby inducing the TdT amplification reaction, forming the G-quadruplex ribozyme nanowire structure, achieving signal amplification, and generating a higher current value. In summary, the constructed electrochemical detection system not only has good target response but also significant signal amplification effect.

[0062] Example 3 Condition Optimization

[0063] To achieve optimal detection performance, key experimental conditions were optimized. These key conditions included ENR incubation time, TdT reaction time, and Hemin concentration.

[0064] The optimization method for ENR incubation time is as follows: Various enrofloxacin electrochemical biosensors were prepared using the method described in Example 1, and electrochemical signals were recorded. The difference from Example 1 is that the ENR incubation time in step (3) was changed to study the effect of ENR incubation time on the electrochemical output signal. The ENR incubation times were set to: 10 min, 20 min, 30 min, 40 min, and 50 min, respectively. Figure 4 As shown in (A), the current value obtained by detecting the same concentration of ENR gradually increases as the incubation time of the target ENR is gradually extended; when the incubation time exceeds 40 min, the current value tends to stabilize. Therefore, 40 min is selected as the optimal target incubation time.

[0065] The optimization method for the TdT reaction time is as follows: Various enrofloxacin electrochemical biosensors were prepared using the method described in Example 1, and electrochemical signals were recorded. The difference from Example 1 is that the TdT reaction time in step (3) was changed to investigate the effect of the TdT reaction time on the current value. The TdT reaction times were set to: 15 min, 30 min, 45 min, 60 min, and 75 min, respectively. Figure 4 As shown in Figure B, as the TdT reaction time increases from 15 min to 75 min, the current value continuously increases and reaches equilibrium at 60 min. Therefore, 60 min is the optimal time for a complete reaction.

[0066] The optimization method for Hemin concentration is as follows: Various enrofloxacin electrochemical biosensors were prepared using the method described in Example 1, and electrochemical signals were recorded. The difference from Example 1 is that the Hemin concentration in step (3) was changed to measure the current values ​​generated at different Hemin concentrations. Figure 4 As shown in Figure C, the electrical signal gradually increases with the increase of Hemin concentration, reaching its maximum value at a concentration of 0.6 mM. Therefore, 0.6 mM is selected as the optimal concentration of Hemin.

[0067] Comparative Example

[0068] To verify the superiority of the proposed electrochemical biosensor, the detection performance of the electrochemical sensor of the present invention was compared with that of the reported ENR biosensor. The reported ENR biosensors are Comparative Examples 1-5. The electrochemical sensor in Comparative Example 1 was prepared using the method described in "SHA JY, LIN H, TIMIRA V, et al. The construction and application of aptamer to simultaneous identification of enrofloxacin and ciprofloxacin residues in fish[J]. Food Analytical Methods, 2021, 14(5): 957-967."; the electrochemical sensor in Comparative Example 2 was prepared using the method described in "DOLATI S, RAMEZANI M, NABAAVINIA MS, et al. Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay[J]. Analytical Biochemistry, 2018, 549: 124-129."; and the electrochemical sensor in Comparative Example 3 was prepared using "LU SY, WANG SL, WU P, et al. A composite prepared from covalent organic framework and gold nanoparticles for the electrochemical determination of The electrochemical sensor of Comparative Example 4 was prepared using the method described in “Neng J, Wang YZ, Zhang YL, et al. MIPs–SERS sensor based on Ag NPs film for selective detection of enrofloxacin in food[J]. Biosensors, 2023, 13(3): 330-345.”; the electrochemical sensor of Comparative Example 5 was prepared using the method described in “GUO XJ, ZHANG LZ, WANG ZW, et al.”.The method described in "Fluorescent carbondots based sensing system for detection of enrofloxacin in water solutions[J]. Spectrochimica Acta Part A, Molecular and Biomolecular Spectroscopy, 2019, 219: 15-22" indicates that the substance was prepared using a method described in this paper.

