Electrochemical sensor for quinolone antibiotics and electrochemical detection method

Abstract

The application discloses a quinolone antibiotic electrochemical biosensor and an electrochemical detection method. 2+ In the presence of quinolone antibiotics, the formation of quinolone antibiotic-Cu 2+ complexes will inhibit the specific DNA ribozyme (Cu 2+ -DNAzyme) activity, resulting in the retention of the intact substrate strand probe on the electrode surface, and then triggering the chain hybridization reaction (HCR), forming long double-stranded DNA nanowires on the electrode surface, signal amplification, and the finally generated electrochemical signal being positively correlated with the concentration of the target quinolone antibiotic. The electrochemical sensor of the application is simple to operate, has outstanding sensitivity and specificity, and is successfully applied to the detection of ciprofloxacin in actual food samples, and has potential practicability in food safety detection.

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 quinolone antibiotics. Background Technology

[0002] Quinolone antibiotics are widely used in agriculture, animal husbandry, and aquaculture due to their broad antibacterial spectrum, strong antibacterial activity, and stability. However, due to antibiotic overuse, their residues in animal and plant-based foods often exceed safe limits, directly harming human health, such as inducing drug resistance and impairing digestive function. Therefore, developing methods for detecting quinolone antibiotic residues is crucial. Existing detection methods (such as immunochromatography, liquid chromatography, and capillary electrophoresis) typically require sophisticated instruments and involve complex and time-consuming procedures. Biosensors, with their advantages of speed, simplicity, high sensitivity, and high selectivity, are widely used in antibiotic residue detection. For example, Hu et al. constructed a sensitive electrochemical nanosensor for quinolone antibiotic residues based on aptamer-specific recognition. Kong et al. developed a colorimetric sensor for pazufloxacin residues based on the aggregation mechanism of gold nanoparticles. However, the synthesis of nanomaterials and the construction of nanosensors are complex and costly. To further meet practical production needs, the development of novel biosensors with high sensitivity, simplicity, and low cost has attracted widespread attention.

[0003] Metal ions play multiple roles in the construction of biosensors. They can serve as recognition elements to specifically identify targets, as signal transduction elements to reduce background signals, and as signal amplification elements to improve the sensitivity of biosensors. 2+ As a common metal ion, Cu can be used to regulate the activity or structure of DNA molecules, mediate the synthesis of nanomaterials, or mediate interactions between different substances. It is a highly promising signal transduction and amplification element to improve the analytical performance of biosensors. Furthermore, Cu... 2+ It exhibits a strong affinity for quinolone antibiotics, thus it has been successfully used as a specific recognition element in the development of quinolone antibiotic biosensors. However, the sensitivity of these biosensors is very limited in practical applications, and further research is needed to develop highly sensitive biosensors. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a Cu-based... 2+ A highly sensitive electrochemical biosensor for detecting quinolone antibiotics by regulating the signal amplification of a chain hybridization reaction; another objective of this invention is to provide an electrochemical detection method for quinolone antibiotics.

[0005] Technical solution: The present invention provides an electrochemical biosensor for quinolone antibiotics, the electrochemical biosensor comprising an electrode, long double-stranded DNA nanowires coated on the electrode, and a signal molecule; wherein the long double-stranded DNA nanowires are obtained by amplification of H1 and H2 on the electrode surface via an HCR reaction;

[0006] The H1 chain sequence is as follows:

[0007] 5'-GGTAAGCCGTATTAGAAAGAAGCTAATATCTCTAATACGG-3';

[0008] The H2 chain sequence is as follows:

[0009] 5'-AGCTTCTTTCTAATACGGCTTACCCCGTATTAGAGATATT-3'.

[0010] As a further improvement to the above scheme, K-Cu is present in the environment of the HCR reaction. 2+ Complex, Cu 2+ - DNAzyme, DNA probe; wherein, K is the quinolone antibiotic to be tested; the DNAzyme chain sequence is:

[0011] 5'-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3';

[0012] The S-DNA strand sequence is: 5'-AGCTTCTTTCTAATACGGCTTACC-SH-3'.

