A method for preparing a TURN ON signal amplification electrode and a method for detecting nucleic acids

Abstract

The present application provides a method for preparing a TURNON signal amplification electrode and a method for detecting nucleic acids. The method uses chitosan, catechol, and nanomaterials (gold nanoparticles (Au-NP), nitrogen-doped carbon nanotubes (N-CNT)) to construct a three-dimensional porous membrane on the electrode surface. When miRNA-141 hybridizes with the probe on the membrane, ferrocene (Fc) will be introduced to the electrode surface, and then a 3D porous membrane containing Fc, ruthenium ions (Ru) will be formed. 3+ ), catechol and electrode pseudocapacitive system. Fc and Ru consumed in the electrode reaction 3+ It quickly returns to its original valence state on the catechol / quinone surface and re-enters the electrode reaction. The "one-to-many" relationship between the target miRNA and the signaling molecule Fc contributes to enhanced sensor sensitivity. The pseudocapacitive system improves the signal intensity and stability of Fc, thereby enhancing the sensitivity and stability of the sensor in detecting miRNA-141.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a method for preparing a TURN ON signal amplification electrode and a method for detecting nucleic acids. Background Art

[0002] In traditional electrochemical methods for detecting nucleic acids, ionic signal amplification molecules mainly induce concentration changes through changes in electrostatic attraction to obtain detection signals, which has problems such as low reliability and insufficient detection limit. Summary of the Invention

[0003] The present application provides a method for preparing a TURN ON signal amplification electrode and a method for detecting nucleic acids to improve the reliability of detection.

[0004] In a first aspect, a method for preparing a TURN ON signal amplification electrode is provided, the method comprising the following steps:

[0005] Chitosan / N-CNTs film was prepared on GCE electrode by electrochemical deposition in chitosan / N-CNTs solution;

[0006] Grafting catechol onto the surface of chitosan / N-CNTs membrane;

[0007] Gold nanoparticles were prepared on the surface of GCE electrode by electrochemical deposition;

[0008] Hairpin capture probes were prepared on the GCE electrode.

[0009] In the above scheme, chitosan, catechol and nanomaterials (gold nanoparticles (Au-NP), nitrogen-doped carbon nanotubes (N-CNT)) are used to construct a three-dimensional porous membrane on the electrode surface. When miRNA-141 hybridizes with the probe on the membrane, ferrocene (Fc) will be introduced to the electrode surface, and then a porous membrane containing Fc, ruthenium ions (Ru 3+ ), catechol and electrode pseudocapacitive system. Fc and Ru consumed in the electrode reaction 3+ It quickly returns to its original valence state on the catechol / quinone surface and re-enters the electrode reaction. The "one-to-many" relationship between the target miRNA and the signaling molecule Fc contributes to enhanced sensor sensitivity. The pseudocapacitive system improves the signal intensity and stability of Fc, thereby enhancing the sensitivity and stability of the sensor in detecting miRNA-141.

[0010] In a specific embodiment, the grafting of catechol onto the surface of the GCE electrode is specifically as follows:

[0011] The GCE electrode was immersed in a 2-10 mM catechol solution and reacted at a potential of 0.5-0.7 V for 300-700 s.

[0012] In a specific embodiment, the method further comprises: polishing the GCE electrode with alumina powder and removing contaminants on the electrode surface before preparing the chitosan / N-CNTs film.

[0013] In a specific embodiment, the GCE electrode is polished with aluminum oxide powder to remove contaminants on the electrode surface; specifically:

[0014] The GCE electrodes were polished continuously using 1.0 μm, 0.3 μm, and 0.05 μm alumina powders;

[0015] The samples were ultrasonically treated in ethanol and ultrapure water for 10 min respectively.

[0016] In a specific embodiment, the concentration of the chitosan / N-CNTs solution is 0.1% to 3%.

[0017] In a specific embodiment, the electrochemical deposition conditions are: -2.6 to -3.5 V voltage, and 100 to 200 s.

