A cortisol aptamer functionalized flexible electrode and its preparation method and application

Abstract

The present invention provides an electrochemical flexible electrode based on cortisol aptamer functionalization and its preparation method and application, belonging to the technical field of cortisol electrochemical detection. The cortisol aptamer functionalized flexible electrode comprises a flexible screen-printed electrode with a three-electrode system, and the working electrode of the flexible screen-printed electrode is modified with a transduction matrix and an aptamer capable of specifically recognizing cortisol in sequence, and the transduction matrix is ​​a graphene aerogel containing gold nanoparticles. The present invention successfully modifies the graphene aerogel containing gold nanoparticles on the working electrode of the screen-printed electrode by a one-step chemical synthesis method, and selects an aptamer with higher stability as the recognition molecule of cortisol. At the same time, the flexible screen-printed electrode is used as the substrate of the transduction matrix and the recognition molecule, which can not only directly realize the construction of a portable or wearable cortisol electrochemical biosensor platform, but also can quickly realize its low-cost mass production, laying a technical foundation for realizing non-invasive, rapid, accurate and real-time cortisol detection.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical detection of cortisol, and in particular to an electrochemical flexible electrode based on functionalization of a cortisol aptamer, and a preparation method and application thereof. Background Art

[0002] Cortisol is extracted from the adrenal cortex and is the adrenal cortex hormone with the strongest effect on carbohydrate metabolism, which is a type of glucocorticoid. Cortisol levels have a vital impact on the body's basic metabolism, organ systems, immune system and bone development. Excessive cortisol levels will lead to weight gain, thin and fragile skin, fertility-related problems, susceptibility to stress, pain, fatigue, high blood sugar and high blood pressure, etc.; too low cortisol levels will lead to fatigue, nausea, vomiting, weight loss, muscle weakness and abdominal pain, etc. Therefore, the detection of cortisol plays a key role in monitoring daily stress, high-pressure workers, sports and fitness management, pregnant women and fetal health, and patients with cardiovascular and cerebrovascular diseases.

[0003] The performance of biosensors is highly dependent on the transduction matrix and recognition molecules. Graphene has attracted much attention as a highly promising transduction matrix due to its high specific surface area, high biocompatibility, and the possibility of improving sensitivity by promoting electron transfer between electroactive substances and the electrode surface, but the current modification methods of graphene on screen-printed electrodes are complex. On the other hand, the current test methods for cortisol mainly use antibodies as recognition molecules, but antibodies cannot be synthesized in vitro, and they show defects in variability and cross-reactivity, and cannot be stored and transported for a long time. In addition, although traditional cortisol detection methods have high sensitivity, the detection process is too lengthy and the detection device is too large to have wearable application value. At the same time, it also requires a lot of human exercise to collect sweat, which often disrupts the cortisol level in the body.

[0004] Aptamers are small oligonucleotide sequences obtained by in vitro screening, which can bind to the corresponding ligand with high affinity and strong specificity. Their emergence provides a new research platform for efficient and rapid identification. In summary, developing a simple and efficient graphene modification method and using suitable aptamers as recognition molecules are crucial for developing reliable, accurate, and quantitative electrochemical cortisol sensors to achieve non-invasive, rapid, accurate, and real-time cortisol detection. Summary of the invention

[0005] To solve the above problems, the present invention discloses an electrochemical flexible electrode functionalized with cortisol aptamers, a preparation method and application thereof, and adopts suitable aptamers as recognition molecules to develop reliable, accurate and quantitative cortisol electrochemical sensors to achieve non-invasive, rapid, accurate and real-time cortisol detection.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for preparing a cortisol aptamer functionalized flexible electrode comprises the following steps:

[0008] Step 10) provides a flexible screen-printed electrode. The flexible screen-printed electrode is a three-electrode working system, including a working electrode, a counter electrode and a reference electrode, wherein the working electrode and the counter electrode use carbon paste, and the reference electrode uses silver chloride paste.

[0009] Step 20) 10 mL of a mixed colloid of graphene oxide, chloroauric acid and L-cysteine ​​is evenly dropped onto the working electrode of the flexible screen-printed electrode;

[0010] Step 30) placing the flexible screen-printed electrode obtained in step 20) in a water bath at 70° C. for 12 hours;

[0011] Step 40) The flexible screen-printed electrode obtained in step 30) is placed in deionized water and soaked for 30 minutes, then taken out and blown dry with a small stream of nitrogen and then frozen in a -20°C refrigerator for 24 hours.

