CN114034748A

CN114034748A - Electrochemical aptamer sensor for detecting insulin and preparation and use methods thereof

Info

Publication number: CN114034748A
Application number: CN202111315217.8A
Authority: CN
Inventors: 常竹; 刘瑾瑾; 朱碧澄; 朱旭; 郝远强; 张银堂; 徐茂田
Original assignee: Shangqiu Normal University
Current assignee: Shangqiu Normal University
Priority date: 2021-11-08
Filing date: 2021-11-08
Publication date: 2022-02-11

Abstract

The invention belongs to the field of sensors, relates to a nano-material modified graphene printing electrode and an aptamer sensor, and particularly relates to an electrochemical aptamer sensor for detecting insulin and a preparation method and a use method thereof. By introducing the aptamer segment Apt, when the target substance insulin exists, the Apt is combined on the surface of the electrode to form a compound, the function of a complete aptamer is shown, and the distance between the gold nanoparticles and the surface of the electrode is changed before and after the target molecule is identified, so that the change of an electrochemical signal is obtained, the detection limit of a laser printing graphene electrode is 22.7fM, and the detection limit of a glassy carbon electrode is 9.8 fM.

Description

Electrochemical aptamer sensor for detecting insulin and preparation and use methods thereof

Technical Field

The invention belongs to the field of sensors, relates to a nano-material modified graphene printing electrode and an aptamer sensor, and particularly relates to an electrochemical aptamer sensor for detecting insulin and a preparation method and a use method thereof.

Background

Insulin is used as a hormone for regulating blood sugar of a human body, and participates in regulating glucose metabolism, maintains blood sugar balance and promotes glucose uptake. Insulin secretion disorders are important causes of diabetes, cancer and neurodegenerative diseases. Diabetes is one of the most common chronic diseases in almost all countries, and the number of diabetics is increasing with the change of life style. Diabetics are often associated with hyperglycemia and can cause serious complications such as renal failure, heart disease, stroke, and the like. The concentration of the insulin can be used as an index for early detection of diabetes and the existence of complications by monitoring the concentration of the insulin, so that the establishment of a sensitive, selective and simple insulin detection method has important significance for diagnosis of the diabetes and prevention of the related complications.

The current methods for detecting insulin are diverse, such as surface enhanced raman scattering, high performance liquid chromatography, electrochemistry, fluorescence, and immunoassay. Among them, electrochemistry is widely concerned by people due to the advantages of simple method, easy miniaturization, fast response speed, high sensitivity, etc. Electrochemical immunosensors and electrochemical aptamer sensors are two major electrochemical biosensors, and have been widely used to detect various disease markers. However, electrochemical immunosensors have disadvantages of high cost, unstable antibodies, and the like. It is therefore necessary to develop an electrochemical aptamer sensor with high sensitivity and selectivity to meet the practical needs of insulin detection.

The aptamer is an oligonucleotide sequence (DNA or RNA) which can be specifically combined with a corresponding target molecule, is obtained by screening an in vitro random oligonucleotide library through an exponential enrichment ligand phylogeny technology (SELEX), and has high affinity and high specificity for the target molecule. The target molecules of the aptamer are very wide, and can be specifically combined with biological macromolecules such as proteins and the like, small molecules such as medicines and metal ions, and even cells and bacteria. After the aptamer is combined with the target molecule, conformation change can occur, which causes the distance between the electrochemical indicator and the surface of the electrode to change, and further causes different electrochemical readings.

In the litting 'construction and application of the metal nanoparticle-graphene composite material-based electrochemical sensor' (Shanxi university, 2016), the insulin aptamer is fixed on an AuNPs/O-GNs modified Glassy Carbon Electrode (GCE) through an Au-S bond, so that the detection of insulin is realized, and the detection range is 1.0 multiplied by 10^-14- 5.0×10^-10mol/L, detection limit is 6.0 multiplied by 10^-5mol/L. GCE has the advantages of good conductivity and high chemical stability, is widely used in electroanalytical chemistry, but has the problems of large volume and no flexibility. Compared with GCE, the laser printing graphene electrode (LSGE) has the advantages of large specific surface area, high conductivity, low cost, easiness in miniaturization and the like. Meanwhile, the LSGE can easily customize geometric figures, dimensions and layout required by experiments, and can be directly used as an electrode without further modification. These characteristics make LSGE a great prospect for use as a disposable electrode in portable electrochemical sensing devices.

