Preparation method and application of electrochemical nano sensor for detecting serotonin

Abstract

The invention provides a preparation method and application of an electrochemical nano sensor for detecting serotonin. The preparation method of the electrochemical nano sensor comprises the following steps: coupling dopamine on a carbon fiber nano electrode through cyclic voltammetry to obtain a CFNE / DA electrode; the CFNE / DA electrode is placed in a dsDNA solution for an electrochemical reaction, and a CFNE / DA / dsDNA electrode is obtained; the CFNE / DA / dsDNA electrode is placed in a methylene blue solution, methylene blue is embedded between double strands of dsDNA through the adsorption effect, and the CFNE / DA / MB / dsDNA electrochemical nano sensor is obtained. According to the invention, an aptamer apt of serotonin is introduced as a recognition element, so that the specific recognition capability of the sensor on a detected target is improved; the detection limit of the electrochemical nano sensor on serotonin is lower than 0.2 nM, and the detection range is 0.5 nM to 400 nM.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensors, and in particular relates to a preparation method and application of an electrochemical nanosensor for detecting serotonin. Background Art

[0002] Serotonin is an important neurotransmitter, and abnormal increases and decreases in its content are closely related to many neurological diseases in the body, such as metabolism, obesity, depression, and hyperactivity. It plays a vital role in both the central and peripheral nervous systems. Due to the regulatory effect of serotonin, an imbalance in serotonin levels can lead to physiological and psychological dysfunction in the human body. In particular, it has been fully confirmed that an imbalance in serotonin may lead to diseases such as depression and cancer. Therefore, rapid, accurate, and in situ detection of serotonin is expected to achieve real-time monitoring and early warning of related diseases such as depression.

[0003] At present, traditional methods for laboratory detection of serotonin include liquid chromatography / mass spectrometry, fluorescence sensing, chemiluminescence and other methods. Although these methods have high sensitivity and specificity in detection, they are not suitable for in situ continuous real-time measurement of analytes from neurons or tissue samples due to the bulky instruments, long measurement time, and the need for sampling. Therefore, it is urgent to develop a method that is simple to operate and can detect serotonin in situ. Electrochemical methods are a good method for detecting serotonin due to their advantages such as fast detection speed, high sensitivity, and convenient detection. Since 5-HT is an electroactive compound, its concentration can be electrochemically quantified by directly oxidizing 5-HT on the electrode surface. However, a key problem in the electrochemical detection of serotonin is that the voltammetric signal may be interfered by other electroactive molecules with similar redox potentials.

[0004] Based on the defects of the current method for detecting serotonin, it is necessary to improve it. Summary of the invention

[0005] In view of this, the present invention proposes an electrochemical nanosensor for detecting serotonin and its application. The method provided by the present invention is low-cost, simple and efficient. It can provide an electrochemical biosensor that meets both low detection limit and high selectivity for in-situ detection of serotonin in samples.

[0006] In a first aspect, the present invention provides a method for preparing an electrochemical nanosensor for detecting serotonin, comprising the following steps:

[0007] Dopamine was coupled to the carbon fiber nanoelectrode by cyclic voltammetry to obtain the CFNE / DA electrode;

[0008] The CFNE / DA electrode is placed in a dsDNA solution for electrochemical reaction to obtain a CFNE / DA / dsDNA electrode;

[0009] The CFNE / DA / dsDNA electrode was placed in a methylene blue solution, and the methylene blue was intercalated between the double strands of dsDNA through adsorption, thus obtaining a CFNE / DA / MB / dsDNA electrochemical nanosensor.

[0010] Preferably, the dsDNA solution is prepared by:

[0011] The serotonin aptamer apt and c-DNA were added to a Tris-HCl buffer solution and incubated at 36-37°C for 4-5 hours to obtain a dsDNA solution;

[0012] Wherein, the gene sequence of the serotonin aptamer apt is:

[0013] 5'-SH-GACTGGTAGGCAGATAGGGGAAGCTGATTTCGATGCGTGGGTC-3';

[0014] The gene sequence of the c-DNA is:

[0015] 5'-GACCCACGCAAGGGTGAGGGCT-3';

[0016] The pH value of the Tris-HCl buffer solution is 7.4-7.5.