[0013] In this invention, in the presence of quinolone antibiotics, the quinolone antibiotic-Cu 2+ The formation of the complex will inhibit Cu 2+ Specific DNA ribozymes (Cu 2+ -DNAzyme) activity leads to the retention of intact substrate strand probes on the electrode surface, subsequently triggering a chain hybridization reaction, forming long double-stranded DNA nanowires on the electrode surface, generating signal amplification, and ultimately the resulting electrochemical signal is positively correlated with the concentration of the target quinolone antibiotic. This invention's electrochemical biosensor uses quinolone antibiotics and Cu... 2+ The complexation reaction is used as the input signal, and HCR is used as the amplified output signal to achieve high-sensitivity detection of quinolones.

[0014] On the other hand, the present invention provides a method for preparing an electrochemical biosensor of a quinolone antibiotic, comprising the following steps:

[0015] (1) DNA probes are modified on the surface of the first electrode to obtain the second electrode;

[0016] (2)Cu 2+ The source was mixed with a quinolone antibiotic solution and incubated, K-Cu 2+ Complex formation, remaining Cu 2+ Combined with DNAzyme to form Cu 2+ -DNAzyme, Cu 2+ - DNAzyme cuts the DNA probe on the surface of the second electrode to obtain the third electrode, where K is the quinolone antibiotic to be tested;

[0017] (3) The third electrode is immersed in a mixed solution containing H1 and H2 and incubated for a period of time. Double-stranded DNA nanowires are formed on the surface of the third electrode, thus obtaining the fourth electrode.

[0018] (4) Introduce signal molecules on the surface of the fourth electrode to obtain an electrochemical biosensor.

[0019] On the other hand, the present invention provides an electrochemical detection method for quinolone antibiotics, comprising the following steps:

[0020] (1) DNA probes are modified on the surface of the first electrode to obtain the second electrode;

[0021] (2)Cu 2+ The source was mixed with a quinolone antibiotic solution and incubated, then DNAzyme solution was added to obtain a mixed solution. The second electrode was immersed in the above mixed solution, and Cu in the mixed solution... 2+ - DNAzyme cuts the DNA probe on the surface of the second electrode to obtain the third electrode, where K is a quinolone antibiotic;

[0022] (3) The third electrode is immersed in a mixed solution containing H1 and H2 and incubated for a period of time. Double-stranded DNA nanowires are formed on the surface of the third electrode, thus obtaining the fourth electrode.

[0023] (4) Introduce signal molecules on the surface of the fourth electrode to obtain an electrochemical biosensor, perform electrochemical detection, and record the electrochemical response value;

[0024] (5) Change the concentration of the quinolone antibiotic solution in step (2) and repeat steps (1)-(4) to obtain the standard curve of the quinolone antibiotic solution and the corresponding electrochemical response value.

[0025] (6) Replace the quinolone antibiotics in step (2) with the test sample, and repeat steps (1)-(4) to obtain the electrochemical response value of the test sample. Compare the electrochemical response value with the standard curve in step (5) to calculate the concentration of quinolone antibiotics in the test sample.

[0026] As a further improvement to the above scheme, step (1) includes a first electrode pretreatment step, which involves dripping a piranha solution onto the surface of a gold electrode and then rinsing it repeatedly with water.

[0027] As a further improvement to the above scheme, in step (1), the DNA probe modification method is to immerse the pretreated first electrode in the DNA probe solution and let it stand for a period of time to obtain the second electrode.

[0028] As a further improvement to the above scheme, in step (2), Cu 2+ It is derived from one or more of copper sulfate, copper chloride, and copper nitrate.

[0029] As a further improvement to the above scheme, in step (2), the second electrode is immersed in a solution containing K-Cu. 2+ Complexes and Cu 2 + The third electrode is obtained by incubating the DNAzyme solution for a period of time.

[0030] As a further improvement to the above scheme, in step (2), Cu 2+ Cu in the source 2+ The concentration is 0.2–0.4 mM. Preferably, Cu 2+ The concentration is 0.1–0.5 mM; more preferably, Cu 2+ The concentration was 0.3 mM;

[0031] As a further improvement to the above scheme, in step (3), the third electrode is incubated in a mixed solution containing H1 and H2 for at least 3 hours; preferably, the incubation time is 0.5-4 hours; more preferably, the incubation time is 1.5-3 hours.