[0012] Step 50) freeze-dry the flexible screen-printed electrode obtained in step 40) for 6 hours, and the transduction matrix is ​​now ready.

[0013] Step 60) 10 μL of 3 μM aptamer solution that can specifically recognize cortisol is evenly added to the working electrode area of ​​the flexible screen-printed electrode obtained in step 50), and then placed in a refrigerator at 0 to 4 degrees Celsius to dry overnight. At this point, a cortisol aptamer functionalized flexible electrode is prepared.

[0014] Furthermore, the method for preparing the mixed colloid of graphene oxide, chloroauric acid and L-cysteine ​​in step 20) comprises the following steps:

[0015] Step 201) 2 mL of a certain concentration of graphene oxide solution is mixed with 1 mL of a certain concentration of chloroauric acid solution, and stirred with a magnetic stirrer for 30 min to make the mixture uniform;

[0016] Step 202) Add 2 mL of L-cysteine ​​of a certain concentration to the solution obtained in step 201), and then use a magnetic stirring for 10 minutes to mix it evenly to obtain a mixed solution of graphene oxide, chloroauric acid and L-cysteine ​​in step 20).

[0017] Furthermore, in step 60), the sequence of the aptamer capable of specifically recognizing cortisol is 5′-SH(CH2)6-ATG GGC AAT GCG GGG TGG AGA ATG GTT GCC GCA CTT CGGC-3′.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Graphene and gold nanoparticles were successfully modified on the working electrode of the screen-printed electrode using a one-step chemical synthesis method. Graphene oxide and chloroauric acid solution were used as precursors of graphene and gold nanoparticles, respectively, and L-cysteine ​​was used as a reducing agent to reduce graphene oxide to obtain reduced graphene oxide, and chloroauric acid was reduced to obtain gold nanoparticles, and finally graphene doped with gold nanoparticles was obtained.

[0020] 2. The graphene is an aerogel structure with a three-dimensional porous network of hierarchical pores, which establishes an interconnected conductive network, is conducive to the efficient transfer of electrons in electrochemical reactions, and shows good electrochemical catalytic activity. After the water bath in step 40), the graphene hydrogel is obtained. After freezing, the freeze-drying technology in step 50) is a drying method that converts ice into water vapor under a relatively high vacuum and removes it. Therefore, the water in the graphene hydrogel can be removed to obtain an aerogel.

[0021] 3. The aptamer with the base sequence of ATG GGC AAT GCG GGG TGG AGA ATG GTT GCC GCA CTT CGGC was used as the recognition molecule of cortisol. Compared with antibodies, the aptamer has higher stability and simpler modification method. The 5′ end of the aptamer is modified with a thiol group, which can form a gold-sulfur bond with the gold nanoparticles in the transduction matrix, and then the aptamer is successfully modified on the working electrode.

[0022] 4. The use of flexible screen-printed electrodes as the substrate for transduction matrix and recognition molecules can not only directly realize the construction of portable or wearable cortisol biosensor platforms, but also quickly realize the mass production of cortisol sensors with low production cost.

[0023] 5. Using flexible screen-printed electrodes as the electrode platform, graphene aerogel doped with gold nanoparticles as the transduction matrix, and an aptamer with a thiol group modified at the 5′ end with the base sequence of ATG GGC AAT GCG GGG TGG AGA ATG GTT GCCGCA CTT CGGC as the recognition molecule of cortisol, a reliable, accurate and quantitative electrochemical cortisol sensor was successfully developed, laying a technical foundation for the non-invasive, rapid, accurate and real-time detection of cortisol. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of a process for preparing a cortisol aptamer functionalized flexible electrode according to the present invention;

[0025] Figure 2 Cyclic voltammograms and comparison of peak currents of oxidation peak and reduction peak of electrodes prepared with different mass ratios of graphene oxide and L-cysteine ​​in 10 mmol / L potassium ferrocyanide;

[0026] Figure 3 Cyclic voltammograms and comparison of peak currents of oxidation peak and reduction peak of electrodes prepared with chloroauric acid solutions of different concentrations in 10 mmol / L potassium ferrocyanide;