[0017] Preferably, the molar ratio of the serotonin aptamer apt and c-DNA is (1-2):(1-2);

[0018] The sum of the molar concentrations of the serotonin aptamer apt and c-DNA in the dsDNA solution is 5 to 10 μM.

[0019] Preferably, dopamine is coupled to a carbon fiber nanoelectrode by cyclic voltammetry to obtain a CFNE / DA electrode, which specifically comprises the following steps:

[0020] Dissolving dopamine in H2SO4 solution to obtain a dopamine solution;

[0021] The carbon fiber nanoelectrode is inserted into the dopamine solution, and the dopamine is coupled to the carbon fiber nanoelectrode by cyclic voltammetry;

[0022] The concentration of the H2SO4 solution is 1-2M, and the concentration of the dopamine solution is 5-10mM;

[0023] When dopamine is coupled to the carbon fiber nanoelectrode by cyclic voltammetry, the potential range is 0.8 to 1.6 V, the scanning speed is 10 to 20 mV / s, and the scanning times are 10 to 20 times.

[0024] Preferably, in the step of placing the CFNE / DA electrode in a dsDNA solution for electrochemical reaction, the reaction is carried out using a chronoamperometry method, and the voltage is controlled to be 0.55 to 0.6 V and the reaction time is 300 to 350 s.

[0025] Preferably, the methylene blue solution is prepared by dissolving methylene blue in PBS buffer to obtain a methylene blue solution;

[0026] The concentration of the subunit blue solution is 1-2 mM;

[0027] Place the CFNE / DA / dsDNA electrode in a methylene blue solution and soak for 2 to 3 hours;

[0028] The pH of the PBS buffer is 7.4-7.5.

[0029] Preferably, the carbon fiber nanoelectrode is in a conical shape with a larger diameter at one end and a smaller diameter at the other end. The diameter of the smaller end of the carbon fiber nanoelectrode is 500-600 nm, and the length is 100-200 μm.

[0030] Preferably, the method for preparing the carbon fiber nanoelectrode comprises the following steps:

[0031] Connect the two ends of a capillary tube to a drawing instrument respectively, and control the drawing instrument to draw the capillary tube, so that the middle part of the capillary tube becomes thinner and is broken into two parts during drawing;

[0032] Take a part of the capillary, insert a carbon fiber into the capillary and let it pass through the end with a smaller diameter;

[0033] The portion of the carbon fiber extending out of the capillary is ablated to make it conical, with the diameter of the smaller end of the carbon fiber being 500-600 nm and the length of the end of the carbon fiber extending out of the capillary being 100-200 μm, thus obtaining a carbon fiber nanoelectrode.

[0034] In a second aspect, the present invention also provides an application of an electrochemical nanosensor prepared by the preparation method in detecting serotonin.

[0035] Preferably, the electrochemical nanosensor is used as a working electrode, and a solution to be tested containing serotonin is used as an electrolyte;

[0036] The working electrode, the counter electrode and the reference electrode are placed in an electrolyte, and the working electrode, the counter electrode and the reference electrode are connected to an electrochemical workstation respectively, and a square wave voltammetry method is used to perform voltammetric detection to detect the oxidation peak current value;

[0037] The serotonin concentration in the test solution was calculated using the oxidation peak current value.

[0038] Preferably, when the voltammetric detection is performed using square wave voltammetry, the potential is -0.5 V to 0 V and the scanning speed is 25 to 30 mV / s.

[0039] The electrochemical nanosensor for detecting serotonin and its application of the present invention have the following beneficial effects compared with the prior art:

[0040] 1. The electrochemical nanosensor for detecting serotonin of the present invention introduces the aptamer apt of serotonin as a recognition element, thereby improving the specific recognition ability of the sensor for the detection target; during the detection process, during the specific recognition of the aptamer apt and serotonin, the double chain opens, and the MB molecules inserted in the middle escape, causing the oxidation peak current to decrease, resulting in different peak currents. According to the linear relationship between the ratio to the original peak current and the concentration, the quantitative detection of serotonin is finally achieved;