[0032] As a further improvement to the above scheme, in step (4), the fourth electrode is immersed in a solution containing methylene blue and incubated for a period of time to introduce signal molecules onto the surface of the fourth electrode, wherein the incubation time is at least 30 min. Preferably, the incubation time is 20 min to 40 min; more preferably, the incubation time is 30 min to 40 min.

[0033] This invention relates to the reaction of quinolone antibiotics with Cu in the presence of the target quinolone antibiotic. 2+ Specific binding to form K-Cu 2+ Complexes, making Cu 2+The activity of the DNAzyme is inhibited, preventing it from catalytically cleaving the substrate DNA probe (S-DNA) modified on the electrode surface. The intact S-DNA then triggers an HCR reaction between nucleic acid probes H1 and H2, amplifying double-stranded DNA nanowires on the electrode surface. This ultimately introduces the electrochemically active substance MB, generating a significant electrochemical signal amplification correlated with the concentration of the target quinolone antibiotic. Conversely, in the absence of quinolone antibiotics, Cu... 2+ This binds to and stimulates DNAzyme activity to catalyze the cleavage of S-DNA, resulting in the inability to trigger HCR to generate an electrochemical signal. This is based on Cu. 2+ Electrochemical biosensors that regulate signal amplification can achieve convenient, rapid, highly sensitive, and highly specific detection of CIP.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The electrochemical biosensor of the present invention uses quinolone antibiotics and Cu 2+ The complexation reaction serves as the input signal, and HCR is used as the amplified output signal to achieve highly sensitive detection of quinolones. Furthermore, the label-free biosensor of this invention is convenient, low-cost, easy to operate, and does not involve complex instruments or expensive nanomaterials. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the electrochemical biosensor of Embodiment 1 of the present invention;

[0036] Figure 2 Figure 1 shows the feasibility verification results of the electrochemical biosensor of Example 1 of this invention; (A) EIS characterization of electrode surface state, (a) bare gold electrode, (b) S-DNA modified gold electrode, (c) with Cu 2+ (a) Electrode after incubation with a mixed solution of CIP and DNAzyme; (b) Electrode after HCR reaction; (c) CV verification of CIP response system, (a) in the absence of CIP; (d) in the presence of CIP but without HCR; (e) in the presence of CIP and with HCR.

[0037] Figure 3 This is a graph showing the quantitative analysis results of CIP using an electrochemical biosensor in Example 1 of the present invention; (A) Differential pulse voltammograms of different concentrations of the target analyte CIP, a~g (ng mL) -1 ):0.1,0.5,2,10,50,200,400. (B) Relationship between peak current and CIP concentration. The inset plot shows the linear relationship between the peak current and the logarithm of the CIP concentration; the error bar represents the standard deviation of three parallel experiments.

[0038] Figure 4This is a graph showing the optimization results of the detection conditions in Example 2 of the present invention. (A) I / I0 with Cu 2+ Concentration variation. I / I0 represents the ratio of peak current in the presence and absence of the target analyte CIP. (B) DPV variation with HCR reaction time. (C) Peak current variation with MB incubation time. The error bar represents the standard deviation of three parallel experiments.

[0039] Figure 5 The figure shows the specificity verification results of the electrochemical biosensor in Example 3 of this invention; blank control: 50 mM HEPES (pH 7.0); KANA: kanamycin, TET: tetracycline, AMP: ampicillin, CM: chloramphenicol, mixture: a mixed solution of KANA, TET, AMP, CM and CIP; the error bar is the standard deviation of three parallel experiments. Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0041] To verify the practicality of the electrochemical biosensor for quinolone antibiotics of the present invention, ciprofloxacin (CIP) was selected as the detection target. CIP is a typical quinolone antibiotic that has been widely used to treat foodborne infections in animals. However, the overuse of CIP can result in its residues in animal-derived foods, which can then enter the human body through the food chain, posing a threat to human health and safety. The electrochemical biosensor for quinolone antibiotics of the present invention not only achieves high sensitivity and high specificity for CIP detection but also has advantages such as low cost and simple operation. Furthermore, the method of the present invention has been preliminarily applied to the detection of actual food samples, demonstrating good detection performance and potential practicality.