[0027] Figure 4 Electrochemical characterization diagram for successful preparation of graphene doped with gold nanoparticles and successful modification of aptamers;

[0028] Figure 5 The relationship between the current difference before and after the electrodes prepared by different concentrations of aptamer solutions bind to different concentrations of cortisol;

[0029] Figure 6 The relationship between the electrode prepared by incubating the same concentration of cortisol solution for different time and the current difference before cortisol binding;

[0030] Figure 7 The current response of a prepared cortisol aptamer functionalized flexible electrode to different concentrations of cortisol;

[0031] Figure 8 This is a graph showing the current response relationship between the prepared cortisol aptamer functionalized flexible electrode and cortisol and common interfering substances in the blood;

[0032] Fig. 9 This is a current response relationship diagram of a prepared cortisol aptamer functionalized flexible electrode after multiple bending at the same angle;

[0033] Fig.10 The current response diagram of a prepared cortisol aptamer functionalized flexible electrode after bending to different degrees. DETAILED DESCRIPTION

[0034] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. It should be noted that the words "front", "rear", "left", "right", "upper" and "lower" used in the following description refer to directions in the accompanying drawings, and the words "inner" and "outer" refer to directions toward or away from the geometric center of a specific component, respectively.

[0035] Reference Figure 1 As shown, according to an embodiment of the present invention, a method for preparing a cortisol aptamer functionalized flexible electrode is provided. In a preferred embodiment, a method for preparing a cortisol aptamer functionalized flexible electrode comprises the following steps:

[0036] Step 10) Provide a flexible screen-printed electrode.

[0037] The flexible screen-printed electrode is obtained by screen printing technology. The flexible screen-printed electrode is a three-electrode working system, including a working electrode, a counter electrode and a reference electrode, wherein the working electrode and the counter electrode use carbon paste, and the reference electrode uses silver chloride paste. Optionally, the electrode substrate uses PET (polyethylene terephthalate) film.

[0038] Step 20) 10 μL of a mixed colloid of graphene oxide, chloroauric acid and L-cysteine ​​is evenly dropped onto the working electrode of the flexible screen-printed electrode.

[0039] Wherein, graphene oxide and chloroauric acid solution are respectively the precursors of graphene and gold nanoparticles, and L-cysteine ​​is used as a reducing agent to reduce graphene oxide to obtain reduced graphene oxide, and chloroauric acid is reduced to obtain gold nanoparticles, and finally graphene doped with gold nanoparticles is obtained. Preferably, the amount of the mixed colloid of graphene oxide, chloroauric acid and L-cysteine ​​added is 10 μL, because the solution of this amount of addition can completely cover the working electrode.

[0040] Step 30) placing the flexible screen-printed electrode obtained in step 20) in a water bath at 70° C. for 12 hours;

[0041] Among them, under a constant temperature environment of 70°C, in a mixed colloid of graphene oxide, chloroauric acid and L-cysteine, L-cysteine ​​reduces graphene oxide to graphene, resulting in partial removal of hydrophilic oxygen-containing functional groups. The hydrophobicity and π-conjugated structure of the reduced graphene oxide increase the hydrophilic-hydrophobic balance, allowing the reduced graphene oxide to assemble into a 3D reduced graphene oxide framework. The absorbed water acts as a continuous template to form a graphene hydrogel.

[0042] Step 40) The flexible screen-printed electrode obtained in step 30) is placed in deionized water and soaked for 30 minutes, then taken out and blown dry with a small stream of nitrogen and then frozen in a -20°C refrigerator for 24 hours.

[0043] The purpose of soaking in deionized water for 30 minutes is to remove excess L-cysteine. The purpose of freezing in a -20°C refrigerator for 24 hours is to freeze the water in the hydrogel into ice for subsequent freeze-drying operations.

[0044] Step 50) freeze-dry the flexible screen-printed electrode obtained in step 40) for 6 hours, and the transduction matrix is ​​now ready.

[0045] The freeze-drying technology is a drying method that converts ice into water vapor under a relatively high vacuum and removes it, so the water in the graphene hydrogel can be removed to obtain an aerogel. The graphene aerogel has a three-dimensional porous network of hierarchical pores, which establishes an interconnected conductive network, which is conducive to the efficient transfer of electrons in electrochemical reactions and shows good electrochemical catalytic activity.

