Electrochemical biosensor based on Y-shaped DNA scaffold and preparation method and application thereof

Abstract

The invention provides an electrochemical biosensor based on a Y-type DNA scaffold as well as a preparation method and application of the electrochemical biosensor. The electrochemical biosensor comprises a Y-type probe, a divalent split nucleic acid aptamer b and a gold electrode. The Y-type probe comprises a DNA connecting chain and a DNA probe chain. The DNA connecting chain and the DNA probe chain are complementarily paired through bases, the extension parts of the DNA connecting chain and the DNA probe chain are split nucleic acid aptamers a of a to-be-detected object, and the split nucleic acid aptamers a and the split nucleic acid aptamers b specifically recognize the to-be-detected object together; one end of the DNA probe chain is modified on the gold electrode, and the other end is modified with the electrochemical signal molecule. The Y-type DNA bracket swings towards the electrode by applying positive potential, and a to-be-detected object can be detected in one step by recording signal molecules by adopting a time current method. According to the sensor, the influence of Debye length is avoided, the sample does not need to be pretreated, the complex modification process can be avoided, the design is simpler, and the sensitivity is higher.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical biosensor detection, and in particular relates to an electrochemical biosensor based on a Y-shaped DNA scaffold and a preparation method and application thereof. Background Art

[0002] The detection of related molecules (including small molecules, nucleic acids and proteins) is of great significance in various fields, especially in clinical medical diagnosis, environmental monitoring and food safety control. In recent years, electrochemical technology using conformational change for one-step detection has become a promising method for monitoring related biomolecules in non-laboratory environments due to its simple probe design, convenient operation and potential applicability in different fields. However, the practical application of most constructed electrochemical biosensors is hindered by the traditional Debye length, because the shielding effect of the double layer at the electrode-electrolyte interface hinders the electron transfer in the complex matrix. Therefore, in view of the limitations of the above sensors, it is necessary to develop a detection method that can circumvent the Debye length limitation to enhance the detection of biomolecular interactions. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art. One of the purposes of the present invention is to design an electrochemical biosensor with a Y-shaped DNA scaffold "pendulum". The second purpose of the present invention is to provide a method for preparing the electrochemical biosensor. The third purpose of the present invention is to provide an application of the electrochemical biosensor. The fourth purpose of the present invention is to provide a universal electrochemical system for detecting enrofloxacin and myoglobin. To this end, the present invention proposes a new strategy for Y-shaped molecular pendulum, introducing a split-type aptamer as a specific recognition element, thereby constructing an electrochemical biosensor based on a Y-shaped DNA scaffold "pendulum".

[0004] In order to solve the above technical problems, the present invention provides an electrochemical biosensor based on a Y-shaped DNA scaffold, wherein the electrochemical biosensor comprises a Y-shaped probe, a bivalent cleavage-type nucleic acid aptamer b, and a gold electrode;

[0005] The Y-shaped probe includes a DNA connecting chain and a DNA probe chain, and the DNA connecting chain and the DNA probe chain are connected by base complementary pairing; the gene sequence of the DNA connecting chain is: 5'-split nucleic acid aptamer a-DNA1-3', and the DNA sequence of the DNA1 is shown in SEQ ID NO.1;

[0006] The gene sequence of the DNA probe chain is: 5'-Probe 1-split nucleic acid aptamer a-3', and the DNA sequence of Probe 1 is shown in SEQ ID NO.2;

[0007] The gene sequence of the bivalent split-type nucleic acid aptamer b is 5'-TTTTTT-split-type nucleic acid aptamer b-TTTTTTTTT-split-type nucleic acid aptamer b-3';

[0008] The split-type nucleic acid aptamer a and the split-type nucleic acid aptamer b together specifically recognize the analyte;

[0009] One end of the DNA probe chain is modified on a gold electrode, and the other end is modified with an electrochemical signal molecule.