Disclosure of Invention

The invention provides an electrochemical aptamer sensor for detecting insulin and a preparation and use method thereof, wherein a nucleic acid aptamer fragment Apt is introduced, when target insulin exists, the Apt is combined on the surface of an electrode to form a compound to show the function of a complete aptamer, and the distance between gold nanoparticles and the surface of the electrode is changed before and after target molecules are identified, so that the change of an electrochemical signal is obtained, the LSGE detection limit is 22.7fM, and the GCE detection limit is 9.8 fM.

The technical scheme of the invention is realized as follows:

a preparation method of an electrochemical aptamer sensor for detecting insulin comprises the following steps:

(1) preparation of AuNPs-Apt probe: adding HAuCl₄Heating the aqueous solution to boiling under stirring, then adding a sodium citrate solution, refluxing and boiling, cooling the obtained AuNPs solution to room temperature, then adding an Apt solution, and incubating at low temperature to obtain an AuNPs-Apt probe solution;

(2) preparing an AuNPs modified electrode: polishing, grinding, cleaning and drying the electrode in sequence, and then using HAuCl₄Preparing electrodes/AuNPs from the solution by an it method;

(3) preparation of electrochemical aptamer sensor: dropping the Apt solution on the electrode/AuNPs, and incubating with a confining liquid to obtain the electrode/AuNPs-Apt.

The sequence of Apt in the step (1) is 5 '-SH-GGT GGT GGG GGG GGT TGG TAG GGT GTC TTC-3', HAuCl₄The mass volume ratio concentration of the water solution is 0.01-0.02 percent, the mass volume ratio concentration of the sodium citrate solution is 2-8 percent, and HAuCl₄The volume ratio of the aqueous solution to the sodium citrate solution is (50-100): 1, boiling for 15-20min, wherein the volume ratio of the AuNPs solution to the Apt solution is (1-2): 19, wherein the concentration of the Apt solution is 50-100 mu M, the incubation temperature is 2-8 ℃, and the incubation time is 16-24 h.

In the step (2), the electrode is LSGE or GCE, and the polishing powder is Al₂O₃Polishing powder of Al₂O₃The grain diameter of the polishing powder is 0.3 and 0.05 mu m, and HAuCl₄The concentration of the aqueous solution is 35-45mM, the potential of the it method is-0.18-0.22V, and the time is 160-200 s.

In the step (3), the concentration of the Apt solution is 4-6 mu M, the electrode is LSGE or GCE, the LSGE confining liquid is bovine serum albumin solution (BSA), the GCE confining liquid is mercaptohexanol solution (MCH), wherein the mass volume ratio concentration of the BSA solution is 1-2%, and the concentration of the MCH solution is 8-10 mM.

The electrochemical aptamer sensor for detecting the insulin, which is prepared by the method, comprises an aptamer Apt for specifically identifying the insulin, exonuclease I for hydrolyzing redundant Apt which is not combined with the insulin on the surface of an electrode, a gold nanoparticle-labeled probe solution AuNPs-Apt and an AuNPs-modified electrode; wherein Apt is modified on the surface of the electrode/AuNPs through Au-S bonds, and the electrode/AuNPs-Apt is formed after the nonspecific active sites are sealed by the sealing liquid.

The use method of the electrochemical aptamer sensor for detecting insulin comprises the following steps:

a. and soaking the electrode/AuNPs-Apt sensor in a standard solution of insulin, an exonuclease I solution and an AuNPs-Apt probe solution in sequence, incubating, and washing to obtain the electrode/AuNPs-Apt/insulin/AuNPs-Apt electrochemical aptamer sensor.

b. Soaking the electrochemical aptamer sensor in Tris-HCl buffer solution containing Methylene Blue (MB), adjusting the pH value, cleaning and using the electrochemical aptamer sensor as a working electrode, carrying out electrochemical DPV detection in the Tris-HCl buffer solution, and drawing a working curve according to the relation between the obtained peak current and the standard solution of insulin;

c. and (c) detecting the concentration of the original sample to be detected, adding insulin into the sample to be detected, detecting according to the operation a and the operation b, and substituting the detected current signal into the working curve obtained in the step b to obtain the concentration of the insulin in the sample to be detected.

