CN112010917B - ex-TTF-aptamer conjugate, preparation method thereof and electrochemical sensor prepared from conjugate - Google Patents
ex-TTF-aptamer conjugate, preparation method thereof and electrochemical sensor prepared from conjugate Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Abstract
The invention provides a preparation method of an ex-TTF-aptamer conjugate, which comprises the following steps: adding a TTF compound and an azide compound into a tert-butyl alcohol solution, then adding N, N-diisopropylethylamine and copper iodide, fully stirring and extracting to obtain an organic phase, washing the organic phase, and then drying, filtering and concentrating to obtain an ex-TTF derivative; adding dimethyl sulfoxide solution into the ex-TTF derivative for dissolving to obtain an ex-TTF derivative solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the ex-TTF derivative solution for ultrasonic dissolving, then adding a modified DNA molecule and sodium bicarbonate solution, carrying out oscillation reaction, centrifuging after the reaction is finished, taking an upper layer solution, and carrying out HPLC separation and purification on the upper layer solution to obtain the ex-TTF-aptamer conjugate. The invention also provides an electrochemical sensor prepared by using the ex-TTF-aptamer conjugate. The electrochemical sensor can effectively realize the detection of small molecule drugs and is not influenced by the pH change of a receptor system.
Description
Technical Field
The invention relates to the technical field of electrochemical sensors, in particular to an ex-TTF-aptamer conjugate, a preparation method thereof and an electrochemical sensor prepared by using the conjugate.
Background
With the development of science and technology and medical technology, people pay more attention to monitoring and management of their health. The effective monitoring of human body activity and physiological change provides useful information for health management and medical care treatment, and plays an important role. Therefore, the electrochemical sensor is a bridge for understanding the activity and physiological change of the human body, and the detection of the physiological environment is also important. Physiological fluids contain large amounts of small molecules and trace analytes relevant to health, which can serve as biomarkers that have proven to be very effective in predicting and diagnosing systemic diseases. Therefore, it becomes especially difficult and important to detect other substances and obtain accurate test results in unstable and complex biological fluids, such as blood, urine, sweat, cells, etc. The research on these influencing factors has been focused by researchers, and the physiological environment samples often face great detection challenges in terms of sample volume, secretion rate, pH change, analyte decomposition and other aspects, and how to develop biosensors applicable to detection under different pH values in the physiological environment is one of the research hotspots in recent years.
Physiological parameters in a physiological environment can provide important information about the health status of a human body and can serve as a warning for early onset and diagnosis of problematic diseases. The pH value in sweat can be used as an indicator of the intensity of the body's motion and the degree of dehydration, so reliable monitoring of the pH value in sweat is closely related to the monitoring of the body's health. Glucose and pH value are used as two important indexes of diabetes, and the glucose and the pH value in the brain of a diabetic rat are simultaneously and quantitatively measured in a ratio method electrochemical biosensor, and the pH value can be accurately measured in a linear range of 5.67-7.65. With the increasing attention on Alzheimer Disease (AD), the simultaneous monitoring of Cu + and pH can better understand the damage of AD on brain functions and the effects of AD on brain pathological and physiological events, and the applicable pH value range of the biosensor is 6.0-8.0. Based on Fluorescence Resonance Energy Transfer (FRET) in living cells as a pH sensor, the pH sensor has a high dynamic range between pH values of 5.8 and 7, and the sensor has great development potential in disease diagnosis and targeted therapy in a living system. The application of biomarker screening technology can reduce the disease prevalence rate, the pH and urea in urine of a patient with renal disease are used as biological detection objects, and the developed pH sensor has certain detection capability in a physiological range. Therefore, the different biosensors have the situation that the pH value dynamically changes in a test system, and the change causes great difficulty in the detection of other target substances by the biosensors and also brings great challenge and influence on the development of new biosensors.
