An electrochemical aptamer sensor for detecting insulin and its preparation and use method

Abstract

The present invention belongs to the field of sensors, and relates to nanomaterial-modified graphene printed electrodes and aptamer sensors, and in particular refers to an electrochemical aptamer sensor for detecting insulin and its preparation and use methods. By introducing the nucleic acid aptamer fragment Apt, when the target insulin is present, Apt combines into a complex on the electrode surface, showing the function of a complete aptamer, and the distance between the gold nanoparticles and the electrode surface changes before and after the target molecule is recognized, thereby obtaining a change in the electrochemical signal. The detection limit of the laser-printed graphene electrode is 22.7 fM, and the detection limit of the glassy carbon electrode is 9.8 fM. The sensor design scheme of the present application is constructed on a disposable, portable, low-cost laser-printed graphene electrode, which will facilitate the commercialization of the sensor and has broad prospects in realizing portable sensing devices for detecting insulin.

Description

Technical Field

[0001] The invention belongs to the field of sensors, and relates to nanomaterial-modified graphene printed electrodes and aptamer sensors, and in particular to an electrochemical aptamer sensor for detecting insulin, and a preparation method and use method thereof. Background Art

[0002] Insulin, as a hormone that regulates blood sugar in the human body, participates in regulating sugar metabolism, maintaining blood sugar balance, and promoting glucose uptake. Impaired insulin secretion is an important cause of diabetes, cancer, and neurodegenerative diseases. Diabetes is one of the most common chronic diseases in almost all countries, and the number of diabetic patients is increasing with the change of lifestyle. Diabetic patients are usually accompanied by high blood sugar, which can lead to serious complications such as kidney failure, heart disease, stroke, etc. Monitoring the concentration of insulin can be used as an indicator for early detection of diabetes and the presence or absence of complications. Therefore, establishing a sensitive, selective, and simple insulin detection method is of great significance for the diagnosis of diabetes and the prevention of related complications.

[0003] There are many methods for detecting insulin, such as surface enhanced Raman scattering, high performance liquid chromatography, electrochemistry, fluorescence and immunoassay. Among them, electrochemistry has attracted widespread attention due to its advantages such as simple method, easy miniaturization, fast response speed and high sensitivity. Electrochemical immunosensors and electrochemical aptamer sensors are two main electrochemical biosensors, which have been widely used to detect various disease markers. However, electrochemical immunosensors have disadvantages such as high cost and unstable antibodies. Therefore, it is necessary to develop an electrochemical aptamer sensor with high sensitivity and selectivity to meet the actual needs of insulin detection.

[0004] An aptamer is an oligonucleotide sequence (DNA or RNA) that can specifically bind to a corresponding target molecule. It is screened from an in vitro random oligonucleotide library by systematic evolution of ligands by exponential enrichment (SELEX) and has high affinity and specificity for the target molecule. The target molecules of aptamers are very broad, and can specifically bind to biological macromolecules such as proteins, small molecules such as drugs and metal ions, and even cells and bacteria. After binding to the target molecule, the aptamer may undergo conformational changes, causing the distance between the electrochemical indicator and the electrode surface to change, which in turn leads to different electrochemical readings.

[0005] Li Tingting’s “Construction and Application of Electrochemical Sensors Based on Metal Nanoparticles-Graphene Composites” (Shanxi University, 2016) mentioned that the insulin aptamer was fixed on the AuNPs / O-GNs modified glassy carbon electrode (GCE) through Au-S bonds, which achieved the detection of insulin with a detection range of 1.0×10 -14 - 5.0×10 -10mol / L, detection limit is 6.0×10 -5 mol / L. GCE has the advantages of good conductivity and high chemical stability and is widely used in electroanalytical chemistry, but it has the problems of large size and lack of flexibility. Compared with GCE, laser-printed graphene electrode (LSGE) has the advantages of large specific surface area, high conductivity, low cost and easy miniaturization. At the same time, LSGE can be easily customized with the geometry, size and layout required for the experiment, and can be directly used as an electrode without further modification. These characteristics make LSGE have great application prospects as a disposable electrode in portable electrochemical sensing devices. Summary of the invention