[0041] 2. The electrochemical nanosensor of the present invention is applied to the detection of serotonin, which can reduce the detection cost, reduce the requirements for experimental equipment and environment, and is simple to operate. By modifying apt, the selectivity is improved, and multiple MB molecules are inserted to amplify the signal value of the probe; the detection limit of the electrochemical nanosensor for serotonin is lower than 0.2nM (the detection limit of serotonin is 0.156nM), and the detection range is: 0.5nM~400nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 The SEM image of the CFNE electrode obtained in Example 1 of the present invention is

[0044] Figure 2 Cyclic voltammograms of the CFNE electrode, CFNE / DA electrode, and CFNE / DA / dsDNA electrode obtained in Example 1 of the present invention;

[0045] Figure 3 Cyclic voltammograms of the CFNE / DA / dsDNA electrode and the CFNE / DA / MB / dsDNA electrode obtained in Example 1 of the present invention;

[0046] Figure 4This is a square wave voltammogram of the electrochemical sensor obtained in Example 1 of the present invention before and after detecting serotonin;

[0047] Figure 5 This is a linear fitting curve diagram of the electrochemical nanosensor obtained in Example 1 of the present invention for detecting serotonin;

[0048] Figure 6 This is a selectivity test diagram of the electrochemical nanosensor obtained in Example 1 of the present invention when detecting serotonin. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0051] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may be in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0052] The present invention provides a method for preparing an electrochemical nanosensor for detecting serotonin, comprising the following steps:

[0053] S1. Dopamine (DA) was coupled to a carbon fiber nanoelectrode (CFNE) by cyclic voltammetry to obtain a CFNE / DA electrode;

[0054] S2, placing the CFNE / DA electrode in a dsDNA solution for electrochemical reaction to obtain a CFNE / DA / dsDNA electrode;

[0055] S3. Place the CFNE / DA / dsDNA electrode in a methylene blue (MB) solution, and insert the methylene blue between the double strands of dsDNA through adsorption, thereby obtaining a CFNE / DA / MB / dsDNA electrochemical nanosensor.

[0056] In some embodiments, the dsDNA solution is prepared by:

[0057] The serotonin aptamer apt and c-DNA were added to a Tris-HCl buffer solution and incubated at 36-37°C for 4-5 hours to obtain a dsDNA solution;

[0058] Among them, the gene sequence of serotonin aptamer apt is:

[0059] 5'-SH-GACTGGTAGGCAGATAGGGGAAGCTGATTTCGATGCGTGGGTC-3', as shown in SEQ ID NO: 1;

[0060] The gene sequence of c-DNA is:

[0061] 5′-GACCCACGCAAGGGTGAGGGCT-3′, as shown in SEQ ID NO:2.

[0062] In some embodiments, the pH of the Tris-HCl buffer solution is 7.4-7.5.

[0063] In some embodiments, the molar ratio of serotonin aptamer apt and c-DNA is (1-2): (1-2);

[0064] The sum of the molar concentrations of the serotonin aptamer apt and c-DNA in the dsDNA solution is 5 to 10 μM.

[0065] In the present invention, compared with a single electrochemical sensor, the present invention introduces the aptamer apt of serotonin as a recognition element, thereby improving the specific recognition ability of the sensor to the detection target; during the detection process, during the specific recognition of the aptamer apt and serotonin, the double chain opens, and the MB molecule inserted in the middle escapes, causing the oxidation peak current to decrease, resulting in different peak currents, and finally achieving quantitative detection of serotonin based on the ratio to the original peak current and the linear relationship between the concentration.

[0066] In some embodiments, dopamine is coupled to a carbon fiber nanoelectrode by cyclic voltammetry to obtain a CFNE / DA electrode, which specifically includes the following steps:

[0067] Dissolving dopamine in H2SO4 solution to obtain a dopamine solution;

[0068] The carbon fiber nanoelectrode was inserted into a dopamine (DA) solution, and dopamine was coupled to the carbon fiber nanoelectrode by cyclic voltammetry;

[0069] The concentration of H2SO4 solution is 1-2M, and the concentration of dopamine solution is 5-10mM;

[0070] When dopamine is coupled to the carbon fiber nanoelectrode by cyclic voltammetry, the potential range is 0.8 to 1.6 V, the scanning speed is 10 to 20 mV / s, and the scanning times are 10 to 20 times.