The electrochemical biosensor has the advantages of simple operation, good specificity, high sensitivity, cheap equipment, easy miniaturization and the like, and is widely applied to the fields of clinical diagnosis, drug analysis, environmental monitoring and the like in the detection of bioactive molecules. The simple and sensitive detection of bioactive molecules is becoming one of the research hotspots at home and abroad in recent years. An Electrochemical aptamer sensor (E-AB) can realize high-precision test on specific active molecules in an untreated complex sample system, can better monitor drugs in a living body in real time, and can understand the pharmacology and kinetics of the drugs. E-AB sensors with redox-active molecular modifications perform well when many drugs are applied directly to complex clinical specimens such as undiluted serum, saliva or primary cell lysates. The results of electrochemical performance tests on 13 common redox active molecules respectively manufactured into a simple E-AB sensor include common redox active molecules such as Methylene Blue (MB), Nile blue, anthraquinone or ferrocene, and show that the performance of the MB redox active molecules in the E-AB sensor is the best, and even include multiple cyclic voltammetry tests and repeated stability tests in serum.
MB redox-active molecules are also widely used in biosensors as very classical signal molecules. In order to improve the performance of the E-AB biosensor, the solution of the interference problem is a research direction to optimize the E-AB sensor. In order to realize real-time monitoring of a specific target in a living body, non-specific adsorption in whole blood is reduced by introduction of a self-assembled membrane (refer to fig. 1), thereby improving the performance of the E-AB biosensor. The carbon graphite electrode is electrochemically modified in an acid solution ((pH 0.5, 5.02), and the immobilization and hybridization processes of the carbon graphite electrode are verified by redox electroactive molecules MB, so that the polymer film has better electrochemical signals than the traditional self-assembled monolayer, in order to have higher sensitivity and interference resistance, double indicators are added to reduce the interference of environmental change to a certain extent, the pH value between 6.0 and 7.5 is the optimal condition of enzyme-catalyzed electrochemical reaction, so the development and application of an enzyme biosensor are limited to a great extent by the problem of pH value change, the performance of the MB active molecules in a specific pH value detection system is more stable, but the current signal output can be changed by the change of the pH value due to the participation of protons in the electrochemical reduction process of the MB (refer to figure 2), so the accurate detection in the pH value change system can not be realized, there is a need to develop new redox-active molecules that are stable in physiological environments of different pH and that enable detection.
Disclosure of Invention
In view of the above, the present invention provides an ex-TTF-aptamer conjugate, a method for preparing the same, and an electrochemical sensor prepared using the same.
The invention provides a preparation method of an ex-TTF-aptamer conjugate, which comprises the following steps:
s101, adding a TTF compound and an azide compound into a tert-butyl alcohol solution, then adding N, N-diisopropylethylamine and copper iodide, fully stirring and extracting to obtain an organic phase, washing the organic phase, and then drying, filtering and concentrating to obtain an ex-TTF derivative;
wherein the TTF compound has the structural formula:
the structural formula of the azide compound is as follows:
the structural formula of the ex-TTF derivative is as follows:
s102, adding dimethyl sulfoxide solution into an ex-TTF derivative for dissolving to obtain an ex-TTF derivative solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the ex-TTF derivative solution, performing ultrasonic dissolution, then adding a modified DNA molecule and a sodium bicarbonate solution, performing oscillation reaction, centrifuging after the reaction is finished, taking an upper layer solution, and performing HPLC (high performance liquid chromatography) separation and purification on the upper layer solution to obtain an ex-TTF-aptamer conjugate; the expression of the modified DNA molecule is as follows:
5'-HO-(CH2)6-S-S-(CH2)6-AGACAAGGAAAATCCTTCAATGAAGTGGGTCG-(CH2)7-NH2-3'
or
5'-HO-(CH2)6-S-S-(CH2)6-GGGACTTGGTTTAGGTAATGAGTCCC-(CH2)7-NH2-3'；
The structural formula of the ex-TTF-aptamer conjugate is as follows:
Further, in the step S102, in the HPLC separation and purification process, triethylamine acetate and cellulose nitrate acetate are used for isocratic elution according to the volume ratio of 1: 4; wherein the pH value of the triethylamine acetate is 6.98.
Further, the modified DNA molecule is obtained by amplifying the corresponding modifying group and the DNA molecular chain.