[0042] Materials and reagents involved in the embodiments of this invention:

[0043] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and mercaptohexanol (MCH) were purchased from Bailingwei Technology Co., Ltd. (Beijing, China). CIP was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Cu(NO3)2 and methylene blue (MB) were ordered from Aladdin Biotechnology Co., Ltd. (Shanghai, China) and TaKaRa Biotechnology Co., Ltd. (Dalian, China), respectively. All DNA strands were synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China). The DNA sequences are shown below:

[0044] S-DNA:5'-AGCTTCTTTCTAATACGGCTTACC-SH-3'

[0045] Cu 2+ -DNAzyme:

[0046] 5'-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3'

[0047] H1:5'-GGTAAGCCGTATTAGAAAGAAGCTAATATCTCTAATACGG-3'

[0048] H2:5'-AGCTTCTTTCTAATACGGCTTACCCCGTATTAGAGATATT-3'

[0049] The milk sample processing procedure involved in this invention is as follows: The milk sample needs to be pretreated to remove protein and fat. First, 5 mL of milk is centrifuged at 3500 g (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 are added, and the mixture is centrifuged at 3500 g (15℃) for 10 min, and the supernatant is collected. Finally, the supernatant is diluted for later use.

[0050] All electrochemical experiments in this invention were performed at room temperature using an electrochemical workstation (CHI660D) with a three-electrode system (gold electrode as the working electrode, saturated calomel electrode as the reference electrode, and platinum electrode as the counter electrode). Electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), and cyclic voltammetry (CV) were used to characterize changes in electrochemical parameters. CV and DPV were performed in 50 mM HEPES buffer containing 500 mM NaCl, and EIS was performed in 5 mM [Fe(CN)6] buffer containing 1 M KNO3. 3- / 4- Measurements were taken in solution. The EIS frequency range was 0.1 Hz to 10 kHz, the bias potential was 0.224 V, and the amplitude was 5 mV. DPV was performed in the range of -0.40 V to -0.1 V, with an amplitude of 50 mV and a pulse width of 50 ms. CV was recorded from -0.5 V to 0 V at a scan rate of 100 m / s. The experiment was performed in triplicate, and the error bars shown in the graphs represent the standard deviation.

[0051] Example 1

[0052] This invention provides an electrochemical biosensor for quinolone antibiotics. The electrochemical biosensor includes an electrode, long double-stranded DNA nanowires coated on the electrode, and a signal molecule. The long double-stranded DNA nanowires are obtained by amplification of H1 and H2 on the electrode surface via an HCR reaction. The HCR reaction environment contains CIP-Cu. 2+ Complex, Cu 2+ -DNAzyme, DNA probe.

[0053] like Figure 1As shown, in the presence of the target CIP, CIP reacts with Cu 2+ Specific binding to form CIP-Cu 2+ Complexes, making Cu 2+ The activity of the DNAzyme is inhibited, preventing it from catalytically cleaving the substrate DNA probe (S-DNA) modified on the electrode surface. Subsequently, the intact S-DNA triggers the HCR reaction of nucleic acid probes H1 and H2, amplifying double-stranded DNA nanowires on the electrode surface. This ultimately introduces the electrochemically active substance MB, generating a significant electrochemical signal amplification correlated with the target CIP concentration. Conversely, in the absence of CIP, Cu... 2+ This binds to and stimulates DNAzyme activity to catalyze the cleavage of S-DNA, resulting in the inability to trigger HCR to generate an electrochemical signal. This is based on Cu. 2+ Electrochemical biosensors that regulate signal amplification can achieve convenient, rapid, highly sensitive, and highly specific detection of CIP.