[0071] During the cyclic voltammetry scanning process, a CN bond is generated between the amine group and the carbon surface, DA forms dopaquinone on the carbon fiber surface, and then is inserted into the dsDNA solution for electrochemical coupling so that dsDNA is connected to the CFNE surface through DA. The obtained CFNE / DA / dsDNA is immersed in a 1M MB solution, and MB is intercalated between dsDNA through adsorption to obtain a CFNE / DA / MB / dsDNA electrochemical nanosensor. Specifically, the cyclic voltammetry scanning is completed in a three-electrode system, in which the carbon fiber nanoelectrode (CFNE) electrode is the working electrode, Ag / AgCl is the reference electrode, the platinum wire is the counter electrode, and the dopamine (DA) solution is the electrolyte. The working electrode, the reference electrode, and the counter electrode are all connected to the electrochemical workstation for cyclic voltammetry scanning.

[0072] In some embodiments, in the step of placing the CFNE / DA electrode in a dsDNA solution for electrochemical reaction, the reaction is carried out using a chronoamperometry method, and the voltage is controlled to be 0.55-0.6V and the time is 300-350s during the reaction. Preferably, the voltage is 0.55V and the time is 300s. Similarly, the electrochemical reaction is carried out in the chronoamperometry method, and is also completed in a three-electrode system, wherein the CFNE / DA electrode is the working electrode, Ag / AgCl is the reference electrode, the platinum wire is the counter electrode, and the dopamine (DA) solution is the electrolyte. The working electrode, the reference electrode, and the counter electrode are all connected to the electrochemical workstation to carry out the chronoamperometry reaction.

[0073] In some embodiments, the methylene blue solution is prepared by: dissolving methylene blue in a PBS buffer to obtain a methylene blue solution;

[0074] The concentration of subunit blue solution is 1-2 mM;

[0075] Place the CFNE / DA / dsDNA electrode in a methylene blue solution and soak for 2 to 3 hours;

[0076] The pH of PBS buffer is 7.4-7.5.

[0077] In some embodiments, the carbon fiber nanoelectrode is in a conical shape with a larger diameter at one end and a smaller diameter at the other end. The smaller diameter end of the carbon fiber nanoelectrode has a diameter of 500-600 nm and a length of 100-200 μm.

[0078] In some embodiments, a method for preparing a carbon fiber nanoelectrode comprises the following steps:

[0079] S1. Connecting two ends of a capillary to a drawing instrument respectively, and controlling the drawing instrument to draw the capillary, so that the middle part of the capillary becomes thinner and is broken into two parts during drawing;

[0080] S2, take a part of the capillary, insert a carbon fiber into the capillary and pass it out from the end with a smaller diameter;

[0081] S3. The portion of the carbon fiber extending out of the capillary is ablated to make it conical, and the diameter of the smaller end of the carbon fiber is 500-600 nm, and the length of the end of the carbon fiber extending out of the capillary is 100-200 μm, thereby obtaining a carbon fiber nanoelectrode.

[0082] Specifically, two ends of a capillary (with an initial inner diameter of 0.5 mm) are connected to a drawing instrument respectively, and the drawing instrument is controlled to stretch the capillary. During stretching, the middle part of the capillary becomes thinner and is broken into two parts; a carbon fiber (with an initial diameter of 7 μm) is cut into a length of about 1 cm by a surgical blade, a copper wire of about 10 cm is selected, and the carbon fiber is bonded to one end of the copper wire by conductive glue; the carbon fiber is inserted into the capillary and passed out from the end with a small diameter (the inner diameter of the end with a small diameter is slightly larger than the diameter of the carbon fiber so that the carbon fiber can pass through), and the opening between the copper wire and the end with a large diameter of the capillary is sealed with AB glue, and the part of the carbon fiber extending out of the capillary is ablated in the outer flame of an alcohol lamp at a frequency of once every 0.5 s to control it to be conical, and the diameter of the end with a small diameter of the carbon fiber is 500-600 nm and the length is 100-200 μm, so as to obtain a carbon fiber nanoelectrode.