In the step a, the electrode is LSGE or GCE, LSGE/AuNPs-Apt is soaked in a series of standard solutions of insulin with the concentration of 0.05pM-1 mu M, GCE/AuNPs-Apt is soaked in a series of standard solutions of insulin with the concentration of 0.02pM-5.0 mu M, the incubation time in the insulin solution is 40-60min, the concentration of exonuclease I solution is 1.25-1.50U/mu L, the incubation temperature is 36-38 ℃, the incubation time is 2.5-3.0h, and the incubation time in the probe solution is 15-25 min.

In the step b, the concentration of the MB solution is 30-50 mu M, the concentration of the Tris-HCl buffer solution is 0.95-0.15M, the pH value of the Tris-HCl buffer solution is 7.3-7.5, and the soaking time is 10-20 min.

The detection principle is as follows: soaking the electrode/AuNPs-Apt in PBS (phosphate buffer solution) containing insulin with different concentrations for 40-60min at room temperature, then soaking the obtained electrode/AuNPs-Apt/insulin electrochemical aptamer sensor in 1.25-1.50U/microliter of exonuclease I solution, incubating for 2.5-3.0h at 36-38 ℃, then taking out and fully washing with ultrapure water, in the process, the exonuclease I hydrolyzes the redundant Apt which is not combined with the insulin on the surface of the electrode, changing the sensor into a signal enhancement type, and then incubating the sensor and the AuNPs-Apt probe solution for 15-25min at room temperature, wherein the formed sandwich structure can be combined with more MB molecules, thereby generating an amplified electrochemical signal. And finally, soaking the constructed sensor in a Tris-HCl buffer solution with the pH value of 7.3-7.5 and containing 30-50 mu M MB for 10-20min, taking out and fully washing the sensor with ultrapure water, carrying out DPV scanning in the Tris-HCl buffer solution with the pH value of 7.3-7.5 by using differential pulse voltammetry DPV, wherein the pulse amplitude is 45-55mV, the pulse width is 45-55ms, the DPV scanning potential is from-0.6V to +0.1V, so that the MB is reduced to generate an electrochemical signal, the DPV peak current at about-0.2V in GCE is regarded as an analysis signal, and the DPV peak current at about-0.26V in LSGE is regarded as an analysis signal.

The invention has the following beneficial effects:

1. the invention relates to the technical field of manufacturing of disposable portable sensors and aptamer sensing, in particular to an electrochemical aptamer sensor for detecting insulin and a preparation method and a use method thereof. The insulin electrochemical sensor is realized by Apt, exonuclease I and a gold nanoparticle labeled probe AuNPs-Apt. And electrodepositing the gold nanoparticles on the LSGE by a potentiostatic method, namely LSGE/AuNPs, wherein Apt is modified on the LSGE/AuNPs through Au-S bonds, and blocking nonspecific active sites by BSA solution to form the LSGE/AuNPs-Apt sensor. After the target substance insulin is identified, exonuclease I is introduced to catalyze and hydrolyze the excessive aptamer Apt which is not combined with the insulin, and the excessive aptamer Apt is converted into a more sensitive signal enhancement sensor. The AuNPs-Apt probe can specifically recognize insulin on the surface of the electrode, and a formed sandwich structure can be combined with more MB molecules, so that an amplified electrochemical signal is generated.

2. According to the invention, the LSGE is adopted, and the graphene-like structure on the surface of the electrode has the advantages of excellent conductivity, low cost, large specific surface area, good mechanical property, easiness in miniaturization and the like. In addition, the LSGE can easily customize geometric figures, sizes and layouts required by experiments, integrates a working electrode, a reference electrode and a counter electrode in the manufacturing process, and can be directly used as electrodes without further modification.