[0054] EIS is widely used to characterize the surface state of working electrodes. To verify... Figure 1 The experimental principle of the electrochemical biosensor shown is illustrated by using EIS to characterize the surface state of the electrode after step-by-step treatment. For example... Figure 2 As shown in Figure A, the EIS plot of the bare electrode is a straight line (curve a), while the curve after S-DNA modification is a semi-circle (curve b), indicating that the formation of a self-assembled monolayer on the electrode surface increases the surface electron transport resistance. The modified electrode contains Cu... 2+ After incubation in a mixed solution of target CIP and DNAzyme, the diameter of the semicircle slightly increased (curve c). Subsequently, S-DNA triggered HCR, amplifying the electrochemical signal and causing a significant increase in the semicircle diameter (curve d). Furthermore, to verify the feasibility of the CIP response system, CV was used to record the electrochemical response values ​​under different conditions. Figure 2 As shown in B. In the absence of CIP, the peak current of CV is very small (curve a) because the DNAzyme is absorbed by Cu. 2+ The electrochemical response resulting from the activation and catalysis of S-DNA cleavage is negligible. In the presence of CIP, Cu... 2+ Coordination with the C3-carboxyl and C4-keto groups of CIP prevents the DNAzyme from being activated, thus preserving the substrate chain modified on the working electrode surface, resulting in a slight increase in the CV current peak (curve b). Further introduction of HCR produces a current peak much larger than that without HCR (curve c), indicating that HCR plays a significant role in signal amplification and improving detection performance.

[0055] The following section details the preparation method of the aforementioned electrochemical biosensor for quinolone antibiotics, as well as the method for electrochemical detection of quinolone antibiotic concentrations using it.

[0056] This invention provides an electrochemical detection method for quinolone antibiotics, comprising preparing an electrochemical biosensor for quinolone antibiotics and performing electrochemical detection on the prepared electrochemical biosensor for quinolone antibiotics. Specifically, the electrochemical detection method for quinolone antibiotics in this embodiment includes the following steps:

[0057] (1) DNA probes are modified onto the surface of a gold electrode to obtain an electrode modified with DNA probes:

[0058] Specifically, the gold electrode underwent pretreatment, following procedures reported in the literature. First, to remove organic matter, a piranha solution (concentrated sulfuric acid: hydrogen peroxide = 3:1) was dropped onto the gold electrode surface for 5 min, followed by repeated rinsing with ultrapure water. Subsequently, the electrode was polished in alumina slurries of different particle sizes (1 μm and 0.3 μm), thoroughly rinsed with ultrapure water, and then ultrasonically immersed in ethanol and ultrapure water for 10 min each to ensure complete removal of residual alumina slurry. Finally, the electrode surface was electrochemically cleaned in 0.5 M H₂SO₄.

[0059] The pretreated gold electrode was immersed in a 1 μM S-DNA solution (containing 1 mM TCEP to prevent disulfide bond formation between S-DNA molecules) and allowed to stand at 25 °C for 1 h. Then, the electrode was immersed in a 0.1 mM MCH solution and allowed to stand at 25 °C for 20 min to prevent nonspecific adsorption.

[0060] (2) CIP-Cu 2+ Complex formation, Cu 2+ -DNAzyme cleaves DNA probes on the electrode surface:

[0061] Specifically, 0.3 mM Cu(NO3)2 was mixed with CIP solution and incubated at 25 °C for 5 min. Then, 200 nM DNAzyme was added, and the modified gold electrode was immersed in the above mixed solution and incubated at room temperature for 30 min.

[0062] (3) Formation of double-stranded DNA nanowires:

[0063] Specifically, 500 nM H1 and 500 nM H2 were heated at 95 °C for 5 min and then cooled to room temperature for 1 h. The gold electrode obtained in step (2) was then immersed in a mixed solution containing H1 and H2 and incubated at 37 °C for 3 h to form double-stranded DNA nanowires.

[0064] (4) Finally, the gold electrode obtained in step (3) is immersed in 0.5 mM MB solution and incubated for a period of time to introduce signal molecules onto the surface of the gold electrode, wherein the incubation time is at least 30 min; an electrochemical biosensor for quinolone antibiotics is obtained, and electrochemical detection is performed using the electrochemical biosensor and the electrochemical response value is recorded.