[0083] Based on the same inventive concept, the present invention also provides an application of an electrochemical nanosensor prepared by the preparation method in detecting serotonin.

[0084] In some embodiments, the electrochemical nanosensor described above is used as a working electrode, and a test solution containing serotonin is used as an electrolyte;

[0085] The working electrode, the counter electrode and the reference electrode are placed in an electrolyte, and the working electrode, the counter electrode and the reference electrode are connected to an electrochemical workstation respectively, and a square wave voltammetry method is used to perform voltammetric detection to detect the oxidation peak current value;

[0086] The serotonin concentration in the test solution was calculated using the oxidation peak current value.

[0087] In some embodiments, the potential is -0.5V to 0V and the scan rate is 25 to 30 mV / s when the voltammetric detection is performed using square wave voltammetry.

[0088] In some embodiments, the counter electrode material is platinum and the reference electrode is an Ag / AgCl reference electrode.

[0089] In some embodiments, the working electrode, the counter electrode, and the reference electrode are connected to an electrochemical workstation, serotonin with different concentration gradients is added to the detection environment, and voltammetric detection is performed using square wave voltammetry. A standard curve is drawn based on the linear relationship between the ratio of the oxidation peak current value to the initial current value and the serotonin concentration. The serotonin concentration in the sample is obtained based on the standard curve and the oxidation peak current value during the detection of the test solution, thereby achieving the detection of serotonin.

[0090] The detection limit of the electrochemical nanosensor for serotonin is lower than 0.2 nM, and the detection range is 0.5 nM to 400 nM.

[0091] Compared with the prior art, the above technology of the present invention can reduce the detection cost, reduce the requirements for experimental equipment and environment, and is simple to operate. By modifying apt, improving selectivity, and inserting multiple MB molecules, the signal value of the probe is amplified to prepare a highly selective and highly sensitive electrochemical nanosensor for use in the detection of serotonin.

[0092] The following further describes the electrochemical nanosensor and application for detecting serotonin of the present application with specific examples. This section further describes the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0093] Example 1

[0094] This embodiment provides a method for preparing an electrochemical nanosensor for detecting serotonin, comprising the following steps:

[0095] S1. Preparation of carbon fiber nanoelectrode (CFNE):

[0096] Connect the two ends of a capillary (with an initial inner diameter of 0.5 mm) to a drawing instrument respectively, and control the drawing instrument to stretch the capillary. During the stretching, the middle part of the capillary becomes thinner and is broken into two parts; cut the carbon fiber (with an initial diameter of 7 μm) into a length of about 1 cm with a surgical blade, select a copper wire of about 10 cm, and bond the carbon fiber to one end of the copper wire with a conductive glue; take a part of the capillary, insert the carbon fiber into the capillary and pass it out from the end with a small diameter (the inner diameter of the end with a small diameter is slightly larger than the diameter of the carbon fiber, so that the carbon fiber can pass through), seal the opening between the copper wire and the end with a large diameter of the capillary with AB glue, and ablate the part of the carbon fiber extending out of the capillary in the outer flame of an alcohol lamp at a frequency of once every 0.5 s, control it to be conical, and the diameter of the end with a small diameter of the carbon fiber is 500 nm and the length is 100 μm, so as to obtain a CFNE electrode;

[0097] S2. Preparation of dsDNA solution: adding serotonin aptamer apt and c-DNA to Tris-HCl buffer solution, incubating at 200 r / min for 4 h at 37°C in a shaker to obtain a dsDNA solution; the pH of the Tris-HCl buffer solution is 7.4, the molar concentration of the serotonin aptamer apt in the dsDNA solution is 5 μM, and the molar concentration of the serotonin aptamer c-DNA in the dsDNA solution is 5 μM;

[0098] The gene sequence of the serotonin aptamer apt is 5′-SH-GACTGGTAGGCAGATAGGGGAAGCTGATTTCGATGCGTGGGTC-3′;

[0099] The gene sequence of c-DNA is:

[0100] 5'-GACCCACGCAAGGGTGAGGGCT-3';