The invention also provides an ex-TTF-aptamer conjugate prepared by the preparation method.
The invention also provides a method for preparing an electrochemical sensor by using the ex-TTF-aptamer conjugate, which comprises the following steps:
s201, adding a tris (2-carboxyethyl) phosphine hydrochloride solution into the ex-TTF-aptamer conjugate, uniformly mixing, and centrifuging to obtain an ex-TTF-aptamer conjugate reduction solution;
s202, adding the ex-TTF-aptamer conjugate reducing solution into a phosphate buffer solution to obtain a mixed solution; and soaking the electrode in the mixed solution, standing at normal temperature, taking out the electrode, and refrigerating the electrode in a 6-mercapto-1-hexanol solution to obtain the electrochemical sensor.
Further, in step S202, the temperature of the cold storage is 4 ℃.
The invention also provides an electrochemical sensor prepared by the preparation method.
The invention firstly uses TTF compound and azide compound to carry out Huisger 1, 3-dipolar cycloaddition reaction to obtain ex-TTF derivative containing carboxyl, then makes the carboxyl of ex-TTF derivative and amino of DNA molecule carry out condensation reaction to obtain ex-TTF-aptamer conjugate, and then uses ex-TTF-aptamer conjugate as redox active molecule to assemble electrochemical sensor.
The technical scheme provided by the invention has the beneficial effects that: the electrochemical sensor assembled by using the ex-TTF-aptamer conjugate provided by the invention as a redox active molecule can effectively realize the detection of small molecule drugs (such as kanamycin and ***e) without the influence of the pH change of a receptor system; the electrochemical test result of the electrochemical sensor obtained by assembling shows that: in PBS solution with pH varying from 4.0 to 8.5, the sensor remained stable for 12 hours after multiple frequency scans (300 scans); also, in artificial sweat systems with pH varying from 4.0 to 8.5, the sensor remains stable after multiple frequency scans, with dissociation constant differences of less than 2-fold within the same order of magnitude.
Drawings
FIG. 1 is a schematic diagram of an aptamer-based electrochemical biosensor.
Fig. 2 is a diagram of MB redox reaction.
FIG. 3 is a schematic of the synthetic route to the ex-TTF-aptamer conjugate.
FIG. 4 is a graph showing the binding reaction of an ex-TTF derivative to a DNA molecule.
FIG. 5 is a drawing of ex-TTF derivatives1H nuclear magnetic resonance characterization (700MHz, DMSO).
FIG. 6 is a drawing of ex-TTF derivatives13C nuclear magnetic resonance characterization (125MHz, DMSO).
FIG. 7 is a mass spectrometric characterization (MALDI-TOF) of ex-TTF derivatives.
FIG. 8 shows the peak time of each substance in the HPLC separation and purification process.
FIG. 9 is a mass spectrometric characterization (MALDI-TOF) of ex-TTF-aptamer conjugates.
FIG. 10 is a graphical representation of the results of testing ex-TTF derivatives and ex-TTF-aptamer conjugates.
FIG. 11 is a graph showing the results of electrochemical measurements of the prepared electrochemical sensor in PBS solutions at different pH values.
Fig. 12 is a graphical representation of the results of electrochemical testing of the manufactured electrochemical sensor in sweat systems at different pH values.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings.