[0046] Step 60) 10 μL of 3 μM aptamer solution that can specifically recognize cortisol is evenly added to the working electrode area of ​​the flexible screen-printed electrode obtained in step 50), and then placed in a refrigerator at 0 to 4 degrees Celsius to dry overnight. At this point, a cortisol aptamer functionalized flexible electrode is prepared.

[0047] Furthermore, the method for preparing the mixed colloid of graphene oxide, chloroauric acid and L-cysteine ​​in step 20) comprises the following steps:

[0048] Step 201) 2 mL of a certain concentration of graphene oxide solution is mixed with 1 mL of a certain concentration of chloroauric acid solution, and stirred with a magnetic stirrer for 30 min to make the mixture uniform;

[0049] Step 202) Add 2 mL of L-cysteine ​​of a certain concentration to the solution obtained in step 201), and then use a magnetic stirring for 10 minutes to mix it evenly to obtain a mixed solution of graphene oxide, chloroauric acid and L-cysteine ​​in step 20).

[0050] Preferably, the optimal mass ratio of graphene oxide to L-cysteine ​​is 1:4. Prepare mixed colloids of graphene oxide and L-cysteine ​​with mass ratios of 1:1, 1:2, 1:4, 1:6, 1:8, and 1:10, take 10 μL and evenly drop it on the working electrode, and perform the treatment of steps 30) to 50). After the preparation is completed, use an electrochemical workstation to measure its cyclic voltammogram in 10 mmol / L potassium ferrocyanide. Figure 2 As shown, when the mass ratio of graphene oxide to L-cysteine ​​is 1:4, the oxidation peak and reduction peak current are basically the same as those at a higher mass ratio. Graphene oxide to L-cysteine ​​at a mass ratio of 1:4 is selected as the optimal ratio for preparing the sensor.

[0051] Preferably, the optimal concentration of the chloroauric acid solution is 6 mg / mL. Prepare mixed colloids of graphene oxide, chloroauric acid and L-cysteine ​​at concentrations of 0 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL and 10 mg / mL of chloroauric acid solution, take 10 μL and evenly drop it on the working electrode, and perform the treatments of step 30) to step 50). After the preparation is completed, use an electrochemical workstation to measure its cyclic voltammogram in 10 mmol / L potassium ferrocyanide. Figure 3 As shown, when the concentration of chloroauric acid solution is 6 mg / mL, its oxidation peak and reduction peak currents are higher than those at other concentrations. The concentration of chloroauric acid solution is 6 mg / mL, which is the optimal concentration for preparing the sensor.

[0052] Furthermore, the sequence of the aptamer that can specifically recognize cortisol in step 60) is 5′-SH(CH2)6-ATG GGC AAT GCG GGG TGG AGA ATG GTT GCC GCA CTT CGGC-3′. The 5′ end of the aptamer is modified with a thiol group, which is placed in a refrigerator at 0 to 4 degrees Celsius to dry overnight. The group can form a gold-sulfur bond with the gold nanoparticles in the transduction matrix, thereby successfully modifying the aptamer on the working electrode.

[0053] Electrochemical characterization of successful preparation of graphene doped with gold nanoparticles and successful modification of aptamers. Graphene oxide, reduced graphene oxide (L-cysteine ​​reduction), reduced graphene oxide with gold nanoparticles added (L-cysteine ​​reduction), and flexible screen-printed electrodes modified with aptamers that can specifically recognize cortisol were prepared. After preparation, the cyclic voltammogram and electrochemical impedance spectroscopy in 10mmol / L potassium ferrocyanide were measured using an electrochemical workstation. Figure 4 As shown, the electrode modified with reduced graphene oxide (L-cysteine ​​reduction) has a larger current response and a smaller impedance value than the electrode modified with graphene oxide, thus proving that graphene oxide can be successfully reduced to reduced graphene oxide by L-cysteine; after the addition of gold nanoparticles, the current increased and the impedance decreased, indicating the successful addition of gold nanoparticles; after modification with the aptamer, the current decreased and the impedance value increased, indicating the successful modification of the aptamer.