[0006] The present invention proposes an electrochemical aptamer sensor for detecting insulin and a method for preparing and using the same. By introducing a nucleic acid aptamer fragment Apt, when the target insulin is present, Apt combines to form a complex on the electrode surface, exhibiting the function of a complete aptamer, and changes the distance between gold nanoparticles and the electrode surface before and after recognizing the target molecule, thereby obtaining a change in the electrochemical signal. The LSGE detection limit is 22.7fM, and the GCE detection limit is 9.8fM. The sensor design scheme of the present application is built on a disposable, portable, low-cost LSGE, which will facilitate the commercialization of the sensor and has broad prospects in realizing portable sensing devices for detecting insulin.

[0007] The technical solution of the present invention is achieved in this way:

[0008] A method for preparing an electrochemical aptamer sensor for detecting insulin, comprising the following steps:

[0009] (1) Preparation of AuNPs-Apt probe: HAuCl4 aqueous solution was heated to boiling under stirring, and then sodium citrate solution was added and refluxed to boil. The obtained AuNPs solution was cooled to room temperature, and then Apt solution was added and incubated at low temperature to obtain AuNPs-Apt probe solution;

[0010] (2) Preparation of AuNPs-modified electrodes: After the electrodes were polished, ground, cleaned and dried in sequence, the electrodes / AuNPs were prepared by the it method using HAuCl4 solution;

[0011] (3) Preparation of electrochemical aptamer sensor: Apt solution was dropped on the electrode / AuNPs and incubated with blocking solution to obtain electrode / AuNPs-Apt.

[0012] In the step (1), the sequence of Apt is 5'-SH-GGT GGT GGG GGG GGT TGG TAG GGT GTCTTC-3', the mass volume ratio concentration of the HAuCl4 aqueous solution is 0.01%-0.02%, the mass volume ratio concentration of the sodium citrate solution is 2%-8%, the volume ratio of the HAuCl4 aqueous solution to the sodium citrate solution is (50-100):1, the boiling time is 15-20 min, 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 μM, the incubation temperature is 2-8° C., and the incubation time is 16-24 h.

[0013] In the step (2), the electrode is LSGE or GCE, the polishing powder is Al2O3 polishing powder, the particle size of Al2O3 polishing powder is 0.3, 0.05 μm, the concentration of HAuCl4 aqueous solution is 35-45 mM, the potential of the it method is -0.18- -0.22 V, and the time is 160-200 s.

[0014] In step (3), the concentration of the Apt solution is 4-6 μM, the electrode is LSGE or GCE, the LSGE blocking solution is bovine serum albumin solution (BSA), and the GCE blocking solution 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.

[0015] The electrochemical aptamer sensor for detecting insulin prepared by the above method includes an aptamer Apt for specifically recognizing insulin, a nuclease I for hydrolyzing excess Apt not bound to insulin on the electrode surface, a probe solution AuNPs-Apt labeled with gold nanoparticles, and an AuNPs-modified electrode; wherein Apt is modified on the electrode / AuNPs surface through an Au-S bond, and the non-specific active sites are blocked by a blocking solution to form an electrode / AuNPs-Apt.

[0016] The method for using the electrochemical aptamer sensor for detecting insulin is as follows:

[0017] a. The electrode / AuNPs-Apt sensor was successively immersed in a standard solution of insulin, a solution of exonuclease I and an AuNPs-Apt probe solution for incubation, and then rinsed to obtain an electrode / AuNPs-Apt / insulin / AuNPs-Apt electrochemical aptamer sensor.

[0018] b. The electrochemical aptamer sensor was immersed in a Tris-HCl buffer solution containing a methylene blue solution (MB), and after adjusting the pH, it was cleaned and used as a working electrode. Electrochemical DPV detection was performed in a Tris-HCl buffer solution, and a working curve was drawn based on the relationship between the peak current obtained and the standard solution of insulin;

[0019] c. Detect the concentration of the original sample to be tested, then add insulin to it, and perform the test according to operations a and b. Substitute the detected current signal into the working curve obtained in step b to obtain the concentration of insulin in the sample to be tested.