Embodiments of the present invention provide a method for preparing an ex-TTF-aptamer conjugate, comprising the steps of:
step S101, under a nitrogen atmosphere, TTF compound (0.05mg,0.16mmol) and azide compound (0.06mg,0.16mmol) were added to a 5mL t-butanol solution, N-diisopropylethylamine (83mol/L,0.48mmol) was added dropwise, CuI (3mg,0.016mmol) was added as a catalyst, and the mixture was stirred at room temperature for 4h with 5% MeOH/CH, respectively2Cl2Extracting for 3 times to obtain organic phase, washing the organic phase with brine, and then extracting with Na2SO4Drying, filtering and concentrating to obtain the ex-TTF derivative; wherein the volume ratio of the tertiary butanol to the water in the tertiary butanol solution is 1: 1; the TTF compound has the structural formula:
the structural formula of the azide compound is as follows:
the structural formula of the ex-TTF derivative is:
step S102, firstly, weighing 5mg of ex-TTF derivative and dissolving the ex-TTF derivative in 100 mu L of dimethyl sulfoxide solution to obtain an ex-TTF derivative solution; weighing 168mgNaHCO3The solid is dissolved in 10mL deionized water to prepare 0.2mol/L NaHCO3A solution; then respectively weighing 2.9mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 3.0mg of N-hydroxysuccinimide, adding the weighed materials into 10 mu L of the ex-TTF derivative solution, carrying out ultrasonic treatment for 30min to completely dissolve the solid, then adding 4 mu L of the modified DNA molecules with the concentration of 500 mu mol/L and 14 mu L of NaHCO with the concentration of 0.2mol/L to the obtained product3Carrying out ultrasonic treatment on the solution for 30s, and then transferring the solution into a shaking table, wherein the parameters of the shaking table are set to be 800rpm and 24 h; centrifuging after the reaction is finished, taking an upper layer solution (light orange), carrying out HPLC (High Performance Liquid Chromatography) separation and purification on the upper layer solution, isocratic elution, and collecting peak samples with retention time of 27min and 39min to obtain an ex-TTF-aptamer conjugate; wherein the volume ratio of dimethyl sulfoxide to water in the dimethyl sulfoxide solution is 1: 1; the expression of the modified DNA molecule is 5' -HO- (CH)2)6-S-S-(CH2)6-AGACAAGGAAAATCCTTCAATGAAGTGGGTCG-(CH2)7-NH2-3'
Or
5'-HO-(CH2)6-S-S-(CH2)6-GGGACTTGGTTTAGGTAATGAGTCCC-(CH2)7-NH2-3'; the structural formula of the ex-TTF-aptamer conjugate is:
In step S102, the rotating speed of the centrifuge in the process of centrifugation is 13000 rpm; in the HPLC separation and purification process, TEAA (triethylamine acetate) and CAN (cellulose nitrate acetate) are used for isocratic elution, wherein the pH value of the TEAA is 6.98, and the volumes of the TEAA and the CAN are 1: 4.
The synthetic route is shown in FIG. 3, and the reaction scheme of binding of ex-TTF derivatives to DNA molecules is shown in FIG. 4.
The ex-TTF derivative (molecular formula is C) obtained above32H31N3O6S4Molecular weight m. ═ 681.11g/mol) was dissolved in dimethylsulfoxide solution (volume ratio of dimethylsulfoxide to water 20:80) and the molecules were preliminarily characterized by nuclear magnetic resonance (1H and 13C) and mass spectrometry as shown in fig. 5, 6 and 7.
The resulting ex-TTF-aptamer conjugate (5. mu.L) was diluted 20-fold with 90. mu.L deionized water and tested by UV/visible spectrophotometry, with a peak at 260nm, as shown in FIG. 8.
The ex-TTF aptamer conjugates obtained above were subjected to mass spectrometric detection, respectively, as shown in FIG. 9.