[0018] In a specific embodiment, the chemical reaction when catechol is grafted onto the surface of the chitosan / N-CNTs membrane is:

[0019]

[0020] In a specific embodiment, the reaction when preparing the chitosan / N-CNTs film on the GCE electrode by electrochemical deposition is:

[0021]

[0022] In a specific embodiment, the hairpin capture probe is prepared on the GCE electrode; specifically:

[0023] Take 10 μL of 10 μM hairpin capture probe and add it to 10 μL of 1 mM TCEP solution for 30 min-120 min;

[0024] Add 10 μL of 10 μM hairpin capture probe solution to the GCE electrode and incubate at 37°C for 2 h;

[0025] The GCE electrode was rinsed with 0.1 M PBS buffer to remove the residual solution on the surface, thereby obtaining an electrode prepared with the hairpin capture probe. The hairpin probe was thus fixed to the GCE electrode surface via an Au-S bond.

[0026] In a second aspect, a method for detecting nucleic acids using a TURN ON signal amplification electrode is provided, the method comprising the following steps:

[0027] Add nucleic acid solution for hybridization;

[0028] P&FcAu-NPs solution was added to the GCE electrode to obtain a GCE electrode containing Fc; the electrode signal was detected by cyclic voltammetry.

[0029] In the above scheme, chitosan, catechol and nanomaterials (gold nanoparticles (Au-NP), nitrogen-doped carbon nanotubes (N-CNT)) are used to construct a three-dimensional porous membrane on the electrode surface. When miRNA-141 hybridizes with the probe on the membrane, ferrocene (Fc) will be introduced to the electrode surface, and then a porous membrane containing Fc, ruthenium ions (Ru 3+ ), catechol and electrode pseudocapacitive system. Fc and Ru consumed in the electrode reaction 3+ It quickly returns to its original valence state on the catechol / quinone surface and re-enters the electrode reaction. The "one-to-many" relationship between the target miRNA and the signaling molecule Fc contributes to enhanced sensor sensitivity. The pseudocapacitive system improves the signal intensity and stability of Fc, thereby enhancing the sensitivity and stability of the sensor in detecting miRNA-141.

[0030] In a specific embodiment, the adding of nucleic acid solution for hybridization is specifically:

[0031] Add miRNA-141 solution to the GCE electrode and hybridize at 37°C for 15 min to 120 min;

[0032] The electrode was washed with 0.1 M PBS buffer to remove miRNA141 that was not effectively hybridized.

[0033] In a specific embodiment, the P&FcAu-NPs solution is added to the GCE electrode to obtain a GCE electrode containing Fc; and the electrode signal is detected by cyclic voltammetry as follows:

[0034] 10 μL of 1 μM SP & FcAu-NPs solution was added to the electrode and reacted at 37°C for 1 hour to obtain an Fc-containing electrode. The electrode was washed with 0.1 M PBS buffer and the electrode signal was detected by cyclic voltammetry. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The following is a flow chart showing a method for preparing a TURN ON signal amplifying electrode according to an embodiment of the present invention;

[0036] Figure 2 The signal response mechanism of the pseudocapacitive structure is shown;

[0037] Figure 3CV curves of 800 pM miRNA-141 and blank groups are shown;

[0038] Figure 4 shows the current-time curves at different potentials provided in the embodiment of the present invention;

[0039] Figure 5 shows the SEM images of gold particles on the GCE electrode at different overpotentials;

[0040] Figure 6 (a) CV test curves of Cat-Chi-N-CNTs electrode at different gold particle deposition times. CV test solution composition: 50μM Fc, 50μM Ru 3+ andPBS, gold was deposited using the CV method, and the potential range of gold deposition was -0.1V-0.2V; (b) is the relationship curve between the Fc peak current and time extracted from Figure (a);

[0041] Figure 7 Shown are the Fc peak currents at different concentrations of miRNA-141. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of this application clearer, this application will be further described in detail below with reference to the accompanying drawings.

[0043] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in one or more embodiments of this specification should have the common meanings understood by people with ordinary skills in the field to which this disclosure belongs.