[0101] S3. Modification of CFNE working electrode:

[0102] DA was dissolved in 0.1 M H2SO4 solution, and the concentration of DA was controlled to be 5 mM to obtain a DA solution;

[0103] The CFNE electrode (as the working electrode) was inserted into the DA solution, with Ag / AgCl as the reference electrode, platinum wire as the counter electrode, and DA solution as the electrolyte. The working electrode, reference electrode, and counter electrode were connected to an electrochemical workstation to perform cyclic voltammetry scanning. During the cyclic voltammetry scanning process, a CN bond was generated between the amine group and the carbon surface, and DA formed dopaquinone on the carbon fiber surface to obtain a CFNE / DA electrode. The cyclic voltammetry potential range was 0.8V to 1.6V, the scanning speed was 10mV / s, and the number of scans was 10 times.

[0104] Then, the CFNE / DA electrode was inserted into the dsDNA solution, with Ag / AgCl as the reference electrode, platinum wire as the counter electrode, and dsDNA solution as the electrolyte. The working electrode, reference electrode, and counter electrode were connected to the electrochemical workstation for a chronoamperometric reaction. The chronoamperometric method was used for electrochemical coupling so that dsDNA was connected to the CFNE surface through DA to obtain a CFNE / DA / dsDNA electrode. During the chronoamperometric reaction, the voltage was 0.55 V and the time was 300 s.

[0105] The CFNE / DA / dsDNA electrode was immersed in 1M MB solution for 2 h, and MB was intercalated between dsDNA through adsorption to obtain the CFNE / DA / MB / dsDNA electrochemical nanosensor.

[0106] The MB solution was prepared by dissolving methylene blue in PBS buffer (pH 7.4) to obtain a methylene blue solution with a concentration of 1 M.

[0107] The morphology of the CFNE electrode prepared in Example 1 was characterized by scanning electron microscopy. The specific test results are as follows: Figure 1 As shown, the prepared CFNE electrode has an elongated cone shape.

[0108] The CFNE electrode, CFNE / DA electrode, and CFNE / DA / dsDNA electrode prepared in Example 1 were used as working electrodes, respectively. Cyclic voltammetry was used to characterize the electrochemical behavior of the working electrode interface. 2 mM FcMeOH was used as a redox probe (i.e., 2 mM FcMeOH (ferrocene methanol) solution was used as the electrolyte). The potential range of cyclic voltammetry detection was -0.2 V to 0.6 V, and the scan rate was 50 mV / s. Figure 2 As shown, the "s"-shaped CV curve unique to nanoelectrodes can be seen, which is consistent with the nonlinear diffusion behavior of microelectrodes. At the same time, the redox signal intensity of the CFNE / DA / dsDNA electrode is significantly lower than that of the CFNE and CFNE / DA electrodes. This is because the electrostatic repulsion between the probe and the negatively charged DNA backbone prevents the probe from reaching the CFNE electrode.

[0109] The working electrode (i.e., the CFNE / DA / dsDNA electrode prepared in Example 1) that was not immersed in the MB solution was used as a reference sample, and a cyclic voltammetry scan was performed in a PBS buffer (pH 7.4) with a potential range of -0.5 V to 0 V and a scan rate of 50 mV / s. Figure 3As shown, the electrode without MB embedding has no redox peak in PBS buffer, while the electrode after MB embedding (i.e., the CFNE / DA / MB / dsDNA finally prepared in Example 1) has a clear redox peak in the cyclic voltammogram near -0.3V, indicating the successful preparation of the CFNE / DA / MB / dsDNA electrochemical nanosensor after immersion.

[0110] Performance Testing

[0111] Detection of serotonin by square wave voltammetry

[0112] Specifically, the electrochemical nanosensor for serotonin analysis and detection operation steps are as follows: serotonin is added to PBS buffer (pH 7.4) to prepare a 500 nM serotonin solution, and a PBS buffer (pH 7.4) without serotonin is used as a comparison, Ag / AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, and the electrochemical nanosensor in Example 1 is used as a working electrode to form a three-electrode system for square wave voltammetry detection. The potential detected by the square wave voltammetry is -0.5V~0V, and the scanning speed is 25mV / s. The test results are as follows: Figure 4 shown.