3. The invention firstly carries out performance detection on the traditional commercial research GCE, the detection range is 0.1pM-1.0 mu M, the detection limit is 9.8fM, and the sensing strategy is successfully transferred to the disposable portable LSGE, the detection range is 0.1pM-0.1 mu M, and the detection limit is 22.7fM, thus having potential broad prospect in the aspect of realizing portable sensing equipment for detecting insulin.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is an SEM topography of the LSGE/AuNPs on the surface of a printed electrode in example 2.

FIG. 2 is a diagram of the process for preparing the GCE sensor of example 1.

FIG. 3 shows 10% denaturing polyacrylamide gel electrophoresis in the examples.

FIG. 4 is a diagram of the AC impedance of the GCE in an embodiment.

FIG. 5 is a graph showing the operation of the GCE sensor according to the embodiment.

Fig. 6 is a schematic diagram of the LSGE fabrication and insulin sensor design in example 2.

Fig. 7 is a graph of the ac impedance of LSGE in an example of an effect.

Fig. 8 is a graph showing the operation of the LSGE sensor according to the embodiment.

Fig. 9 is a diagram of the repeatability test of the LSGE sensor in the example of the embodiment.

Fig. 10 is a stability test chart of the LSGE sensor in the example of the embodiment.

Fig. 11 is a diagram of the selectivity test of the LSGE sensor in the embodiment.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

(1) preparation of AuNPs-Apt Probe

50mL of 0.01% (w/v) HAuCl₄The aqueous solution was heated to boiling with stirring, then 1 mL of 2% (w/v) sodium citrate solution was rapidly added, and then the mixed solution was boiled under reflux for 15min, the solution changed in color from initially pale yellow to purple and finally to wine red, indicating the synthesis of AuNPs. The AuNPs solution was cooled to room temperature, transferred to a brown glass bottle, and stored in a refrigerator at 4 ℃ for further use. Adding 30 mu L of 100 mu M Apt solution into 570 mu L of AuNPs solution, incubating for 16h at 4 ℃, centrifugally washing for three times, re-dispersing into 600 mu L of ultrapure water to obtain AuNPs-Apt probe solution, and storing in a refrigerator at 4 ℃. The sequence recognizing the insulin aptamer Apt is: 5 '-SH-GGT GGT GGG GGG GGT TGG TAG GGT GTC TTC-3';

(2) preparation of AuNPs modified GCE

a. The bare GCE with a diameter of 3.0mm is sequentially coated with 0.3 and 0.05 mu m of Al₂O₃Polishing the electrode surface to obtain mirror surface, and cleaning with ultra-water to remove residual Al₂O₃Drying the powder at room temperature;

b. with 40mM HAuCl₄The aqueous solution is electrolyte, gold nanoparticles are electrodeposited on the surface of the GCE by an it method (potential: 0.2V, time: 180 s) to obtain GCE/AuNPs;

(3) preparation of insulin sensor

The fabrication process of the sensor is shown in fig. 2.

a. mu.L of 5. mu.M Apt solution was dropped onto GCE/AuNPs, incubated for 5h at 4 ℃ and then the unbound gold sites were blocked by incubation with 10mM MCH for 40min, resulting in an insulin sensor electrode, recorded as GCE/AuNPs-Apt.

b. And (b) incubating the electrode prepared in the step (a) with insulin with different concentrations for 40min, then incubating with exonuclease I solution for 3h at 37 ℃, then adding AuNPs-Apt probe solution, incubating for 20min, and finally rinsing with ultrapure water for 3 times to obtain the insulin electrochemical aptamer sensor.