[0044] The use of "first", "second" and similar words does not indicate any order, quantity or importance, but is only used to distinguish different components. "Include" or "comprising" and similar words mean that the elements or objects preceding the word include the elements or objects listed after the word and their equivalents, but do not exclude other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative position relationships. When the absolute position of the object being described changes, the relative position relationship may also change accordingly.

[0045] To facilitate understanding of the TURN ON signal amplification electrode preparation method provided in the embodiments of the present application, the application scenario of the TURN ON signal amplification electrode preparation method provided in the embodiments of the present application is first explained. The TURN ON signal amplification electrode preparation method provided in the embodiments of the present application is used to improve the effect of nucleic acid detection. Currently, nucleic acids have relatively low sensitivity and stability when detected by electrodes. For this reason, the embodiments of the present application provide a TURN ON signal amplification electrode preparation method, which is described in detail below.

[0046] The TURN ON signal amplification electrode provided in the embodiment of the present application is a three-dimensional porous membrane constructed in situ on the electrode surface by chitosan, catechol and nanomaterials (gold nanoparticles (Au-NP), nitrogen-doped carbon nanotubes (N-CNT)). During nucleic acid detection, when miRNA-141 hybridizes with the probe on the membrane, ferrocene (Fc) will be introduced to the electrode surface, and then a ion containing Fc, ruthenium ions (Ru 3+ ), catechol and electrode pseudocapacitive system. Fc and Ru consumed in the electrode reaction 3+ It quickly returns to its original valence state on the catechol / quinone surface and re-enters the electrode reaction. A "one-to-many" relationship exists between the target and the signaling molecule Fc, which helps enhance sensor sensitivity. The improved signal intensity and stability of the Fc enhance the sensitivity and stability of miRNA-141 detection. This is explained in detail below.

[0047] refer to Figure 1 The present invention provides a method for preparing a TURN ON signal amplifying electrode, which comprises the following steps:

[0048] Step 001: Prepare chitosan / N-CNTs film on GCE electrode by electrochemical deposition in chitosan / N-CNTs solution;

[0049] Specifically, the concentration of the chitosan / N-CNTs solution is 0.1% to 3%. Exemplarily, the concentration is 0.1%, 0.5%, 1.25%, 2%, 3%, and other concentrations. During electrochemical deposition, the electrochemical deposition conditions are: -2.6 to -3.5V voltage, 100 to 200 seconds. Exemplarily, the reaction can be performed at -2.6V for 200 seconds, at -3V for 150 seconds, and at -3.5V for 100 seconds.

[0050] In addition, before preparing the chitosan / N-CNTs film, the GCE electrode was polished with alumina powder to remove surface contaminants and then ultrasonically treated in ethanol and ultrapure water for 10 min, respectively.

[0051] For example, a GCE electrode was polished sequentially with 1.0 μm, 0.3 μm, and 0.05 μm alumina powders and ultrasonically treated in ethanol and ultrapure water for 10 min, respectively, to remove surface contaminants. The electrode was dried with a nitrogen stream. A chitosan / N-CNTs (25:14, w / w) film was then prepared on the GCE electrode by electrochemical deposition (-3 V vs. SCE, 120 s) in a 1.25% (w / w) chitosan / N-CNTs solution.

[0052] The reaction when chitosan / N-CNTs film is prepared on GCE electrode by electrochemical deposition is:

[0053]

[0054] In addition, after the chitosan / N-CNTs film was prepared, the GCE electrode was cleaned with ultrapure water to remove the chitosan solution that was not effectively attached to the surface, and then dried with N2 flow to obtain a clean GCE electrode.

[0055] Step 002: Grafting catechol onto the surface of chitosan / N-CNTs membrane;

[0056] Specifically, the GCE electrode is immersed in a 2-10 mM catechol solution and reacted at a potential of 0.5-0.7 V for 300-700 seconds. For example, the catechol solution may be 2 mM, 4 mM, 6 mM, 8 mM, or 10 mM. The potential can be 0.5 V, 0.6 V, or 0.7 V. The reaction time can be 300 seconds, 400 seconds, 600 seconds, or 700 seconds.