[0065] (5) Change the concentration of CIP solution in step (2) and repeat steps (1)-(4) to obtain the standard curve of quinolone antibiotic solution and corresponding electrochemical response value; the results of quantitative analysis of CIP using electrochemical biosensors are as follows: Figure 3 As shown. Figure 3 As shown, the peak current gradually increases with increasing CIP concentration, and is highest at 0.1 ng / mL. -1 ~200ng / mL -1 The peak current within the range shows a linear relationship with the logarithm of the CIP concentration (y = 1.575x + 2.049, R). 2 =0.997). Based on the S:N = 3:1 criterion, the limit of detection (LOD) was calculated to be 0.052 ng / mL. -1 The above results indicate that CIP has a low detection limit and a wide linear range.

[0066] (6) Replace the CIP solution in step (2) with the milk sample and repeat steps (1)-(4) to obtain the electrochemical response value of the milk sample. Compare the electrochemical response value with the standard curve in step (5) to calculate the CIP concentration in the milk sample.

[0067] CIP, a potent antibacterial antibiotic, is widely used in the prevention and treatment of animal diseases. However, excessive or inappropriate use can lead to antibiotic residues in animal products. Besides its toxic side effects on humans, a more serious concern is that long-term consumption of animal products containing low concentrations of CIP can easily induce drug resistance, thus affecting the clinical efficacy of these drugs. Therefore, detecting CIP antibiotic residues in food is urgently needed. To verify the practicality of this method, the method provided in this embodiment was used to analyze CIP in milk samples. Different concentrations of standard CIP were added to the milk samples, and the recovery rate was determined using the constructed biosensor. As shown in Table 1, within the allowable relative error range, the recovery rate was between 96.3% and 115%, demonstrating the potential practicality of the electrochemical biosensor of this invention in complex food samples.

[0068] Table 1. Detection results of CIP in milk samples by the electrochemical biosensor in Example 1

[0069]

[0070]

[0071] Three independent parallel experiments were conducted for each concentration.

[0072] Comparative Example

[0073] 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 a previously reported CIP biosensor. The reported CIP biosensors are Comparative Examples 1-5. The electrochemical sensor in Comparative Example 1 was prepared using the method described in "Jalal NR, Madrakian T, Afkhami A, et al. Polyethylenimine@Fe3O4@carbon nanotubes nanocomposite as a modifier inglassy carbon electrode for sensitive determination of ciprofloxacin inbiological samples, Journal of Electroanalytical Chemistry, 2019, 833: 281-289."; the electrochemical aptamer sensor in Comparative Example 2 was prepared using the method described in "Abnous K, Danesh NM, Alibolandi M, et al. Anovel electrochemical aptamer sensor for ultrasensitive detection of fluoroquinolones based on single-stranded DNA-binding protein[J]. Sensors and Actuators B: Chemical, 2017, 240: 100-106."; and the photoelectrochemical sensor in Comparative Example 3 was prepared using the method described in "Yan PC, Xu...". L, Jiang DS, et al. Photoelectrochemical monitoring of ciprofloxacin based on metallic Bi self-doping BiOBr nanocomposites[J]. Electrochimica Acta, 2018, 259: 873-881.” The fluorescent sensor of Comparative Example 4 was prepared using the method described in “Fu B, Zheng XY, Li HH, et al. A highly stable, rapid and sensitive fluorescent probe for ciprofloxacin based on Al 3+The electrochemiluminescence sensor of Comparative Example 5 was prepared using the method described in “Wang YW, Sun XD, Cai L, et al. A “signalon / off” biomimetic electrochemiluminescence sensor using titanium carbidenanodots as co-reaction accelerator for ultra-sensitive detection of ciprofloxacin[J]. Analytica Chimica Acta, 2022, 1206: 339690.”

[0074] Table 2. Comparison of detection performance between the electrochemical sensor of the present invention and reported CIP biosensors.

[0075]

[0076]

[0077] The electrochemical biosensor established in Example 1 was compared with reported CIP 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.

[0078] Example 2: Optimization of Detection Conditions

[0079] To achieve optimal detection performance, this embodiment uses DPV to optimize key conditions in the electrochemical detection method for quinolone antibiotics. Key conditions include Cu 2+ Concentration, HCR reaction time, and MB incubation time. In this example, the CIP solution concentration was 200 ng / mL. -1 .