Example 2

(1) preparation of AuNPs-Apt Probe

50mL of 0.01% (w/v) HAuCl₄The aqueous solution was heated to boiling with stirring, then 1 mL of 2% (w/v) sodium citrate solution was rapidly added, and then the mixed solution was boiled under reflux for 15min, the solution changed in color from initially pale yellow to purple and finally to wine red, indicating the synthesis of AuNPs. The AuNPs solution was cooled to room temperature, transferred to a brown glass bottle, and stored in a refrigerator at 4 ℃ for further use. Adding 30 mu L of 100 mu M Apt solution into 570 mu L of AuNPs solution, incubating for 16h at 4 ℃, centrifugally washing for three times, re-dispersing into 600 mu L of ultrapure water to obtain AuNPs-Apt probe solution, and storing in a refrigerator at 4 ℃. The sequence for identifying the insulin aptamer Apt is 5 '-SH-GGT GGT GGG GGG GGT TGG TAG GGT GTC TTC-3';

(2) preparation of AuNPs modified LSGE

Reference is made to summary (2) for the preparation of LSGE. The LSGE was immersed in 40mM HAuCl₄In the water solution, depositing gold nanoparticles for 180s by an it method at a potential of-0.2V to prepare LSGE/AuNPs for later use;

the morphology of the LSGE/AuNPs graphene electrode material synthesized by the method is shown in figure 1. Fig. 1 shows a thin layer, surface multi-fold structure of the graphene electrode, while it can be seen that AuNPs are uniformly distributed on the graphene thin layer, and exhibit a network distribution. The LSGE/AuNPs composite graphene is used as a substrate electrode, so that on one hand, the electron transfer rate (higher than that of simple LSGE) can be improved; on the other hand, the composite material has higher electrochemical active surface area and can bind more insulin aptamers.

(3) Preparation of the sensor

a. The insulin electrochemical aptamer sensor design is shown in figure 6. The LSGE/AuNPs were immersed in 5. mu.M Apt solution for 10min, incubated at 4 ℃ for 5h, and then incubated with 1% BSA solution for 40min to block unbound gold sites, yielding insulin sensor electrodes, recorded as LSGE/AuNPs-Apt.

b. And (b) incubating the electrode manufactured in the step a with insulin with different concentrations for 40min, then incubating with exonuclease I solution for 3h at 37 ℃, then adding AuNPs-Apt probe solution, acting for 20min, and finally rinsing with ultrapure water for 3 times to obtain the insulin electrochemical aptamer sensor.

The embodiment has the following effects: performance detection of biosensors

(1) Verification of cleavage of insulin aptamer by exonuclease I in solution

The catalytic hydrolysis capacity of the exonuclease I on the insulin aptamer is verified by using 10% deformed polyacrylamide gel electrophoresis (PAGE). After incubation of 5. mu.M of aptamer (PBS buffer) with different concentrations of insulin from 0.1. mu.M to 100. mu.M, respectively, and hydrolysis with 0.1U/. mu.L exonuclease I at 37 ℃ in the presence of different concentrations of insulin as shown in FIG. 3, lanes 3-6 indicate that the aptamer is hydrolyzed and that the amount of hydrolyzed aptamer increases as the concentration of insulin decreases. Indicating that the aptamer was protected from exonuclease I catalyzed hydrolysis when bound to insulin to form a complex.

(2) Electrochemical impedance detection of GCE

Experiment the electrochemical AC impedance EIS was used to characterize the electrode preparation process at 5mm [ Fe (CN) ] with 0.1M KCl₆]^3-/[Fe(CN)₆]^4-Each step of assembly was verified in solution as shown in figure 4. When AuNPs are decorated on bare GCE, the impedance value is reduced from 308.5Ohm (curve a) to 23.02Ohm (curve b) because GCE/AuNPs have stronger conductivity and higher specific surface area than bare electrodes. After assembly of the negatively charged aptamer Apt onto GCE/AuNPs, the Apt pairs [ Fe (CN) ] in solution₆]^3-/[Fe(CN)₆]^4-The repulsion of the electrons at the electrode surface is hindered and the resistance increases to 209.1Ohm (curve c). After the electrode was blocked with a poorly conducting MCH solution, the impedance value was further increased to 645.9Ohm (curve d). Following incubation with the target insulin, the impedance value was again increased to 1610.1Ohm (curve e). The aptamer, unbound to the target, was then hydrolyzed by exonuclease I, and the impedance value was reduced to 1307.3Ohm (curve f). Finally, after the AuNPs-Apt probe is combined with the insulin on the surface of the electrode, the impedance value is increased to 1834.2Ohm (curve g), which proves that the AuNPs load more phosphate skeletons containing negative charges and block the transfer process of electrons. The impedance map may indicate successful construction of the aptamer sensor.