[0020] In step a, the electrode is LSGE or GCE, LSGE / AuNPs-Apt is immersed in a series of standard insulin solutions with concentrations of 0.05pM-1μM, GCE / AuNPs-Apt is immersed in a series of standard insulin solutions with concentrations of 0.02pM-5.0μM, the incubation time in the insulin solution is 40-60min, the concentration of the nuclease I solution is 1.25-1.50U / μL, the incubation temperature is 36-38°C, the incubation time is 2.5-3.0h, and the incubation time in the probe solution is 15-25min.

[0021] In the step b, the concentration of the MB solution is 30-50 μM, the concentration of the Tris-HCl buffer solution is 0.95-0.15 M, the pH value of the Tris-HCl buffer solution is 7.3-7.5, and the immersion time is 10-20 min.

[0022] The detection principle is: immerse the electrode / AuNPs-Apt in a PBS solution containing different concentrations of insulin and incubate at room temperature for 40-60 minutes, then immerse the obtained electrode / AuNPs-Apt / insulin electrochemical aptamer sensor in a 1.25-1.50U / μL nuclease I solution, incubate at 36-38°C for 2.5-3.0h, then take it out and wash it thoroughly with ultrapure water. During this process, nuclease I hydrolyzes the excess Apt on the electrode surface that is not bound to insulin, turning the sensor into a "signal enhancement" type. Then the sensor is incubated with the AuNPs-Apt probe solution at room temperature for 15-25 minutes. The sandwich structure formed can bind more MB molecules, thereby generating an amplified electrochemical signal. Finally, the constructed sensor was immersed in a Tris-HCl buffer solution with a pH of 7.3-7.5 containing 30-50 μM MB for 10-20 min, taken out and rinsed thoroughly with ultrapure water, and DPV scanning was performed in a Tris-HCl buffer solution with a pH of 7.3-7.5 using differential pulse voltammetry (DPV). The pulse amplitude was 45-55 mV, the pulse width was 45-55 ms, and the DPV scanning potential was from -0.6 V to +0.1 V, so that MB reduction produced an electrochemical signal. In GCE, the DPV peak current at around -0.2 V was regarded as the analytical signal, and in LSGE, the DPV peak current at around -0.26 V was regarded as the analytical signal.

[0023] The present invention has the following beneficial effects:

[0024] 1. The present invention relates to the field of disposable portable sensor manufacturing and aptamer sensing technology, and in particular to an electrochemical aptamer sensor for detecting insulin and its preparation and use methods. The insulin electrochemical sensor is realized by Apt, exonuclease I and gold nanoparticle labeled probe AuNPs-Apt. Gold nanoparticles are electro-deposited on LSGE by constant potential method, i.e. LSGE / AuNPs, wherein Apt is modified on LSGE / AuNPs by Au-S bonds, and non-specific active sites are blocked by BSA solution to form LSGE / AuNPs-Apt sensor. After identifying the target insulin, exonuclease I is introduced to catalyze the hydrolysis of excess aptamer Apt that is not bound to insulin, and converted into a more sensitive "signal enhancement" sensor. The AuNPs-Apt probe can specifically identify insulin on the electrode surface, and the sandwich structure formed can bind more MB molecules, thereby generating an amplified electrochemical signal.

[0025] 2. The present invention uses LSGE, the graphene-like structure on the electrode surface has the advantages of excellent conductivity, low cost, large specific surface area, good mechanical properties, easy miniaturization, etc. In addition, LSGE can be easily customized to the geometry, size and layout required for the experiment, and integrates the working electrode, reference electrode and counter electrode in the manufacturing process, and can be directly used as an electrode without further modification.

[0026] 3. The present invention firstly carried out performance testing on traditional commercial research-grade GCE with a detection range of 0.1pM-1.0μM and a detection limit of 9.8fM, and successfully transferred the sensing strategy to a disposable portable LSGE with a detection range of 0.1pM-0.1μM and a detection limit of 22.7fM, which has potential broad prospects in realizing portable sensing devices for detecting insulin. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 This is the SEM morphology of LSGE / AuNPs on the printed electrode surface in Example 2.