[0069] Table 2. Comparison of detection performance between the established electrochemical biosensor and reported ENR biosensors.

[0070]

[0071] The electrochemical biosensor established in Example 1 was compared with reported ENR biosensors (Comparative Examples 1-5). As shown in Table 2, the electrochemical sensor of Example 1 exhibits a lower LOD and a wider linear range, demonstrating better detection performance.

[0072] Example 4: Specificity Verification of Electrochemical Biosensors

[0073] To examine the specificity of this method, different antibiotics were selected for control experiments. The test samples were as follows: blank control: 10 mM HEPES (pH 8.0), CM: chloramphenicol, KANA: kanamycin, OFL: ofloxacin, TET: tetracycline, mixture: a mixed solution of CM, KANA, OFL, TET, and ENR. The concentration of all antibiotics was 50 ng / mL. The error bars represent the standard deviation of three parallel experiments.

[0074] Under the optimal detection conditions obtained in Example 3, different antibiotics were quantitatively detected. The specific steps are as follows:

[0075] (1) Pretreatment of gold electrodes

[0076] Same as Example 1.

[0077] (2) DNA modification and fixation

[0078] Same as Example 1.

[0079] (3) ENR detection

[0080] 20 μL each of the ENR aptamer sequence and R-DNA probe were mixed in a centrifuge tube and heated at 95 °C for 5 min, followed by cooling to room temperature for 1 h. Then, 20 μL of the test sample was added to the centrifuge tube and incubated at room temperature for 30 min. After incubation, the gold electrode was immersed in the above mixture and incubated at room temperature for 30 min to allow the released R-DNA to hybridize with the S-DNA. Subsequently, the gold electrode was immersed in TdT reaction buffer (containing 0.2 U μL). -1 The reaction mixture (TdT, 0.4 mM dATP, 0.6 mM dGTP) was reacted at 37 °C for 60 min to amplify a continuous G-quadruplex sequence. Finally, 0.6 mM Hemin was introduced onto the gold electrode surface, and the electrochemical signal was recorded.

[0081] The results are as follows Figure 5 As shown, the current values ​​obtained by this method for detecting ENR are significantly higher than those obtained for detecting other antibiotics, and the target ENR can be accurately identified in mixed samples. This indicates that this method has good selectivity for ENR detection.

[0082] This invention establishes an electrochemical detection method for residual ENR based on TdT-synergistic G-quadruplex ribozyme signal amplification. This method employs an ENR-triggered dual-probe separation-recognition-transfer strategy, using single-stranded DNA with a 3'-OH terminus as the signal conversion probe. TdT is introduced onto the electrode surface to catalyze amplification, generating a G-quadruplex sequence and forming G-quadruplex ribozyme nanowires, ultimately amplifying the signal and achieving highly sensitive and specific detection of ENR. This method has been preliminarily applied to the detection and analysis of antibiotics in actual milk samples, showing good application potential. This sensing and detection system does not involve complex DNA strand design, is simple, rapid, and cost-effective. Furthermore, this method has good universality; by simply changing the corresponding nucleic acid aptamer sequence, the detection and analysis of various other targets can be achieved.

Claims

1. An electrochemical biosensor for enrofloxacin, characterized in that, The electrochemical biosensor includes electrodes and G-quadruplex ribozyme nanowires coated on the electrodes. The G-quadruplex ribozyme nanowires are obtained by amplification and extension after complementary hybridization of a single-stranded R-DNA probe with an S-DNA probe pre-modified on the electrode surface. The single-stranded R-DNA probe is obtained by releasing a DNA double-stranded probe, which is obtained by partial complementary hybridization of an R-DNA probe with a free 3'-OH end with an enrofloxacin aptamer sequence. In the presence of enrofloxacin, the DNA double-stranded probe separates, releasing the single-stranded R-DNA probe, thereby inducing an amplification reaction and forming the G-quadruplex ribozyme nanowire structure. The amplification reaction utilizes terminal deoxynucleotidyl transferase for amplification and extension at the 3'-OH end. The S-DNA sequence is: 5'-TTA GTG CCC TG-SH-3'; the R-DNA sequence is: 5'-CAG GGC ACT AAG GTA-3'; the enrofloxacin aptamer sequence is: 5'-GCT GTG TGA CTC CTG CAAGTC CGA CAT ACC. TTA GTG CCC TGA TAT AAT GTA ACA CTA TTG AGC AGC TGT ATC TTGTCT CC-3'.