(3) Detection curve of insulin in GCE

According to example 1, the electrochemical signals of the aptamer sensor of the invention in different concentrations of insulin are detected, and a standard curve graph of the peak current change value and the insulin level is drawn, wherein the specific detection steps are as follows:

1) the constructed GCE/AuNPs-Apt sensor was immersed in a series of standard solutions of insulin at concentrations of (0.02pM, 0.05pM, 0.1pM, 1pM, 10pM, 100pM, 0.1nM, 1nM, 10nM, 100nM, 0.1 μ M, 1.0 μ M, 2.0 μ M, 5.0 μ M), incubated at room temperature for 40min, taken out and washed with ultrapure water 3 times, the sensor was immersed in a 1.25U/μ L exonuclease i solution, incubated at 37 ℃ for 3h, taken out and washed with ultrapure water 3 times, then the sensor was incubated with an AuNPs-Apt probe solution at room temperature for 20min and washed with ultrapure water 3 times, and finally the sensor was immersed in a 0.1M-Tris-HCl buffer solution (pH 7.4) containing 50 μ M MB for 10min and then taken out and washed with ultrapure water 3 times.

2) Connecting the sensor to an electrochemical instrumentation device: electrochemical DPV detection was performed in 0.1M Tris-HCl buffer at pH 7.4, with a scanning potential of-0.6V to +0.1V, a pulse amplitude of 50mV, and a pulse width of 50 ms. Based on the DPV peak current electrochemical signal at a potential of-0.2V, a standard curve was established based on the DPV signal and the corresponding insulin concentration, as shown in FIG. 5A.

According to the concentration and the concentration of insulinThe corresponding current signal is plotted as a linear correlation between the insulin concentration and Δ I in FIG. 5B, and the linear regression equation is-0.86 l g [ insulin]1.70, (0.1pM to 1. mu.M), the linear correlation is R₂The limit of detection was 9.8fM (3Sb/m, Sb is the standard deviation of the blank signal and m is the slope of the standard curve) at 0.9969.

(4) LSGE fabrication

A schematic diagram of the process for making the LSGE and designing the sensor according to example 2 is shown in fig. 6.

(5) Electrochemical impedance detection of LSGE

According to example 2, the experiment used an electrochemical AC impedance test to characterize the preparation of the electrodes, with the sensing interface at 5mM [ Fe (CN)₆]^3-/[Fe(CN)₆]^4-Tested in solution (containing 0.1M KCl) as shown in FIG. 7. The impedance value of bare LSGE (curve a) is small, indicating that the transfer of electrons at the graphene printing electrode surface is not impeded. When the AuNPs of the gold nanoparticles are modified on the electrode (curve b), the impedance value is lower, and for the reason, the AuNPs have strong conductivity and can accelerate the transfer of electrons on the surface of the electrode. After LSGE/AuNPs bind Apt (curve c), the impedance value increases because Apt is a negatively charged nucleic acid backbone, for [ Fe (CN) ] in solution₆]^3-/[Fe(CN)₆]^4-The repulsion hinders the transfer of electrons at the electrode surface. After blocking the electrodes with BSA solution (curve d), the impedance value increases again, followed by incubation of the electrodes with insulin (curve e), which further increases because BSA and insulin are both proteins and are poorly conductive. The electrode was then treated with exonuclease I, which reduced the impedance value as a result of the exonuclease I hydrolyzing the aptamer that did not bind insulin (curve f). And finally, an AuNPs-Apt probe (curve g) is introduced, and because a large amount of Apt containing a phosphate skeleton with negative charges is loaded on AuNPs, the transfer process of electrons is blocked, and the impedance value of the AuNPs-Apt probe is increased. The impedance map may indicate successful construction of the aptamer sensor.