[0029] Figure 2 This is a diagram of the preparation process of the GCE sensor in Example 1.

[0030] Figure 3 This is 10% denaturing polyacrylamide gel electrophoresis in the implementation example.

[0031] Figure 4 This is the AC impedance diagram of GCE in the implementation example.

[0032] Figure 5 It is the working curve diagram of the GCE sensor in the implementation effect example.

[0033] Figure 6 Schematic diagram of LSGE production and insulin sensor design in Example 2.

[0034] Figure 7 This is the AC impedance diagram of LSGE in the implementation effect example.

[0035] Figure 8 It is the working curve diagram of the LSGE sensor in the implementation effect example.

[0036] Fig. 9This is a repeatability test chart of the LSGE sensor in the implementation example.

[0037] Fig.10 This is a stability test diagram of the LSGE sensor in the implementation example.

[0038] Fig.11 This is a test diagram of the selectivity of the LSGE sensor in the implementation example. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in 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.

[0040] Example 1

[0041] A method for preparing an electrochemical aptamer sensor for detecting insulin, comprising the following steps:

[0042] (1) Preparation of AuNPs-Apt probe

[0043] 50mL of 0.01% (w / v) HAuCl4 aqueous solution was heated to boiling under stirring, and then 1 mL of 2% (w / v) sodium citrate solution was quickly added. The mixed solution was then refluxed and boiled for 15 minutes. The color of the solution changed from light yellow at the beginning to purple and finally to wine red, indicating the synthesis of AuNPs. The resulting AuNPs solution was cooled to room temperature, transferred to a brown glass bottle, and stored in a 4°C refrigerator for later use. 30μL, 100 μM Apt solution was added to 570μL AuNPs solution, incubated at 4°C for 16h, centrifuged and washed three times, and redispersed in 600μL ultrapure water to obtain AuNPs-Apt probe solution, which was stored in a 4°C refrigerator. The sequence for recognizing insulin nucleic acid aptamer Apt is: 5'-SH-GGT GGT GGG GGG GGT TGG TAG GGT GTCTTC-3';

[0044] (2) Preparation of AuNPs-modified GCE

[0045] a. The bare GCE with a diameter of 3.0 mm was treated with 0.3 and 0.05 μm Al2O3 polishing powders, the electrode surface was polished to a mirror surface, cleaned with super water to remove the residual Al2O3 powder, and dried at room temperature;

[0046] b. Using 40 mM HAuCl4 aqueous solution as electrolyte, gold nanoparticles were electrodeposited onto the GCE surface by the it method (potential: -0.2 V, time: 180 s) to obtain GCE / AuNPs;

[0047] (3) Preparation of insulin sensor

[0048] The sensor manufacturing process is as follows Figure 2 shown.

[0049] a. 5 μL of 5 μM Apt solution was dropped on GCE / AuNPs and incubated at 4°C for 5 h. Unbound gold sites were then blocked by incubation with 10 mM MCH for 40 min to obtain an insulin sensor electrode, which was recorded as GCE / AuNPs-Apt.

[0050] b. The electrode prepared in a was incubated with different concentrations of insulin for 40 minutes, followed by incubation with nuclease I solution at 37°C for 3 hours, and then the AuNPs-Apt probe solution was added and incubated for 20 minutes. Finally, it was rinsed with ultrapure water for 3 times to obtain an insulin electrochemical aptamer sensor.

[0051] Example 2

[0052] A method for preparing an electrochemical aptamer sensor for detecting insulin, comprising the following steps:

[0053] (1) Preparation of AuNPs-Apt probe

[0054] 50mL of 0.01% (w / v) HAuCl4 aqueous solution was heated to boiling under stirring, and then 1 mL of 2% (w / v) sodium citrate solution was quickly added. The mixed solution was then refluxed and boiled for 15 minutes. The color of the solution changed from light yellow at the beginning to purple and finally to wine red, indicating the synthesis of AuNPs. The resulting AuNPs solution was cooled to room temperature, transferred to a brown glass bottle, and stored in a 4°C refrigerator for later use. 30μL, 100 μM Apt solution was added to 570μL AuNPs solution, incubated at 4°C for 16h, centrifuged and washed three times, and redispersed in 600μL ultrapure water to obtain AuNPs-Apt probe solution, which was stored in a 4°C refrigerator. The sequence for recognizing insulin nucleic acid aptamer Apt is: 5'-SH-GGT GGT GGG GGG GGT TGG TAG GGT GTCTTC-3';