[0010] The electrochemical biosensor described above, further, the split-type nucleic acid aptamer a and the split-type nucleic acid aptamer b together specifically recognize enrofloxacin;

[0011] The gene sequence of the DNA connecting strand is shown in SEQ ID NO.3; the gene sequence of the DNA probe strand is shown in SEQ ID NO.4;

[0012] The gene sequence of the bivalent split-type nucleic acid aptamer b is shown in SEQ ID NO.5.

[0013] The electrochemical biosensor described above, further, the split-type nucleic acid aptamer a and the split-type nucleic acid aptamer b together specifically recognize myoglobin;

[0014] The gene sequence of the DNA connecting strand is shown in SEQ ID NO.6; the gene sequence of the DNA probe strand is shown in SEQ ID NO.7;

[0015] The gene sequence of the bivalent split-type nucleic acid aptamer b is shown in SEQ ID NO.8.

[0016] In the above electrochemical biosensor, further, one end of the DNA probe chain is connected to the gold electrode via the SH-C6 group; and the signal molecule modified at the other end is ferrocene.

[0017] Based on a general technical concept, the present invention provides a method for preparing the electrochemical biosensor, comprising the following steps:

[0018] S1, mixing and incubating the DNA connection strand and the DNA probe strand;

[0019] S2. dropping the mixture onto the surface of the gold electrode, incubating to form a Y-shaped probe, and blocking with a blocking agent to obtain an electrochemical biosensor.

[0020] In the above preparation method, further, the incubation temperature in S1 is 37° C., and the incubation time is 10 min to 30 min.

[0021] In the above preparation method, further, the incubation time in S2 is 3 to 12 hours; and the blocking agent is MCH.

[0022] Based on a general technical concept, the present invention provides an application of the electrochemical biosensor in detecting enrofloxacin.

[0023] In the above application, further, the method of the application includes:

[0024] (1) After mixing the bivalent split-type nucleic acid aptamer b and the analyte, incubating them on the probe surface of the electrochemical biosensor to form a Y-shaped DNA scaffold through bivalent recognition;

[0025] (2) After cleaning the electrodes with buffer, the three-electrode system was placed in a PBS solution for time-current scanning to obtain a detection signal.

[0026] Based on a general technical concept, the present invention provides an application of the electrochemical biosensor in detecting myoglobin.

[0027] In the above application, further, the method of the application includes:

[0028] (1) After mixing the bivalent split-type nucleic acid aptamer b and the analyte, incubating them on the probe surface of the electrochemical biosensor to form a Y-shaped DNA scaffold through bivalent recognition;

[0029] (2) After cleaning the electrodes with buffer, the three-electrode system was placed in a buffer solution for time current scanning, and the current value at 20 ms was recorded to obtain the detection signal.

[0030] In the above application, further, the buffer solution is PBS.

[0031] In the above application, further, the scanning voltage of the time current scanning is +0.5V, the scanning time is 30ms, and the scanning frequency is 1000Hz.

[0032] Compared with the prior art, the advantages of the present invention are:

[0033] The present invention provides an electrochemical biosensor based on a Y-shaped DNA scaffold, the probe design is simple and the target can be detected in one step, and the sample can be directly (or diluted) detected without pretreatment, avoiding the influence of the Debye length. In addition, compared with other electrochemical biosensors, the sensor has higher sensitivity, can avoid complex modification processes, and makes the design simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0035] Figure 1 Schematic diagram of the construction process of the electrochemical biosensor with a Y-shaped DNA support "pendulum" in Example 1 of the present invention.

[0036] Figure 2 This is the standard curve for enrofloxacin detection in Example 1 of the present invention.

[0037] Figure 3 This is a flow chart of the application of the electrochemical biosensor with the Y-shaped DNA support "pendulum" in Example 1 of the present invention to the detection of enrofloxacin.

[0038] Figure 4 This is a curve of the sensor of Example 1 of the present invention detecting the concentration of enrofloxacin in an actual sample.

[0039] Figure 5 This is the result of the effect of the length of the base pairs constituting the DNA scaffold on the performance of the sensor in Experiment 1 of the present invention.