(6) Drawing of standard curve

The electrochemical aptamer sensor detects electrochemical signals in insulin with different concentrations, and draws a standard curve graph of a peak current change value and the insulin concentration, wherein the specific detection steps are as follows:

1) the LSGE/AuNPs-Apt sensor was immersed in a series of standard solutions of insulin at concentrations (0.05pM, 0.1pM, 1pM, 10pM, 100pM, 0.1nM, 1nM, 10nM, 100nM, 0.1 μ M, 0.5 μ M, 1 μ M), taken out after incubation at room temperature for 40min and washed with ultrapure water 3 times, immersed in 1.25U/μ L exonuclease i solution, taken out after incubation at 37 ℃ for 3h and washed with ultrapure water 3 times, followed by incubating the sensor with AuNPs-Apt probe solution at room temperature for 20min and washing with ultrapure water 3 times, and finally, after immersing the sensor in 0.1M Tris-HCl (pH 7.4) containing 50 μ M MB 10min, taken out and washed with ultrapure water 3 times.

2) Connecting the sensor to an electrochemical instrumentation device: electrochemical DPV detection was performed in 0.1M Tris-HCl buffer at pH 7.4, with a scanning potential of-0.6V to +0.1V, a pulse amplitude of 50mV, and a pulse width of 50 ms. Based on the DPV peak current electrochemical signal at a potential of-0.26V, a standard curve was established based on the DPV signal and the corresponding insulin concentration, as shown in FIG. 8A.

From the insulin concentration and the corresponding current signal, a linear correlation between the insulin concentration and Δ I is plotted as shown in fig. 8B, and the linear regression equation is-2.28 l g [ insulin]-3.10, (0.1pM to 0.1. mu.M), linear correlation is R₂The detection limit was 22.7fM (3Sb/m) at 0.9941.

(7) Reproducibility test

The relative standard deviation between different electrodes was 1.80% for 0.1 μ M of insulin measured under the same experimental conditions using 6 different LSGEs, as shown in fig. 9, indicating that the electrodes prepared have good reproducibility.

(8) Stability test

The prepared electrochemical aptamer sensor was placed at 4 ℃ and 0.1 μ M insulin was detected after 5, 10, 15 and 20 days, respectively, as shown in fig. 10, and the signal after 20 days of sensor storage was 90.93% of the signal of the first day, indicating that the prepared electrochemical aptamer sensor had good stability.

(9) Selective testing

The selectivity of the electrochemical aptamer sensor was studied and L-cysteine, glucose, uric acid and lysozyme were selected for the interference test experiments, as shown in fig. 11. The LSGE was incubated with a mixture containing 1 μ M L-cysteine, glucose, uric acid and lysozyme (column a), the change in DPV peak current was 3.59 μ A, and the change in current signal was 14.77 μ A when 0.1 μ M insulin was incubated (column b), and when insulin was mixed with the interferent (column c), the difference in signal obtained by electrode detection was only slightly greater than that of insulin alone (17.43 μ A vs 14.77 μ A), indicating that the sensor prepared according to the invention had better selectivity.

Application example: detection of insulin in serum samples

The LSGE/AuNPs-Apt sensor detects insulin in real samples evaluated by the sensing property of detecting insulin concentration in human serum samples. First, the concentration of insulin in the original human serum samples was measured directly at 0.04nM (relative standard deviation RSD ═ 4.25%) due to the low concentration of insulin in serum. Insulin at 0.10pM, 0.50pM and 1.00pM was added to the original serum sample using standard addition methods (insulin to serum sample volume ratio 1: 1). The DPV signal was recorded before and after the addition of different concentrations of insulin. The results showed that the spiked concentrations were 0.10pM, 0.52pM and 1.03pM, respectively, and the spiked recoveries were 100.0%, 103.7% and 102.9%, respectively. This result indicates that the LSGE/AuNPs-Apt sensor of the present invention is a promising method for detecting insulin in real samples.

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.