[0057] For example, Chi-N-CNTs-GCE was immersed in a 50 mM catechol solution and reacted at a potential of 0.6 V for 500 s to graft catechol onto the electrode surface (vs. SCE). The electrode was cleaned with ultrapure water to remove the residual catechol solution on the electrode surface and dried with a N2 stream.

[0058] Among them, the chemical reaction when catechol is grafted onto the surface of chitosan / N-CNTs membrane is:

[0059]

[0060] Step 003: Prepare gold nanoparticles on the surface of the GCE electrode by electrochemical deposition.

[0061] Specifically, gold nanoparticles (E-Au-NPs) were prepared on the electrode surface by electrochemical deposition.

[0062] Step 004: preparing a hairpin capture probe on the GCE electrode;

[0063] Specifically, 10 μL of 10 μM hairpin capture probe is added to 10 μL of 1 mM TCEP solution for 30 min to 120 min; for example, the duration is 30 min, 60 min, 90 min, or 120 min.

[0064] 10 μL of 10 μM hairpin capture probe solution was added to the GCE electrode and incubated at 37° C. for 2 h. The GCE electrode was rinsed with 0.1 M PBS buffer to remove the residual solution on the surface, thereby obtaining an electrode prepared with the hairpin capture probe.

[0065] For example, 10 μL of a 10 μM hairpin capture probe was added to 10 μL of a 1 mM TCEP solution for 1 hour. Then, 10 μL of the 1 μM hairpin capture probe solution was added to the Cat-Chi-N-CNTs-GCE-E-Au NPs electrode and incubated at 37°C for 15 to 120 minutes, such as 15 minutes, 60 minutes, 90 minutes, or 120 minutes.

[0066] The electrode was rinsed with 0.1 M PBS buffer to remove the residual solution on the surface, thereby obtaining an electrode modified with a hairpin capture probe.

[0067] In the above scheme, chitosan, catechol and nanomaterials (gold nanoparticles (Au-NP), nitrogen-doped carbon nanotubes (N-CNT)) are used to construct a three-dimensional porous membrane on the electrode surface. When miRNA-141 hybridizes with the probe on the membrane, ferrocene (Fc) will be introduced to the electrode surface, and then a porous membrane containing Fc, ruthenium ions (Ru 3+ ), catechol and electrode pseudocapacitive system. Fc and Ru consumed in the electrode reaction 3+ It quickly returns to its original valence state on the catechol / quinone surface and re-enters the electrode reaction. The "one-to-many" relationship between the target miRNA and the signaling molecule Fc contributes to enhanced sensor sensitivity. The pseudocapacitive system improves the signal intensity and stability of the Fc, thereby enhancing the sensitivity and stability of the sensor in detecting miRNA-141. This is explained in detail below in conjunction with nucleic acid detection methods.

[0068] The present application also provides a method for detecting nucleic acids using a TURN ON signal amplification electrode, the method comprising the following steps:

[0069] Step 01: Add nucleic acid solution for hybridization;

[0070] Specifically, the miRNA-141 solution was added to the GCE electrode and hybridized at 37° C. for 1 h; the electrode was washed with 0.1 M PBS buffer to remove the miRNA141 that was not effectively hybridized.

[0071] Step 02: Add the P&FcAu-NPs solution to the GCE electrode to obtain a GCE electrode containing Fc; use cyclic voltammetry to detect the electrode signal.

[0072] Specifically, 10 μL of a 1 μM SP & FcAu-NPs solution was added to the electrode and reacted at 37°C for 1 hour to obtain an Fc-containing electrode. The electrode was then washed with 0.1 M PBS buffer and the electrode signal was detected using cyclic voltammetry.

[0073] refer to Figure 2 During hybridization, catechol polymerized on the electrode can switch between two chemical states, namely, catechol (oxidized state) and catechol (reduced state). 3+ Reduction to Ru 2 + Then, quinone will Ru 2+ Oxidized to Ru 3+ , while o-phthaloquinone is converted to o-phthaloquinone. When Fc is introduced into the electrode, a complete redox cycle is formed, that is, the pseudocapacitive structure is "on". In the forward scan, Fc is oxidized to Fc + Then, the obtained catechol will Fc + Reduced to Fc, and catechol is oxidized to o-phthaloquinone. 3+ , o-catechol and o-catechol will participate in the next cycle of reaction.