[0113] Figure 4 Here, with ST indicates PBS buffer containing 500 nM serotonin, and without ST indicates PBS buffer without serotonin;

[0114] like Figure 4 As shown in the figure, in the presence of 500 nM concentration of serotonin (ST), the specific binding with the aptamer apt causes the MB molecules to escape, and the peak of the square wave voltammetry curve decreases significantly compared with the PBS buffer without serotonin, indicating that the electrochemical nanosensor can detect serotonin in solution.

[0115] The concentrations of serotonin solutions prepared with PBS buffer (pH 7.4) were: 0 nM, 0.5 nM, 1 nM, 10 nM, 20 nM, 100 nM, 200 nM, and 400 nM; Ag / AgCl was used as a reference electrode, a platinum wire was used as a counter electrode, and corresponding serotonin solutions of different concentrations were used as electrolytes. The electrochemical nanosensor in Example 1 was used as a working electrode to form a three-electrode system for square wave voltammetry detection. The potential detected by the square wave voltammetry was -0.5 V to 0 V, and the scanning rate was 25 mV / s; the peak current measured in PBS buffer (i.e., the serotonin concentration was 0 nM and did not contain serotonin) was recorded as I0, and the peak current detected at the corresponding concentration was recorded as I, and I / I0 was used to characterize the change in current. At least three parallel experiments were performed for each concentration of solution.

[0116] This method is used to calculate the linear fitting curve, such as Figure 5 As shown, within the serotonin concentration range of 0.5 nM and 400 nM, I / I0 and the logarithm of serotonin concentration show a good linear relationship, and the linear curve y = -0.151x + 0.664, R 2 =0.997, x is the logarithm of serotonin concentration, y is the value of I / I0 at the corresponding serotonin concentration, R 2 The correlation is a linear curve, and the detection limit of serotonin is 0.156nM.

[0117] Selectivity test for serotonin detection by square wave voltammetry

[0118] Specifically, the selective detection operation steps of the electrochemical nanosensor for serotonin are as follows: different detection objects are added to PBS buffer (pH 7.4), wherein the concentration of L-tyrosine (L-Tyr) is 1 μM, the concentration of uric acid (UA) is 1 μM, the concentration of ascorbic acid (AA) is 1 μM, the concentration of acetylcholine (ACH) is 1 μM, the concentration of dopamine (DA) is 1 μM, and the concentration of serotonin (ST) is 0.25 μM. Ag / AgCl is used as a reference electrode, platinum wire is used as a counter electrode, and the PBS buffer of the corresponding detection object with different concentrations is used as an electrolyte. The three-electrode system composed of the electrochemical nanosensor in Example 1 as a working electrode is subjected to square wave voltammetry detection. The potential detected by square wave voltammetry is -0.5V~0V, the scanning speed is 25mV / s, the peak current measured in PBS buffer is recorded as I0 (does not contain any antibiotics), and the peak current detected in the corresponding solution is recorded as I, and I / I0 is used to characterize the change of current. At least three parallel experiments were performed for each concentration of solution. Figure 6 As shown in the figure, except for the serotonin solution, the I / I0 of other substances detected is greater than that of serotonin and close to 1. The larger the I / I0 detected, the less selective the electrochemical nanosensor is to the substance, which shows the specific recognition ability of the electrochemical nanosensor to serotonin.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical nanosensor for detecting serotonin, characterized in that: The following steps are involved: Dopamine was coupled to the carbon fiber nanoelectrode by cyclic voltammetry to obtain the CFNE / DA electrode; The CFNE / DA electrode is placed in a dsDNA solution for electrochemical reaction to obtain a CFNE / DA / dsDNA electrode; The CFNE / DA / dsDNA electrode was placed in a methylene blue solution, and the methylene blue was intercalated between the double strands of dsDNA through adsorption, thus obtaining a CFNE / DA / MB / dsDNA electrochemical nanosensor.