[0080] Cu 2+ The optimization method for concentration conditions is as follows: Various CIP electrochemical biosensors were prepared using the method described in Example 1, and their electrochemical response values ​​were measured using DPV. The difference from Example 1 is that in step (2), Cu... 2+ The concentrations were 0.1 mM, 0.15 mM, 0.2 mM, 0.3 mM, 0.4 mM and 0.5 mM, respectively.

[0081] Due to Cu 2+ The concentration is closely related to the background signal and the output value of the CIP concentration response. Therefore, at different Cu... 2+ At certain concentrations, the ratio of peak currents in CIP with (I) and without (I0) target analytes was recorded. For example... Figure 4 As shown in A, with Cu 2+ With increasing concentration, the I / I0 value peaks at 0.3 mM; therefore, 0.3 mM is chosen as the optimal concentration for Cu. 2+ concentration.

[0082] To ensure sufficient complementary hybridization between nucleic acid probes H1 and H2, the effect of HCR reaction time on the electrochemical response value was investigated. The optimization method for HCR reaction time is as follows: Various CIP electrochemical biosensors were prepared using the method described in Example 1, and their electrochemical response values ​​were measured using DPV. The difference from Example 1 is that in step (3), each gold electrode was immersed in a mixed solution containing H1 and H2 and incubated at 37°C for 0.5 h, 1 h, 2 h, 3 h, and 4 h, respectively. Figure 4 As shown in Figure B, the peak current continuously increases with the reaction time increasing from 0.5 h to 4 h, and reaches equilibrium at 3 h. Therefore, 3 h is selected as the optimal reaction time for HCR.

[0083] The method for optimizing MB incubation time is as follows: various CIP electrochemical biosensors were prepared using the method described in Example 1, and their electrochemical response values ​​were measured by DPV respectively; the difference from Example 1 is that in step (4), the MB incubation time is 5 min, 10 min, 20 min, 30 min, and 40 min respectively.

[0084] Finally, the effect of MB incubation time on signal output was investigated. Figure 4 As shown in Figure C, the electrochemical signal increases with the increase of MB incubation time, and almost reaches equilibrium at a concentration of 30 min. Therefore, 30 min was selected as the optimal incubation time for MB.

[0085] Example 3: Specificity Verification of Electrochemical Biosensors

[0086] Under the optimal detection conditions obtained in Example 2, different concentrations of CIP were quantitatively detected. The specific steps are as follows:

[0087] The pretreated gold electrode was immersed in a 1 μM S-DNA solution (containing 1 mM TCEP to prevent disulfide bond formation between S-DNA molecules) and allowed to stand at 25 °C for 1 h. Then, the electrode was immersed in a 0.1 mM MCH solution and allowed to stand at 25 °C for 20 min to prevent nonspecific adsorption.

[0088] 0.3 mM Cu(NO3)2 was mixed with CIP solutions of different concentrations and incubated at 25 °C for 5 min. Then, 200 nM DNAzyme was added, and the modified gold electrode was immersed in the mixture and incubated at room temperature for 30 min. 500 nM H1 and 500 nM H2 were heated at 95 °C for 5 min, followed by cooling at room temperature for 1 h. The gold electrode was then immersed in a mixture containing H1 and H2 and incubated at 37 °C for 3 h to form double-stranded DNA nanowires. Finally, 0.5 mM MB was introduced as a signaling molecule onto the gold electrode surface, and the electrochemical response was recorded, with MB incubation time being 30 min.

[0089] In addition, to verify the specificity of the proposed electrochemical biosensor, a control experiment was conducted to compare the electrochemical responses of the biosensor to different types of antibiotics under the same detection conditions. The different antibiotics included KANA (kanamycin), TET (tetracycline), AMP (ampicillin), CM (chloramphenicol), and a mixture of KANA, TET, AMP, CM, and CIP. All antibiotics were at a concentration of 200 ng / mL. -1 Blank control: 50 mM HEPES (pH 7.0).

[0090] like Figure 5 As shown, the peak current generated in the presence of CIP is significantly higher than that generated in the presence of other antibiotics, and the peak current is less affected when other interfering antibiotics are present. These results fully demonstrate the high specificity of the electrochemical biosensor of this invention.