2. An electrochemical detection method for enrofloxacin, characterized in that, The electrochemical detection method includes the following steps: (1) The gold electrode is pretreated to obtain the pretreated gold electrode; (2) Modify S-DNA on the surface of the pretreated gold electrode; (3) Mix the ENR aptamer sequence and R-DNA probe evenly and react for a period of time; then add enrofloxacin solution and incubate for a period of time to obtain a mixed solution; (4) Change the concentration of enrofloxacin solution and repeat steps (1) to (3) several times to obtain several mixed solutions containing gradient concentrations of enrofloxacin; immerse several gold electrodes modified with S-DNA in the above mixed solutions respectively; then immerse the gold electrodes in terminal deoxynucleotidyl transferase reaction buffer to amplify continuous G-quadruplex sequences; finally introduce Hemin onto the surface of the gold electrodes and record the electrochemical signals; obtain the standard curve of enrofloxacin concentration and corresponding electrochemical signals. (4) Replace the enrofloxacin solution in step (3) with the sample to be tested, and repeat steps (1) to (3) to obtain a mixed solution containing the sample to be tested; immerse the gold electrode modified with S-DNA in the above mixed solution; then immerse the gold electrode in terminal deoxynucleotidyl transferase reaction buffer to amplify a continuous G-quadruplex sequence; finally, introduce Hemin onto the surface of the gold electrode and record the electrochemical signal; compare the electrochemical signal with the standard curve in step (3) to calculate the concentration of enrofloxacin in the sample to be tested; The S-DNA sequence is: 5'-TTA GTG CCC TG-SH-3'; the R-DNA sequence is: 5'-CAG GGC ACT AAGGTA-3'; and the ENR aptamer sequence is: 5'-GCT GTG TGA CTC CTG CAA GTC CGA CAT ACC TTA GTG CCCTGA TAT AAT GTA ACA CTA TTG AGC AGC TGT ATC TTG TCT CC-3'.

3. The electrochemical detection method for enrofloxacin according to claim 2, characterized in that, In step (1), the preprocessing includes: The gold electrode was polished and then soaked in a piranha solution for a period of time to obtain the first gold electrode. The first gold electrode was polished to obtain the second gold electrode with a mirror-like surface. The second gold electrode was then subjected to ultrasonic cleaning and piranha solution soaking to obtain the third gold electrode. The third gold electrode was then subjected to electrochemical scanning cleaning and then the electrode surface was thoroughly rinsed with ultrapure water to obtain the pretreated gold electrode.

4. The electrochemical detection method for enrofloxacin according to claim 3, characterized in that, The polishing process uses an alumina slurry solution for polishing.

5. The electrochemical detection method for enrofloxacin according to claim 2, characterized in that, In step (2), the incubation time for enrofloxacin is 30 min to 50 min.

6. The electrochemical detection method for enrofloxacin according to claim 2, characterized in that, In steps (4) and (5), the gold electrode is immersed in the terminal deoxynucleotidyl transferase reaction buffer for 45 min to 75 min.

7. The electrochemical detection method for enrofloxacin according to claim 2, characterized in that, In steps (4) and (5), the concentration of Hemin is 0.4 mM to 0.8 mM.

8. The electrochemical detection method for enrofloxacin according to claim 2, characterized in that, In steps (4) and (5), electrochemical signals are measured using electrochemical analysis methods, such as chronoamperometry, electrochemical impedance spectroscopy, or cyclic voltammetry.

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