[0040] Figure 6 This is the result of the effect of the length of the probe chain support on the sensor performance in Experiment 2 of the present invention.

[0041] Figure 7 This is the result of investigating the electron transfer of signal molecules by the components of the buffer solution in Experiment 3 of the present invention.

[0042] Figure 8 This is the standard curve for myoglobin detection in Example 2 of the present invention.

[0043] Fig. 9 This is a curve of the sensor of Example 2 of the present invention detecting the myoglobin concentration in an actual sample. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0045] The materials, reagents and instruments used in the following examples can all be purchased from commercial channels. The experimental methods in the following examples are all conventional methods in the art unless otherwise specified.

[0046] Example

[0047] An electrochemical biosensor based on a Y-shaped DNA scaffold "pendulum" comprises a Y-shaped DNA scaffold, a divalent cleavage-type nucleic acid aptamer b, and a gold electrode.

[0048] The Y-shaped DNA scaffold includes a DNA connecting chain (DNA1) and a DNA probe chain (Probe 1), the extension parts of the DNA connecting chain and the DNA probe chain are the split-type nucleic acid aptamer a of the object to be detected, and the DNA connecting chain 1 and the DNA probe chain are connected through base complementary pairing to form a Y-shaped probe.

[0049] The gene sequence of DNA1 is shown in SEQ ID NO.1, specifically: 5'-split nucleic acid aptamer a-TTTTCT TAT TAG ATA CAA TAG CTG GTA-3'.

[0050] The gene sequence of Probe 1 is shown in SEQ ID NO. 2, specifically: 5'-SH-C6-TAC CAG CTA TTGTAT CTA ATA AGATTT-split-type nucleic acid aptamer a-Fc-3'. Split-type nucleic acid aptamer a is a partial sequence of a nucleic acid aptamer that specifically recognizes the object to be detected, and two split-type nucleic acid aptamers a are two identical sequences.

[0051] SH-C6 is designed to be connected to the gold electrode surface via Au-S bonds, and Fc is a redox substance used for signal generation.

[0052] The gene sequence of the bivalent split-type aptamer b is specifically: 5'-TTTTTT-split-type aptamer b-TTTTTTTTT-split-type aptamer b-3'. The two split-type aptamers b are two identical sequences connected together by T9. The split-type aptamer b is another part of the sequence of the aptamer that specifically recognizes the object to be detected.

[0053] Only when the split-type nucleic acid aptamer a and the split-type nucleic acid aptamer b exist at the same time can they specifically identify the analyte.

[0054] Figure 1 This is a schematic diagram of the structure of the Y-shaped DNA scaffold. It can be seen from the figure that the yellow part of DNA1 is complementary to the blue part of Probe1, and the green part is the split-type aptamer a. The purple sequence is a bivalent split-type aptamer b, which is formed by connecting two split-type aptamers b with the same sequence through the black part, and can specifically recognize the target together with the split-type aptamer a.

[0055] When a target is present, the bivalent split-type aptamer b forms a Y-shaped DNA structure with the split-type aptamer a on the electrode surface. A positive voltage is applied to the electrode surface using the time current method, and the signal molecule ferrocene (Fc) undergoes redox reaction on the electrode surface to generate an electrical signal. We detect the concentration of the target based on the change in current when there is a target or not.

[0056] In the present invention, the role of Fc is to generate an electrochemical signal, but all signal molecules that can generate an electrochemical signal can achieve the same or similar technical effects. The split-type nucleic acid aptamers a and b can be adjusted according to the nucleotide sequence of the analyte. Therefore, the electrochemical biosensor of the present invention can detect various analytes, and Examples 1 and 2 are only examples of the present invention to demonstrate its detection effect.

[0057] Example 1

[0058] An electrochemical biosensor based on a Y-shaped DNA scaffold "pendulum" for detecting enrofloxacin comprises a Y-shaped DNA scaffold, a divalent cleavage-type nucleic acid aptamer b, and a gold electrode.