2. The method for preparing an electrochemical nanosensor for detecting serotonin according to claim 1, characterized in that: The configuration method of the dsDNA solution is: The serotonin aptamer apt and c-DNA were added to a Tris-HCl buffer solution and incubated at 36-37°C for 4-5 hours to obtain a dsDNA solution; Wherein, the gene sequence of the serotonin aptamer apt is: 5'-SH-GACTGGTAGGCAGATAGGGGAAGCTGATTTCGATGCGTGGGTC-3'; The gene sequence of the c-DNA is: 5'-GACCCACGCAAGGGTGAGGGCT-3'; The pH value of the Tris-HCl buffer solution is 7.4-7.

5.

3. The method for preparing an electrochemical nanosensor for detecting serotonin according to claim 1, characterized in that: The molar ratio of the serotonin aptamer apt and c-DNA is (1-2): (1-2); The sum of the molar concentrations of the serotonin aptamer apt and c-DNA in the dsDNA solution is 5-10 μM.

4. The method for preparing an electrochemical nanosensor for detecting serotonin according to claim 1, characterized in that: Dopamine is coupled to a carbon fiber nanoelectrode by cyclic voltammetry to obtain a CFNE / DA electrode, which specifically includes the following steps: Dissolving dopamine in H2SO4 solution to obtain a dopamine solution; The carbon fiber nanoelectrode is inserted into the dopamine solution, and the dopamine is coupled to the carbon fiber nanoelectrode by cyclic voltammetry; The concentration of the H2SO4 solution is 1-2M, and the concentration of the dopamine solution is 5-10mM; When dopamine was coupled to the carbon fiber nanoelectrode by cyclic voltammetry, the potential range was 0.8~1.6V, the scanning speed was 10~20mV / s, and the number of scans was 10~20 times.

5. The method for preparing an electrochemical nanosensor for detecting serotonin according to claim 1, characterized in that: In the step of placing the CFNE / DA electrode in a dsDNA solution for electrochemical reaction, the reaction was carried out using a chronoamperometry method, with the voltage controlled at 0.55-0.6 V and the reaction time at 300-350 s; The methylene blue solution is prepared by: dissolving methylene blue in PBS buffer to obtain a methylene blue solution; The concentration of the subunit blue solution is 1-2 mM; Place the CFNE / DA / dsDNA electrode in the methylene blue solution and soak for 2-3 hours; The pH of the PBS buffer is 7.4-7.

5.

6. The method for preparing an electrochemical nanosensor for detecting serotonin according to claim 1, characterized in that: The carbon fiber nanoelectrode is in a conical shape with a large diameter at one end and a small diameter at the other end. The diameter of the small end of the carbon fiber nanoelectrode is 500-600nm, and the length is 100-200μm.

7. The method for preparing an electrochemical nanosensor for detecting serotonin according to claim 6, characterized in that: The method for preparing the carbon fiber nanoelectrode comprises the following steps: Connect the two ends of a capillary tube to a drawing instrument respectively, and control the drawing instrument to draw the capillary tube, so that the middle part of the capillary tube becomes thinner and is broken into two parts during drawing; Take a part of the capillary, insert a carbon fiber into the capillary and let it pass through the end with a smaller diameter; The portion of the carbon fiber extending out of the capillary is ablated to make it conical, with the diameter of the smaller end of the carbon fiber being 500-600 nm and the length of the end of the carbon fiber extending out of the capillary being 100-200 μm, thereby obtaining a carbon fiber nanoelectrode.

8. Use of an electrochemical nanosensor prepared by the preparation method according to any one of claims 1 to 7 in detecting serotonin.

9. The use according to claim 8, characterized in that The electrochemical nanosensor is used as a working electrode and a solution to be tested containing serotonin is used as an electrolyte; The working electrode, the counter electrode and the reference electrode are placed in an electrolyte, and the working electrode, the counter electrode and the reference electrode are connected to an electrochemical workstation respectively, and a square wave voltammetry method is used to perform voltammetric detection to detect the oxidation peak current value; The serotonin concentration in the test solution was calculated using the oxidation peak current value.

10. The use according to claim 9, characterized in that The potential was -0.5V~0V and the scanning speed was 25~30 mV / s when the square wave voltammetry was used for voltammetric detection.

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