[0055] (2) Preparation of AuNPs-modified LSGE

[0056] The preparation of LSGE refers to the invention content (2). LSGE was immersed in a 40 mM HAuCl4 aqueous solution, and gold nanoparticles were deposited at a potential of -0.2 V for 180 s using the it method to prepare LSGE / AuNPs for use;

[0057] The morphology of the LSGE / AuNPs graphene electrode material synthesized in the present invention is as follows: Figure 1 shown. Figure 1 The graphene electrode has a thin layer and a multi-fold structure on the surface. It can be seen that AuNPs are evenly distributed on the graphene thin layer and present a network distribution. Using LSGE / AuNPs composite graphene as the base electrode can, on the one hand, increase the electron transfer rate (higher than simple LSGE); on the other hand, it has a higher electrochemically active surface area and can bind more insulin aptamers.

[0058] (3) Preparation of sensor

[0059] a. Design of Insulin Electrochemical Aptasensor Figure 6 LSGE / AuNPs were immersed in 5 μM Apt solution for 10 min, incubated at 4°C for 5 h, and then incubated with 1% BSA solution for 40 min to block the unbound gold sites, thus obtaining an insulin sensor electrode, which was recorded as LSGE / AuNPs-Apt.

[0060] b. The electrode prepared in a was incubated with different concentrations of insulin for 40 minutes, followed by incubation with exonuclease I solution at 37°C for 3 hours, and then the AuNPs-Apt probe solution was added and allowed to act for 20 minutes. Finally, it was rinsed three times with ultrapure water to obtain an insulin electrochemical aptamer sensor.

[0061] Implementation effect example: Biosensor performance testing

[0062] (1) Verification of exonuclease I cleavage of insulin aptamers in solution

[0063] The ability of exonuclease I to catalyze the hydrolysis of insulin aptamers was verified by 10% polyacrylamide gel electrophoresis (PAGE). 5 μM aptamers (PBS buffer solution) were incubated with different concentrations of insulin ranging from 0.1 μM to 100 μM, and then hydrolyzed with 0.1 U / μL exonuclease I at 37°C. Figure 3 As shown, in the presence of different concentrations of insulin, lanes 3-6 show that the aptamer is hydrolyzed, and as the insulin concentration decreases, the amount of the hydrolyzed aptamer increases, indicating that when the aptamer binds to insulin to form a complex, the aptamer can be protected from being hydrolyzed by exonuclease I.

[0064] (2) Electrochemical impedance spectroscopy of GCE

[0065] Electrochemical impedance spectroscopy (EIS) was used to characterize the preparation process of the electrode. 3- / [Fe(CN)6] 4- Each assembly step is verified in solution, e.g. Figure 4 As shown. When AuNPs are modified onto bare GCE, its impedance value decreases from 308.5Ohm (curve a) to 23.02Ohm (curve b). This is because, compared with bare electrodes, GCE / AuNPs have stronger conductivity and higher specific surface area. After the negatively charged aptamer Apt is assembled onto GCE / AuNPs, due to the affinity of Apt to [Fe(CN)6] 3- / [Fe(CN)6] 4- The repulsive effect hindered the transfer of electrons on the electrode surface, and its impedance value increased to 209.1Ohm (curve c). After the electrode was sealed with the poorly conductive MCH solution, the impedance value further increased to 645.9Ohm (curve d). After incubation with the target insulin, the impedance value increased again to 1610.1Ohm (curve e). Then, the aptamer that did not bind to the target was hydrolyzed by exonuclease I, and the impedance value decreased to 1307.3Ohm (curve f). Finally, after the AuNPs-Apt probe bound to insulin on the electrode surface, the impedance value increased to 1834.2Ohm (curve g), proving that AuNPs loaded more negatively charged phosphate skeletons, which hindered the electron transfer process. The impedance diagram can show the successful construction of the aptamer sensor.