[0059] The Y-shaped DNA scaffold includes a DNA connecting chain (DNA1) and a DNA probe chain (Probe 1), the extension parts of the DNA connecting chain and the DNA probe chain are the split-type nucleic acid aptamer a of the object to be detected, and the DNA connecting chain 1 and the DNA probe chain are connected through base complementary pairing to form a Y-shaped probe.

[0060] The gene sequence of DNA1 is shown in SEQ ID NO.3, specifically: 5'- TCTCTGAGCCCG GGTTAT TTC AGG GGG A TTT TCT TAT TAG ATA CAATAG CTG GTA-3'.

[0061] The gene sequence of Probe 1 is shown in SEQ ID NO.4, specifically: 5'-SH-C6-TAC CAG CTA TTGTAT CTA ATA AGA TTT TCT CTG AGC CCG GGT TAT TTC AGG GGG A -Fc-3'.

[0062] The underlined part in the DNA1 and Probe 1 gene sequences is the split-type nucleic acid aptamer a of enrofloxacin.

[0063] SH-C6 is designed to be connected to the gold electrode surface via Au-S bonds, and Fc is a redox substance used for signal generation.

[0064] The gene sequence of the bivalent split-type nucleic acid aptamer b is shown in SEQ ID NO.5, specifically: 5'-TTTTTT CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GC T TTTTTTTTT CCC ATC AGG GGG CTA GGC TAACAC GGT TCG GC -3'.

[0065] A method for constructing the electrochemical biosensor of this embodiment comprises the following steps:

[0066] (1) Surface pretreatment of gold electrode.

[0067] A gold electrode with a diameter of 2 mm was polished with alumina powder, and the polished gold electrode was placed in ethanol and ultrapure water for ultrasonic treatment for 5 min respectively, and then immersed in freshly prepared piranha solution (H2SO4:30% H2O2=7:3) for 10 min, rinsed with distilled water and the electrode surface was blown dry with nitrogen.

[0068] (2) Modification of the Y-shaped probe on the gold electrode surface.

[0069] After incubating 1 μM DNA1, 1 μM Probe 1 and 9 μM MCH at 37°C for 10 min, the mixture was dropped onto the treated gold electrode surface and incubated overnight to form a Y-shaped probe with Fc. The electrode was then thoroughly washed with 1 mM PBS to prepare an electrochemical biosensor.

[0070] An application of the electrochemical biosensor of this embodiment in detecting enrofloxacin, wherein the application method is as follows:

[0071] (1) Draw a standard curve for enrofloxacin detection: Add 7 μL of a mixture of 1 μM DNA3 and different concentrations of enrofloxacin to the sensor interface. The final concentrations of enrofloxacin are 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL. Wait for 30 min at room temperature before performing electrochemical testing. The measurement method is the time current method, with a scanning voltage of +0.5 V, a scanning time of 30 ms, and a scanning frequency of 1000 Hz. Record the current value at 20 ms and analyze it. Figure 2 The calibration curve shown:

[0072] △I=-2.85×lgC-15.658.

[0073] In the formula, △I is the measured value of the test group minus the measured value of the blank group, C is the concentration of enrofloxacin, R 2 =0.989.

[0074] (2) 1 μM DNA3 and 100 pg / mL enrofloxacin were added to the probe surface and incubated for 30 min to form a Y-shaped DNA scaffold through divalent recognition. The electrode was then thoroughly cleaned with 1 mM PBS and the three-electrode system (gold electrode as working electrode, Pt as counter electrode, and Ag / AgCl as reference electrode) was placed in a 10 mM PBS (pH 7.4) solution for time current scanning with a scanning voltage of +0.5 V, a scanning time of 30 ms, and a scanning frequency of 1000 Hz.

[0075] Figure 3This is a flow chart of the application of the electrochemical biosensor of the Y-shaped DNA scaffold "pendulum" prepared in this embodiment to the detection of enrofloxacin.