Embodiments of the present invention also provide a method for preparing a biosensor using the ex-TTF-aptamer conjugate obtained above, comprising the steps of:
preparing an electrode: respectively cutting polytetrafluoroethylene tubes with the diameter of 2mm by 1.5cm and 7cm, gold wires with the diameter of 2mm by 1.2cm and tungsten wires with the diameter of 2mm by 8cm, sewing with epoxy resin, setting a heat gun at 400 ℃, dipping a proper amount of epoxy resin, heating to connect the gold wires, the tungsten wires and the polytetrafluoroethylene tubes together, and straightening the tungsten wires as much as possible to obtain electrodes;
cleaning the electrode: opening CHI 1040C software, putting the electrode into an electrolytic cell, taking Ag/AgCl as a reference electrode and a platinum wire as a counter electrode; operating CHI 1040C software correctly and connecting the electrodes in the correct order; washing with 0.5M NaOH to remove sulfide remaining on the surface of the electrode, and washing with ultrapure water to remove the NaOH solution remaining on the surface of the electrode; with 0.5M H2SO4Washing to oxidize organic pollutants, simultaneously forming a metal oxide layer and increasing the specific surface area of the working electrode so as to load more aptamers; after washing, 0.5M H of the surface is washed away by deionized water2SO4；
Preparing an electrochemical sensor: taking out the subpackaged ex-TTF-aptamer conjugate from a refrigerator at the temperature of-20 ℃, recovering to room temperature, centrifuging, adding 1 mu L of tris (2-carboxyethyl) phosphine hydrochloride solution (Tcep) with the concentration of 20mM to reduce the ex-TTF-aptamer conjugate, uniformly mixing, centrifuging to obtain ex-TTF-aptamer conjugate reducing solution, and standing at room temperature for 1 h; diluting 2 μ L of ex-TTF-aptamer conjugate reducing solution in 40 μ L of Phosphate Buffer Solution (PBS) to obtain a mixed solution, wherein the concentration of the mixed solution is 2 μ M; washing the cleaned electrode with water, soaking the electrode in the mixed solution, standing at room temperature for 4h, taking out the electrode, transferring the electrode into 1mL of a 20mM 6-mercapto-1-hexanol (MCH) solution, and placing the electrode in a refrigerator at 4 ℃ overnight to obtain the electrochemical sensor; the electrochemical sensor prepared by using DNA (deoxyribonucleic acid) molecules with the nucleotide sequence of AGACAAGGAAAATCCTTCAATGAAGTGGGTCG is marked as an electrochemical sensor A, and a target object corresponding to the electrochemical sensor A is ***e (***e); an electrochemical sensor prepared by using a DNA molecule with the nucleotide sequence of GGGACTTGGTTTAGGTAATGAGTCCC is marked as an electrochemical sensor B, and a target corresponding to the electrochemical sensor B is kanamycin (kanamycin).
And carrying out electrochemical test on the prepared electrochemical sensor A and the electrochemical sensor B, wherein the specific process comprises the following steps:
firstly, in a 10mLPBS solution, scanning for 15 times by using a Square Wave Voltammetry (SWV) macro command, and then superposing the pre-scanning times until the peak value is well overlapped, thereby indicating that the electrode is relatively stable; the area is measured again using Cyclic Voltammetry (CV), and finally the induced current is scanned at multiple frequencies using macros. And sequentially converting the sweat system into sweat systems with different pH values (pH 4-8) for subsequent testing: the stability time in different systems is measured or the current response degree to the corresponding target substances ***e and kanamycin, and the test results are shown in figures 10-12.
FIG. 10A shows the redox property test results of the ex-TTF derivatives in the solution, which shows that the half-wave potential of the ex-TTF derivatives in the aqueous solution with pH of 4-8 is maintained at about 0V, and the redox property ensures the stability of the prepared electrochemical sensor; FIG. 10B is a schematic representation of HPLC separation of ex-TTF-aptamer conjugates; FIG. 10C is a representation of the mass spectrum of ex-TTF-aptamer conjugates, and the results of the mass spectrum show that the ex-TTF derivatives successfully bind to DNA molecules to form ex-TTF-aptamer conjugates, which are the target products.
Fig. 11A is a schematic diagram of the current response of the prepared electrochemical sensor a to the target ***e, fig. 11B is a schematic diagram of the response degree of the prepared electrochemical sensor a to the target ***e in PBS solutions with different pH values, fig. 11C is a schematic diagram of the stability test result of the prepared electrochemical sensor a to the target ***e in PBS solutions with different pH values, fig. 11D is a schematic diagram of the current response of the prepared electrochemical sensor B to the target kanamycin, fig. 11E is a schematic diagram of the response degree of the prepared electrochemical sensor B to the target kanamycin in PBS solutions with different pH values, and fig. 11F is a schematic diagram of the stability test result of the prepared electrochemical sensor B to the target kanamycin in PBS solutions with different pH values. As can be seen from FIG. 11, the prepared electrochemical sensor has better current response to the target substances ***e and kanamycin, is not influenced by pH value change, and finally can have higher current value under different pH conditions and keep continuous stability for 12 hours.