[0066] (3) Insulin detection curve in GCE

[0067] According to Example 1, the electrochemical signals of the nucleic acid aptamer sensor of the present invention in different concentrations of insulin are detected, and a standard curve diagram of the peak current change value and insulin concentration is drawn. The specific detection steps are as follows:

[0068] 1) The constructed GCE / AuNPs-Apt sensor was immersed in a series of standard solutions of insulin with different concentrations (0.02pM, 0.05pM, 0.1pM, 1pM, 10pM, 100pM, 0.1nM, 1nM, 10nM, 100nM, 0.1μM, 1.0μM, 2.0μM, 5.0μM), incubated at room temperature for 40min, then taken out and washed 3 times with ultrapure water. The sensor was immersed in 1.25U / μL exonuclease I solution, incubated at 37°C for 3h, then taken out and washed 3 times with ultrapure water. Then, the sensor was incubated with AuNPs-Apt probe solution at room temperature for 20min, and washed 3 times with ultrapure water. Finally, the sensor was immersed in 0.1M Tris-HCl buffer solution (pH=7.4) containing 50μM MB for 10min, then taken out and washed 3 times with ultrapure water.

[0069] 2) Connect the sensor to an electrochemical instrument: perform electrochemical DPV detection in a 0.1M Tris-HCl buffer solution at pH 7.4, scan the potential from -0.6V to +0.1V, with a pulse amplitude of 50mV and a pulse width of 50ms. According to the DPV peak current electrochemical signal at a potential of -0.2V, Figure 5 A, A standard curve was established based on the DPV signal and the corresponding insulin concentration.

[0070] According to the insulin concentration and the corresponding current signal, the following is drawn: Figure 5 The linear correlation graph between insulin concentration and ΔI of B, the linear regression equation is ΔI=-0.86lg[insulin]-1.70, (0.1pM to 1 μM), the linear correlation is R2=0.9969, and the detection limit is 9.8fM (3Sb / m, Sb is the standard deviation of the blank signal, and m is the slope of the standard curve).

[0071] (4)LSGE production

[0072] According to Example 2, the schematic diagram of the manufacturing process of LSGE and the design process of the sensor is as follows Figure 6 shown.

[0073] (5) Electrochemical impedance spectroscopy of LSGE

[0074] According to Example 2, the experiment uses electrochemical impedance spectroscopy to characterize the preparation process of the electrode. The sensing interface at different preparation stages is 5 mM [Fe(CN)6] 3- / [Fe(CN)6] 4- Solution (containing 0.1M KCl) was tested, such as Figure 7As shown. The impedance value of bare LSGE (curve a) is very small, indicating that the transfer of electrons on the surface of the graphene printed electrode is not hindered. When gold nanoparticles AuNPs are modified on the electrode (curve b), the impedance value is even lower. The reason is that AuNPs have strong conductivity and will accelerate the transfer of electrons on the electrode surface. After LSGE / AuNPs are combined with Apt (curve c), the impedance value increases. This is because Apt is a negatively charged nucleic acid skeleton, which is sensitive to [Fe(CN)6] 3- / [Fe(CN)6] 4- The repulsive effect of the electrode hinders the transfer of electrons on the electrode surface. After the electrode was sealed with BSA solution (curve d), the impedance value increased again. Then the electrode was incubated with insulin (curve e), and the impedance value increased further. This is because BSA and insulin are both proteins with poor conductivity. Then the electrode was treated with exonuclease I. Since exonuclease I hydrolyzed the aptamer that was not bound to insulin (curve f), the impedance value decreased. Finally, the AuNPs-Apt probe was introduced (curve g). Since AuNPs were loaded with a large amount of Apt with a negatively charged phosphate backbone, the electron transfer process was hindered, and its impedance value increased. The impedance diagram can show the successful construction of the aptamer sensor.