Claims

1. A preparation method of an electrochemical aptamer sensor for detecting insulin is characterized by comprising the following steps:

(1) preparation of AuNPs-Apt probe: adding HAuCl₄Heating the aqueous solution to boiling under stirring, adding sodium citrate solutionBoiling under reflux, cooling the AuNPs solution to room temperature, then adding Apt solution, and incubating at low temperature to obtain AuNPs-Apt probe solution;

(2) preparing an AuNPs modified electrode: polishing, grinding, cleaning and drying the electrode in sequence, and then using HAuCl₄Preparing electrodes/AuNPs from the aqueous solution by an it method;

2. The method of claim 1, wherein: the sequence of Apt in the step (1) is 5 '-SH-GGT GGT GGG GGG GGT TGG TAG GGT GTC TTC-3', HAuCl₄The mass volume ratio concentration of the water solution is 0.01-0.02 percent, the mass volume ratio concentration of the sodium citrate solution is 2-8 percent, and HAuCl₄The volume ratio of the aqueous solution to the sodium citrate solution is (50-100): 1, boiling for 15-20min, wherein the volume ratio of the AuNPs solution to the Apt solution is (1-2): 19, wherein the concentration of the Apt solution is 50-100 mu M, the incubation temperature is 2-8 ℃, and the incubation time is 16-24 h.

3. The method of claim 1, wherein: in the step (2), the electrode is a laser printing graphene electrode or a glassy carbon electrode, and the polishing powder is Al₂O₃Polishing powder of Al₂O₃The grain diameter of the polishing powder is 0.3 and 0.05 mu m, and HAuCl₄The concentration of the aqueous solution is 35-45mM, the potential of the it method is-0.18-0.22V, and the time is 160-200 s.

4. The method of claim 1, wherein: in the step (3), the Apt solution concentration is 4-6 mu M, the electrode is a laser printing graphene electrode or a glassy carbon electrode, the laser printing graphene electrode sealing solution is a bovine serum albumin solution, the glassy carbon electrode sealing solution is a mercaptohexanol solution, wherein the mass volume ratio concentration of the bovine serum albumin solution is 1% -2%, and the concentration of the mercaptohexanol solution is 8-10 mM.

5. An electrochemical aptamer sensor for detecting insulin prepared by the method of claim 1, wherein: the kit comprises an aptamer Apt for specifically identifying insulin, exonuclease I for hydrolyzing redundant Apt which is not combined with insulin on the surface of an electrode, a probe solution AuNPs-Apt marked by gold nanoparticles and an electrode modified by AuNPs; wherein Apt is modified on the surface of the electrode/AuNPs through Au-S bonds, and the electrode/AuNPs-Apt is formed after the nonspecific active sites are sealed by the sealing liquid.

6. The method for using the electrochemical aptamer sensor for detecting insulin according to claim 5, wherein the steps are as follows:

b. Soaking the electrode/AuNPs-Apt/insulin/AuNPs-Apt electrochemical aptamer sensor in Tris-HCl buffer solution containing methylene blue solution, adjusting pH, cleaning and using the sensor as a working electrode, carrying out electrochemical DPV detection in the Tris-HCl buffer solution, and drawing a working curve according to the relation between the obtained peak current and the standard solution of insulin;

7. The method for using an electrochemical aptamer sensor for detecting insulin according to claim 6, wherein: in the step a, the electrode is a laser printing graphene electrode or a glassy carbon electrode, the laser printing graphene electrode/AuNPs-Apt is soaked in a series of standard solutions of insulin with the concentration of 0.05pM-1 mu M, the glassy carbon electrode/AuNPs-Apt is soaked in a series of standard solutions of insulin with the concentration of 0.02pM-5.0 mu M, the incubation time in the insulin solution is 40-60min, the concentration of exonuclease I solution is 1.25-1.50U/mu L, the incubation temperature is 36-38 ℃, the incubation time is 2.5-3.0h, and the incubation time in the probe solution is 15-25 min.

8. The method for using an electrochemical aptamer sensor for detecting insulin according to claim 6, wherein: in the step b, the concentration of the methylene blue solution is 30-50 mu M, the concentration of the Tris-HCl buffer solution is 0.95-0.15M, the pH value of the Tris-HCl buffer solution is 7.3-7.5, and the soaking time is 10-20 min.