[0074] Detection process: The prepared electrode was placed in a 3+ In a PBS solution, a platinum wire was used as the counter electrode and a saturated calomel electrode was used as the reference electrode. Cyclic voltammetry was performed at 50 mVs -1 The scanning speed was tested in the range of -0.5 V to +0.5 V. The composition of the PBS solution was: 0.1 M K2HPO4 and 0.025 M KH2PO4, pH = 7.2.

[0075] refer to Figure 3 , the signal amplification effect of the pseudocapacitive structure is as follows Figure 3 As shown. Among them, Figure 3 The CV curves of 800 pM miRNA-141 and blank group are shown in FIG. Solution composition: 50 μM Ru 3+ and PBS buffer. Figure 4 It can be seen that with the introduction of Fc on the electrode, not only the Fc signal is detected, but also the original Ru 3+ The signal is also enhanced. This indicates that the Ru reduced on the electrode surface 3+It is oxidized by o-phthaloquinone and quickly participates in the next cycle reaction, as does Fc. Therefore, the pseudocapacitive structure can enhance the signal intensity, which is beneficial to improving the detection performance of the sensor.

[0076] refer to Figure 4 , SEM images of gold particles on the GCE electrode at different overpotentials, with a deposition time of 180 s. Potential step experiments were conducted using a three-electrode system in a 1 g / L HAuCl4 / (100 mg / L KNO3) solution.

[0077] At all potential values, a monotonic decrease in transient current was observed within the first few milliseconds. This experimental phenomenon is consistent with Report on UPD of copper on Au(111) The results are very consistent with those of Zle et al., 1994a. The large initial current is caused by double-layer charging. After a short period of charging, the current decays rapidly. The current-time curve then exhibits different shapes with different potentials and can be divided into three types, as shown in Figure S1: T Ⅰ - monotonically decreasing and gradually increasing, T Ⅱ - Initially decreases, then increases, and then decreases to a stable state, T Ⅲ -Monotonically decreasing and then stable.

[0078] Depend on Figure 5 It can be seen that under the same deposition time, the particle density increases with the increase of overpotential, and the particle diameter decreases with the increase of overpotential. In order to ensure that the number density of gold particles on the electrode surface remains high, the potential deposition range is selected to be -0.1V-0.2V, combined with Figure 6 According to the test results, when the gold particle growth time is 60s, the Fc peak current signal is the strongest, so the deposition time is positioned at 60s.

[0079] Figure 6 (a) CV test curves of Cat-Chi-N-CNTs electrode at different gold particle deposition times. CV test solution composition: 50 μM Fc, 50 μM Ru 3+ andPBS, gold was deposited using the CV method, and the gold potential range was -0.1 V to 0.2 V. (b) is the relationship curve between the Fc peak current and time extracted from Figure (a).

[0080] Figure 7Peak current of the Fc at different miRNA-141 concentrations, and the linear relationship between the current and the logarithm of the miRNA-141 concentration (a). Selectivity of the biosensor: Changes in the Fc current before and after CV analysis of miRNA-141, single-base mismatched RNA (SM), triple-base mismatched RNA (TM), and non-complementary RNA (NC). All analytes were tested at a concentration of 100 fM (b). The linear detection range was 0.5 fM-800 pM, and the detection limit was calculated to be 43.73 aM.

[0081] From the above description, it can be seen that chitosan, catechol and nanomaterials (gold nanoparticles (Au-NP), nitrogen-doped carbon nanotubes (N-CNT)) are used to construct a three-dimensional porous membrane on the electrode surface. When miRNA-141 hybridizes with the probe on the membrane, ferrocene (Fc) will be introduced to the electrode surface, and then a porous membrane containing Fc, ruthenium ions (Ru 3+ ), catechol and electrode pseudocapacitive system. Fc and Ru consumed in the electrode reaction 3+ It quickly returns to its original valence state on the catechol / quinone surface and re-enters the electrode reaction. A "one-to-many" relationship exists between the target and the signaling molecule Fc, which helps enhance sensor sensitivity. The improved signal intensity and stability of Fc enhance the sensitivity and stability of miRNA-141 detection.