[0075] (6) Drawing of standard curve

[0076] The electrochemical signals of the electrochemical aptamer sensor of the present invention in different concentrations of insulin are detected, and a standard curve diagram of the peak current change value and the insulin concentration is drawn. The specific detection steps are as follows:

[0077] 1) The LSGE / AuNPs-Apt sensor was immersed in a series of insulin standard solutions (0.05pM, 0.1pM, 1pM, 10pM, 100pM, 0.1nM, 1nM, 10nM, 100nM, 0.1μM, 0.5μM, 1μM), incubated at room temperature for 40min, then taken out and washed 3 times with ultrapure water. The sensor was immersed in 1.25U / μL exonuclease I solution, incubated at 37°C for 3h, then taken out and washed 3 times with ultrapure water. The sensor was then incubated with the AuNPs-Apt probe solution at room temperature for 20min and washed 3 times with ultrapure water. Finally, the sensor was immersed in 0.1M Tris-HCl (pH=7.4) containing 50μM MB for 10min, then taken out and washed 3 times with ultrapure water.

[0078] 2) Connect the sensor to an electrochemical instrument: perform electrochemical DPV detection in a 0.1M Tris-HCl buffer solution at pH 7.4, scan the potential from -0.6V to +0.1V, with a pulse amplitude of 50mV and a pulse width of 50ms. According to the DPV peak current electrochemical signal at a potential of -0.26V, Figure 8 A, A standard curve was established based on the DPV signal and the corresponding insulin concentration.

[0079] According to the insulin concentration and the corresponding current signal, the following is drawn: Figure 8 The linear correlation diagram between insulin concentration and ΔI of B, the linear regression equation is ΔI=-2.28lg[insulin]-3.10, (0.1pM to 0.1 μM), the linear correlation is R2=0.9941, and the detection limit is 22.7fM (3Sb / m).

[0080] (7) Reproducibility test

[0081] Six different LSGEs were used to detect 0.1 μM insulin under the same experimental conditions, such as Fig. 9 , the relative standard deviation between different electrodes is 1.80%, indicating that the prepared electrode has good reproducibility.

[0082] (8) Stability test

[0083] The prepared electrochemical aptasensor was placed at 4 °C and detected 0.1 μM insulin after 5, 10, 15, and 20 days, respectively. Fig.10 As shown, the signal of the sensor after 20 days of storage is 90.93% of the signal on the first day, indicating that the prepared electrochemical aptasensor has good stability.

[0084] (9) Selective testing

[0085] The selectivity of electrochemical aptamer sensors was studied, and L-cysteine, glucose, uric acid and lysozyme were selected for interference test experiments. Fig.11 As shown. When LSGE was first incubated with a mixture containing 1 μM L-cysteine, glucose, uric acid and lysozyme (column a), the change in DPV peak current was 3.59 μA. When incubated with 0.1 μM insulin (column b), the change in current signal was 14.77 μA. When insulin was mixed with interference (column c), the signal difference obtained by electrode detection was only slightly larger than that of insulin alone (17.43 μA vs 14.77 μA). It can be seen that the sensor prepared by the present invention has good selectivity.

[0086] Application example: Detection of insulin in serum samples

[0087] The sensing performance of the LSGE / AuNPs-Apt sensor for detecting insulin in actual samples was evaluated by detecting the insulin concentration in human serum samples. First, since the insulin concentration in serum is low, the insulin concentration in the original human serum sample is directly detected, which is 0.04nM (relative standard deviation RSD=4.25%). Using the standard addition method, 0.10pM, 0.50pM and 1.00pM insulin are added to the original serum sample (the volume ratio of insulin to serum sample = 1:1). The DPV signals before and after the addition of different concentrations of insulin are recorded. The results show that the spiked concentrations are 0.10pM, 0.52pM and 1.03pM, respectively, and the spiked recoveries are 100.0%, 103.7% and 102.9%, respectively. This result shows that the LSGE / AuNPs-Apt sensor of the present invention for detecting insulin is a promising method for detecting insulin in actual samples.