[0076] Example 2

[0077] An application of the sensor of Example 1 in detecting actual samples. The specific application method is: respectively mix artificial urine, cosmetics, milk, PBS and different concentrations of enrofloxacin, take 7 μL of the mixture and drop it onto the sensing interface, wait for 30 minutes at room temperature and then perform electrochemical testing.

[0078] The measurement method is the time current method, the scanning voltage is +0.5V, the scanning time is 30ms, and the scanning frequency is 1000Hz. The current value at 20ms is recorded and analyzed. Figure 4 The curve shown is: △I=-3.345×lgC-16.006, △I is the measured value of the test group minus the measured value of the blank group, and C is the concentration value of enrofloxacin.

[0079] Figure 4 The results of the sensor in Example 1 in detecting actual samples are shown in the figure. From the figure, we can see that the slope is not much different from the slope detected in PBS. According to the above steps, the sensor also has good sensitivity in milk and cosmetics, indicating that the performance of the sensor is good and can be directly measured in complex samples diluted to a certain multiple.

[0080] Experiment 1: Investigate the effect of different lengths of DNA scaffolds on the detection of enrofloxacin in the biosensor interface:

[0081] 14, 24 and 34 pairs of complementary bases were selected to construct the Y-shaped DNA scaffold to detect a series of enrofloxacin concentrations.

[0082] The DNA sequence of DNA1-14 is: TCT CTG AGC CCG GGT TAT TTC AGG GGG ATT T TA CAA TAG CTG GTA .

[0083] The DNA sequence of Probe 1-14 is: SH-C6- TAC CAG CTA TTG TA TTT TCT CTG AGC CCGGGT TAT TTC AGG GGG A-Fc.

[0084] The DNA sequence of DNA1-24 is: TCT CTG AGC CCG GGT TAT TTC AGG GGG ATT T TC TTA TTA GAT ACA ATA GCT GGT A .

[0085] The DNA sequence of Probe 1-24 is: SH-C6- TAC CAG CTA TTG TAT CTA ATA AGA TTT TCTCTG AGC CCG GGT TAT TTC AGG GGG A-Fc.

[0086] The DNA sequence of DNA1-34 is: TCT CTG AGC CCG GGT TAT TTC AGG GGG ATT T CT ATA TGT GAT CTT ATT AGA TAC AAT AGC TGG TA .

[0087] The DNA sequence of Probe 1-34 is: SH-C6- TAC CAG CTA TTG TAT CTA ATA AGA TCA CAT ATA GTT TTC TCT GAG CCC GGG TTA TTT CAG GGG GA-Fc.

[0088] The underlined parts are complementary bases.

[0089] Results Figure 5 In the figure, A indicates the change of the current signal of DNA scaffolds with different complementary lengths as the concentration of enrofloxacin increases, and B indicates the conversion of the current signal into signal gain based on A. It can be seen from the figure that the length of the DNA scaffold will affect the degree of downward swing after applying a positive potential. We found that the Y-shaped DNA scaffold composed of 24 pairs of complementary bases has a greater signal change when detecting different concentrations of enrofloxacin, and the signal gain is also more obvious. This shows that the 24 pairs do not hinder the electron transfer of adjacent probes and can approach the electrode under the induction of a positive potential.

[0090] Experiment 2: Optimize and investigate the probe bracket length.

[0091] To verify that a stable Y-shaped DNA scaffold is more conducive to movement under positive potential, we extended the bottom of Probe1 modified on the gold electrode surface by 3 or 6 T bases (the bold part is the extended T base) and investigated the effect of assembling it into a Y-shaped DNA scaffold on the performance of the biosensor.

[0092] The DNA sequence of Probe1-3T is: SH-C6-TTT TAC CAG CTA TTG TAT CTA ATA AGA TTTTCT CTG AGC CCG GGT TAT TTC AGG GGG A-Fc.

[0093] The DNA sequence of Probe1-6T is: SH-C6-TTT TTT TAC CAG CTA TTG TAT CTA ATA AGATTT TCT CTG AGC CCG GGT TAT TTC AGG GGG A-Fc.