[0082] The one or more embodiments of this specification are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this specification shall be included within the scope of protection of this disclosure.

[0083] The above are only specific embodiments of the present application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of this application. Therefore, the scope of protection of this application should be based on the scope of protection of the claims.

Claims

1. A method for detecting nucleic acid using a TURN ON signal amplification electrode, characterized in that: The following steps are involved: Add miRNA solution for hybridization; Adding the P&FcAu-NPs solution to the modified GCE electrode to obtain a modified GCE electrode containing Fc; detecting the electrode signal by cyclic voltammetry; The modified GCE electrode was prepared in the following manner: Chitosan / N-CNTs film was prepared on GCE electrode by electrochemical deposition in chitosan / N-CNTs solution; Grafting catechol onto the surface of chitosan / N-CNTs membrane; Gold nanoparticles were prepared on the surface of GCE electrode by electrochemical deposition, and a three-dimensional porous membrane was constructed in situ on the electrode surface using chitosan, catechol, gold nanoparticles, and N-CNTs; The hairpin capture probe was prepared on the GCE electrode to obtain a modified GCE electrode.

2. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 1, wherein: The miRNA solution is added for hybridization; specifically: Add miRNA-141 solution to the modified GCE electrode and hybridize at 37°C for 15 min to 120 min; The electrode was washed with 0.1 M PBS buffer to remove miRNA141 that was not effectively hybridized.

3. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 1, wherein: The P&FcAu-NPs solution is added to the modified GCE electrode to obtain a modified GCE electrode containing Fc; and the electrode signal is detected by cyclic voltammetry as follows: 10 μL of 1 μM SP&FcAu-NPs solution was added to the modified GCE electrode and reacted at 37°C for 1 h to obtain a modified GCE electrode containing Fc; the modified GCE electrode containing Fc was washed with 0.1 M PBS buffer, and the signal of the modified GCE electrode containing Fc was detected by cyclic voltammetry.

4. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 1, wherein: The step of grafting catechol onto the surface of chitosan / N-CNTs is specifically as follows: The GCE electrode was immersed in a 2-10 mM catechol solution and reacted at a potential of 0.5-0.7 V for 300-700 s.

5. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 1, wherein: Also includes: Before preparing the chitosan / N-CNTs film, the GCE electrode was polished with alumina powder to remove contaminants on the electrode surface.

6. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 5, wherein: The GCE electrode is polished with alumina powder and contaminants on the electrode surface are removed; specifically: The GCE electrode was polished continuously using 1.0μm, 0.3μm, and 0.05μm alumina powders; The samples were ultrasonically treated in ethanol and ultrapure water for 10 min respectively.

7. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 1, wherein: The concentration of the chitosan / NCNTs solution is 0.1% to 3%.

8. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 1, wherein: The conditions of the electrochemical deposition are: voltage of -2.6 to -3.5 V, and time of 100 to 200 s.

9. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 1, wherein: The chemical reaction when catechol is grafted onto the surface of the chitosan / N-CNTs membrane is: 。 10. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to claim 1, wherein: The reaction when preparing chitosan / N-CNTs film on GCE electrode by electrochemical deposition is: 。 11. The method for detecting nucleic acid using a TURN ON signal amplifying electrode according to any one of claims 1 to 10, wherein: The hairpin capture probe is prepared on the modified GCE electrode; specifically: Take 10 μL of 10 μM hairpin capture probe and add it to 10 μL of 1 mM TCEP solution for 30 min-120 min; 10 μL of 10 μM TCEP-treated hairpin capture probe solution was added to the GCE electrode and incubated at 37°C for 2 h; The GCE electrode was rinsed with 0.1 M PBS buffer to remove the residual solution on the surface, thereby obtaining an electrode modified with a hairpin capture probe.

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