[0091] In summary, this study established a Cu-based... 2+ A quinolone antibiotic residue electrochemical biosensor that modulates HCR signal amplification. Based on Cu 2+ Interactions with quinolone antibiotics and DNA zymes; designing quinolone antibiotics and Cu 2+ The complexation reaction is used as the input signal, and HCR is used as the amplified output signal, thereby achieving high sensitivity and high specificity detection of quinolone antibiotics. Secondly, the label-free biosensor of this invention is low in cost and simple to operate, requiring no expensive nanomaterials or complex instruments. Furthermore, the electrochemical biosensor of this invention has been successfully applied to the detection of CIP in actual food samples and has demonstrated good analytical performance, indicating its potential practicality in food safety testing.

Claims

1. An electrochemical biosensor for detecting ciprofloxacin, characterized in that, The biosensor includes an electrode, long double-stranded DNA nanowires coated on the electrode, and a signal molecule; wherein the signal molecule is methylene blue, and the long double-stranded DNA nanowires are obtained by amplifying H1 and H2 chains on the electrode surface through an HCR reaction. The H1 chain sequence is as follows: 5'-GGTAAGCCGTATTAGAAAGAAGCTAATATCTCTAATACGG-3'; The H2 chain sequence is as follows: 5'-AGCTTCTTTCTAATACGGCTTACCCCGTATTAGAGATATT-3'; CIP-Cu is present in the environment of the HCR reaction. 2+ Complex, Cu 2+ - DNAzyme, DNA probe; wherein, CIP is ciprofloxacin; the DNAzyme strand sequence is: 5'-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3'; The DNA probe strand sequence is: 5'-AGCTTCTTTCTAATACGGCTTACC-SH-3'.

2. A method for electrochemical detection of ciprofloxacin using the electrochemical biosensor according to claim 1, characterized in that, Includes the following steps: (1) First, the first electrode is pretreated, and then the DNA probe is modified on the surface of the first electrode to obtain the second electrode; the first electrode is a gold electrode; (2) Cu 2+ The source was mixed with ciprofloxacin solution and incubated, then DNAzyme solution was added to obtain a mixed solution. The second electrode was immersed in the above mixed solution, and the Cu in the mixed solution... 2+ - DNAzyme cleaves the DNA probe on the surface of the second electrode to obtain the third electrode; Cu 2+ Cu in the source 2+ Concentration of 0.2~0.4 mM (3) The third electrode is immersed in a mixed solution containing H1 and H2 chains and incubated for a period of time. Double-stranded DNA nanowires are formed on the surface of the third electrode, thus obtaining the fourth electrode. (4) Signal molecules are introduced onto the surface of the fourth electrode to obtain an electrochemical biosensor, which is used for electrochemical detection and recording of electrochemical response values. (5) Change the concentration of ciprofloxacin solution in step (2) and repeat steps (1)-(4) to obtain the standard curve of ciprofloxacin solution and corresponding electrochemical response value; (6) Replace the ciprofloxacin in step (2) with the sample to be tested, and repeat steps (1)-(4) to obtain the electrochemical response value of the sample to be tested. Compare the electrochemical response value with the standard curve in step (5) to calculate the concentration of ciprofloxacin in the sample to be tested.

3. The electrochemical detection method according to claim 2, characterized in that, In step (1), the first electrode pretreatment step is to drop the piranha solution onto the surface of the gold electrode and then rinse the first electrode repeatedly with water.

4. The electrochemical detection method according to claim 3, characterized in that, In step (1), the DNA probe modification method is to immerse the pretreated first electrode in the DNA probe solution and let it stand for a period of time to obtain the second electrode.

5. The electrochemical detection method according to claim 2, characterized in that, In step (2), Cu 2+ The source is one or more of copper sulfate, copper chloride, and copper nitrate.

6. The electrochemical detection method according to claim 2, characterized in that, In step (3), the third electrode is incubated in a mixed solution containing H1 and H2 chains for at least 3 hours.

7. The electrochemical detection method according to claim 2, characterized in that, In step (4), the fourth electrode is immersed in a solution containing methylene blue and incubated for a period of time to introduce signal molecules onto the surface of the fourth electrode, wherein the incubation time is at least 30 minutes.

Images