FIG. 12A is a graph showing the stability test results of the electrochemical sensor A prepared in a sweat system with different pH values for the target ***e, FIG. 12B is a graph showing the titration curve test results of the electrochemical sensor A prepared in a sweat system with different pH values for the target ***e, and FIG. 12C is a graph showing the K value of the electrochemical sensor A prepared in a sweat system with different pH values for the target ***edFig. 12D is a schematic diagram of a stability test result of the manufactured electrochemical sensor B on the target kanamycin in a sweat system with different pH values, fig. 12E is a schematic diagram of a titration curve test result of the manufactured electrochemical sensor B on the target kanamycin in the sweat system with different pH values, and fig. 12F is a schematic diagram of a K test result of the manufactured electrochemical sensor B on the target kanamycin in the sweat system with different pH valuesdThe value size test results are shown schematically. As can be seen from FIG. 12, the pH change had no effect on the test signal, i.e., as prepared in this exampleThe electrochemical sensor is not affected by the pH change of the test system.
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> China geological university (Wuhan)
<120> ex-TTF-aptamer conjugate, preparation method thereof and electrochemical sensor prepared by using same
<160> 2
<210> 1
<211> 32
<212> DNA
<213> Artificial Synthesis (Artificial)
<400> 1
AGA CAA GGA AAA TCC TTC AAT GAA GTG GGT CG 32
<210> 2
<211> 26
<212> DNA
<213> Artificial Synthesis (Artificial)
<400> 2
GGG ACT TGG TTT AGG TAA TGA GTC CC 26
Claims (6)
1. A method of preparing an ex-TTF-aptamer conjugate, comprising the steps of:
s101, adding a TTF compound and an azide compound into a tert-butyl alcohol solution, then adding N, N-diisopropylethylamine and copper iodide, fully stirring and extracting to obtain an organic phase, washing the organic phase, and then drying, filtering and concentrating to obtain an ex-TTF derivative; the TTF compound has the structural formula:
the structural formula of the azide compound is as follows:
the structural formula of the ex-TTF derivative is as follows:
s102, adding dimethyl sulfoxide solution into an ex-TTF derivative for dissolving to obtain an ex-TTF derivative solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the ex-TTF derivative solution, performing ultrasonic dissolution, then adding a modified DNA molecule and a sodium bicarbonate solution, performing oscillation reaction, centrifuging after the reaction is finished, taking an upper layer solution, and performing HPLC (high performance liquid chromatography) separation and purification on the upper layer solution to obtain an ex-TTF-aptamer conjugate; the structural formula of the ex-TTF-aptamer conjugate is as follows:
wherein R1 represents a DNA having a nucleotide sequence of AGACAAGGAAAATCCTTCAATGAAGTGGGTCG or a DNA having a nucleotide sequence of GGGACTTGGTTTAGGTAATGAGTCCC.
2. The method of preparing ex-TTF-aptamer conjugate according to claim 1, wherein in step S102, the separation and purification by HPLC is performed by isocratic elution using triethylamine acetate and nitrocellulose acetate at a volume ratio of 1: 4.
3. An ex-TTF-aptamer conjugate prepared by the preparation method of any one of claims 1 to 2.
4. A method for preparing an electrochemical sensor, comprising the steps of:
s201, adding a tris (2-carboxyethyl) phosphine hydrochloride solution into the ex-TTF-aptamer conjugate, uniformly mixing, and centrifuging to obtain an ex-TTF-aptamer conjugate reduction solution; wherein the ex-TTF-aptamer conjugate is prepared by the preparation method according to any one of claims 1 to 2;
s202, adding the ex-TTF-aptamer conjugate reducing solution into a phosphate buffer solution to obtain a mixed solution; and soaking the electrode in the mixed solution, standing at normal temperature, taking out the electrode, and refrigerating the electrode in a 6-mercapto-1-hexanol solution to obtain the electrochemical sensor.
5. The method for manufacturing an electrochemical sensor according to claim 4, wherein the temperature for cold storage is 4 ℃ in step S202.
6. An electrochemical sensor produced by the production method according to any one of claims 4 or 5.
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