[0094] Results Figure 6 In the figure, A indicates the change of the current signal when the length of Probe1 is extended as the concentration of enrofloxacin increases, and B indicates the conversion of the current signal into signal gain based on A. It can be seen from the figure that extending the length of Probe1 does not improve the change of the electrical signal, indicating that the stable Y-shaped DNA structure will approach the electrode under the drive of the potential to transfer electrons, and the extended structure will reduce the overall stability. The induced downward swing of the Y-shaped DNA scaffold under the positive potential is limited, thus causing lower current changes and signal gains within a certain range.

[0095] Experiment 3: Investigate the effect of different ionic strengths on ferrocene electron transfer.

[0096] 0.1M NaCl, 0.5M NaCl, and 1M NaCl were added to the PBS solution to investigate the effect of different ionic strengths on the electron transfer of ferrocene.

[0097] Results Figure 7 : At high ionic strength, the signal gain gradually increases with increasing target concentration. We found that the best sensitivity was obtained at 1M NaCl.

[0098] Example 2

[0099] An electrochemical biosensor based on a Y-shaped DNA scaffold "pendulum" for detecting myoglobin comprises a Y-shaped DNA scaffold, a divalent cleavage-type nucleic acid aptamer b, and a gold electrode.

[0100] The Y-shaped DNA scaffold includes a DNA connecting chain 1 (DNA1) and a DNA probe chain (Probe 1), the extension parts of the DNA connecting chain and the DNA probe chain are the split-type nucleic acid aptamer a of the object to be detected, and the DNA connecting chain 1 and the DNA probe chain are connected through base complementary pairing to form a Y-shaped probe;

[0101] The gene sequence of DNA1 is shown in SEQ ID NO.6, specifically: 5'- TCTAG ATGCA GCTTC CTTTC CTCCC TTT TCT TAT TAG ATA CAA TAG CTG GTA-3'.

[0102] The gene sequence of Probe 1 is shown in SEQ ID NO.7, specifically: 5'-SH-C6-TAC CAG CTA TTGTAT CTA ATAAGA TTT CCC TCC TTT CCT TCG ACG TAG ATC T -Fc-3'.

[0103] The underlined part in the DNA1 and Probe 1 gene sequences is the myoglobin cleavage-type nucleic acid aptamer a.

[0104] The gene sequence of the bivalent cleavage-type nucleic acid aptamer b is shown in SEQ ID NO.8, specifically: 5'-TTTTTT GCTGCG TTG TTC CGATTTTTTTTTT AGC CTT GTT GCG TCG-3'.

[0105] Its construction method is consistent with that of Example 1.

[0106] An application of the electrochemical biosensor of this embodiment in detecting myoglobin, wherein the application method is as follows:

[0107] (1) Establishment of myoglobin standard curve: Add 7 μL of a mixture of 1 μM DNA3 and different concentrations of myoglobin to the sensor interface. The final concentration of myoglobin is 0.1, 1, 10, 100, 1000 and 10000 pM. Wait for 50 min at room temperature before conducting electrochemical testing. The measurement method is the time current method, with a scanning voltage of +0.5 V, a scanning time of 30 ms, and a scanning frequency of 1000 Hz. Record the current value at 20 ms and analyze it. Figure 8 The calibration curve shown:

[0108] △I=-5.963×lgC-16.045.

[0109] In the formula, △I is the measured value of the test group minus the measured value of the blank group, C is the concentration of enrofloxacin, R 2 =0.996.

[0110] (2) Artificial urine, 10% serum and different concentrations of myoglobin were mixed respectively, and the mixture was added to the working electrode for incubation. After waiting for 30 minutes at room temperature, the electrochemical test was performed. The measurement method was the time current method, with a scanning voltage of +0.5V, a scanning time of 30ms, and a scanning frequency of 1000Hz. The current value at 20ms was recorded and analyzed.