[0088] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical aptamer sensor for detecting insulin, characterized in that: Here are the steps: (1) Preparation of AuNPs-Apt probe: heating a HAuCl4 aqueous solution to boiling under stirring, then adding a sodium citrate solution, reflux 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; the sequence of the Apt is 5'-SH-GGT GGT GGG GGG GGT TGG TAG GGT GTC TTC-3', and the mass volume ratio concentration of the HAuCl4 aqueous solution is 0.01%-0.02%; (2) Preparation of AuNPs-modified electrodes: After the electrodes were polished, ground, cleaned and dried in sequence, the electrodes / AuNPs were prepared by the it method using HAuCl4 aqueous solution; (3) Preparation of electrochemical aptamer sensor: Apt solution is dropped onto the electrode / AuNPs and incubated with a blocking solution, including the aptamer Apt for specific recognition of insulin, nuclease I for hydrolyzing excess Apt on the electrode surface that is not bound to insulin, a probe solution AuNPs-Apt labeled with gold nanoparticles, and an AuNPs-modified electrode; wherein Apt is modified on the electrode / AuNPs surface through Au-S bonds, and the nonspecific active sites are blocked by the blocking solution to form an electrode / AuNPs-Apt.

2. The preparation method according to claim 1, characterized in that: The mass volume ratio concentration of the sodium citrate solution is 2%-8%, the volume ratio of the HAuCl4 aqueous solution to the sodium citrate solution is 50-100:1, the boiling time is 15-20min, the volume ratio of the AuNPs solution to the Apt solution is 1-2:19, the concentration of the Apt solution is 50-100μM, the incubation temperature is 2-8°C, and the incubation time is 16-24h.

3. The preparation method according to claim 1, characterized in that: In the step (2), the electrode is a laser-printed graphene electrode or a glassy carbon electrode, the polishing powder is Al2O3 polishing powder, the particle size of Al2O3 polishing powder is 0.3, 0.05 μm, the concentration of HAuCl4 aqueous solution is 35-45 mM, the potential of the it method is -0.18--0.22 V, and the time is 160-200 s.

4. The preparation method according to claim 1, characterized in that: In the step (3), the concentration of the Apt solution is 4-6 μM, the electrode is a laser-printed graphene electrode or a glassy carbon electrode, the laser-printed graphene electrode sealing liquid is a bovine serum albumin solution, and the glassy carbon electrode sealing liquid 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. A method for using the electrochemical aptamer sensor prepared by the preparation method according to claim 1, characterized in that: Here are the steps: a. The electrode / AuNPs-Apt sensor was successively immersed in a standard solution of insulin, a solution of exonuclease I and an AuNPs-Apt probe solution for incubation, and then rinsed to obtain an electrode / AuNPs-Apt / insulin / AuNPs-Apt electrochemical aptamer sensor; b. The electrode / AuNPs-Apt / insulin / AuNPs-Apt electrochemical aptamer sensor was immersed in a Tris-HCl buffer solution containing a methylene blue solution, and after adjusting the pH, it was cleaned and used as a working electrode. Electrochemical DPV detection was performed in a Tris-HCl buffer solution, and a working curve was drawn based on the relationship between the peak current obtained and the standard solution of insulin; c. Detect the concentration of the original sample to be tested, then add insulin to it, and perform the test according to operations a and b. Substitute the detected current signal into the working curve obtained in step b to obtain the concentration of insulin in the sample to be tested.

6. A method for using the electrochemical aptamer sensor obtained by the preparation method according to claim 5, characterized in that: In the step a, the electrode is a laser-printed graphene electrode or a glassy carbon electrode, the laser-printed graphene electrode / AuNPs-Apt is immersed in a series of standard insulin solutions with a concentration of 0.05pM-1μM, the glassy carbon electrode / AuNPs-Apt is immersed in a series of standard insulin solutions with a concentration of 0.02pM-5.0μM, the incubation time in the insulin solution is 40-60min, the concentration of the nuclease I solution is 1.25-1.50U / μL, the incubation temperature is 36-38°C, the incubation time is 2.5-3.0h, and the incubation time in the probe solution is 15-25min.

7. A method for using the electrochemical aptamer sensor obtained by the preparation method according to claim 5, characterized in that: In the step b, the concentration of the methylene blue solution is 30-50 μM, the concentration of the Tris-HCl buffer solution is 0.95-0.15 M, the pH value of the Tris-HCl buffer solution is 7.3-7.5, and the immersion time is 10-20 min.

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