[0111] like Fig. 9 As shown, we found that the sensor has good application ability in mixed 50% artificial urine and 5% serum.

[0112] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. An electrochemical biosensor based on a Y-shaped DNA scaffold, characterized in that: The electrochemical biosensor comprises a Y-shaped probe, a divalent cleavage-type nucleic acid aptamer b, and a gold electrode; The Y-shaped probe includes a DNA connecting chain and a DNA probe chain, and the DNA connecting chain and the DNA probe chain are paired by base complementation; the gene sequence of the DNA connecting chain is: 5'-split nucleic acid aptamer a-DNA1-3', and the DNA sequence of the DNA1 is shown in SEQ ID NO.1; The gene sequence of the DNA probe chain is: 5'-Probe 1-split nucleic acid aptamer a-3', and the DNA sequence of Probe 1 is shown in SEQ ID NO.2; The gene sequence of the bivalent split-type nucleic acid aptamer b is 5'-TTTTTT-split-type nucleic acid aptamer b-TTTTTTTTT-split-type nucleic acid aptamer b-3'; The split-type nucleic acid aptamer a and the split-type nucleic acid aptamer b together specifically recognize the analyte; One end of the DNA probe chain is modified on a gold electrode, and the other end is modified with an electrochemical signal molecule.

2. The electrochemical biosensor according to claim 1, characterized in that: The split-type nucleic acid aptamer a and the split-type nucleic acid aptamer b together specifically recognize enrofloxacin; The gene sequence of the DNA connecting strand is shown in SEQ ID NO.3; the gene sequence of the DNA probe strand is shown in SEQ ID NO.4; The gene sequence of the bivalent split-type nucleic acid aptamer b is shown in SEQ ID NO.

5.

3. The electrochemical biosensor according to claim 1, characterized in that: The split-type nucleic acid aptamer a and the split-type nucleic acid aptamer b together specifically recognize myoglobin; The gene sequence of the DNA connecting strand is shown in SEQ ID NO.6; The gene sequence of the DNA probe chain is shown in SEQ ID NO.7; The gene sequence of the bivalent split-type nucleic acid aptamer b is shown in SEQ ID NO.

8.

4. The electrochemical biosensor according to any one of claims 1 to 3, characterized in that: One end of the DNA probe chain is connected to the gold electrode via the SH-C6 group; the signal molecule modified at the other end is ferrocene.

5. A method for preparing an electrochemical biosensor according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: S1, mixing and incubating the DNA connection strand and the DNA probe strand; S2, dripping on the surface of the gold electrode, incubating to form a Y-shaped probe, and blocking with a blocking agent to obtain an electrochemical biosensor.

6. The preparation method according to claim 5, characterized in that: The incubation temperature in S1 is 37° C., and the incubation time is 10 min to 30 min; the incubation time in S2 is 3 to 12 h; and the blocking agent is MCH.

7. Use of the electrochemical biosensor according to claim 2 or 4 in detecting enrofloxacin, characterized in that: The method of application includes: (1) After mixing the bivalent split-type nucleic acid aptamer b and the analyte, incubating them on the probe surface of the electrochemical biosensor to form a Y-shaped DNA scaffold through bivalent recognition; (2) After cleaning the electrodes with buffer, the three-electrode system was placed in a PBS solution for time-current scanning to obtain a detection signal.

8. Use of the electrochemical biosensor according to claim 3 or 4 in detecting myoglobin, characterized in that: The method of application includes: (1) After mixing the bivalent split-type nucleic acid aptamer b and the analyte, incubating them on the probe surface of the electrochemical biosensor to form a Y-shaped DNA scaffold through bivalent recognition; (2) After cleaning the electrodes with buffer, the three-electrode system is placed in a buffer solution for time current scanning, and the current value at 20 ms is recorded to obtain the detection signal.

9. The use according to claim 7 or 8, characterized in that: The buffer solution is PBS, the scanning voltage of the time current scanning is +0.5V, the scanning time is 30ms, and the scanning frequency is 1000Hz.

Images