[0054] Preferably, the optimal concentration of the aptamer solution capable of specifically recognizing cortisol is 3 μM. The concentrations of the aptamer solution capable of specifically recognizing cortisol are respectively prepared to be 2 μM, 3 μM, and 4 μM, 10 μL of which is evenly dripped onto the working electrode and placed in a refrigerator at 0 to 4 degrees Celsius to dry overnight. Figure 5 As shown, when the concentration of the aptamer solution is 3 μM, the current response change caused by the same concentration of cortisol is the largest. In order to save production costs, this concentration is selected for the preparation of the cortisol test electrode.

[0055] Preferably, the optimal incubation time for cortisol testing is 20 min. 10 ng mL-1 of cortisol solution was evenly dripped onto WE for incubation, and the incubation time was set to 5, 10, 15, 20, and 25 min, respectively. After the incubation, DPV test was performed to obtain the current response change |Ic-I0| result. Figure 6 As shown, when the incubation time reaches 20 min, the current response caused by the same concentration of cortisol changes the most, so this time is selected as the incubation time for subsequent cortisol tests.

[0056] The cortisol test performance of the cortisol test electrode based on MB@Aptamer-Au@GA was studied under the optimal experimental parameters. PBS was selected as the electrolyte, and DPV was used to quantitatively analyze cortisol of different concentrations. The actual test results were consistent with the theoretical analysis. As the cortisol concentration increased, the peak current of the DPV curve decreased regularly. The logarithm of the cortisol concentration was used as the horizontal axis, and its value was linearly fitted with the corresponding current response (N=3), and the calibration curve was obtained as shown in the figure. Figure 7 As shown in Figure 2, the logarithmic value of cortisol concentration and the peak value of DPV current response showed an excellent linear relationship in the range of 0.5-100 ng / mL (R2=0.0.98242), and the regression equation was: cor =-3.38069lg[C cor ]+12.9674, where I cor is the peak value of the DPV current response (μA), C cor is the logarithm of cortisol concentration (lg[ng mL -1 ]).

[0057] Several common substances in blood were studied, including glucose (Glu), lactic acid (LA), ascorbic acid (AA), sodium chloride (NaCl), urea (UA), and potassium chloride (KCl). Figure 8 As shown, cortisol was the only substance that caused a sharp current response, while the other substances studied consistently showed lower current changes, indicating that the electrode was able to specifically recognize cortisol.

[0058] First, the DPV test was performed on the cortisol test electrode bent at different angles. By observing the DPV curve, it can be found that compared with the current response results of the electrode without bending, the current response of the electrode after bending at different angles remained basically unchanged. Then, the glucose test electrode was bent repeatedly 50 times (bending angle 60°), and a DPV test was performed every 5 times. Fig. 9As shown in the figure, the relationship curve between the current response and the current response of the glucose test electrode without bending fluctuates, but basically remains horizontal, that is, the current response of the electrode remains basically unchanged after multiple bendings at the same angle. In addition, the cortisol test electrode with a bending angle of 60° was used to test 10 ng mL-1 of cortisol, as shown in the figure. Fig.10 The results of the current response changes corresponding to different bending angles |Ic-I0| show that the bending of the electrode does not affect the electrode's ability to detect cortisol. The above results all show that the cortisol test electrode has excellent flexibility and can meet the application scenarios of wearable skin biosensors.

[0059] In this example, the performance of the biosensor is improved by using graphene aerogel embedded with gold nanoparticles as the transduction matrix to improve the sensitivity, and using nucleic acid aptamers to efficiently identify cortisol. In order to enable fast and convenient detection, flexible electrodes are made for portable wear, and screen printing technology can support future mass production.

[0060] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above technical features.

Claims

1. A method for preparing a cortisol aptamer functionalized flexible electrode, characterized in that: The steps include: Step 10) providing a flexible screen-printed electrode; the flexible screen-printed electrode is a three-electrode working system, including a working electrode, a counter electrode and a reference electrode, wherein the working electrode and the counter electrode use carbon paste, and the reference electrode uses silver chloride paste; Step 20) 10 μL of a mixed colloid of graphene oxide, chloroauric acid and L-cysteine ​​is evenly dropped onto the working electrode of the flexible screen-printed electrode; the method for preparing the mixed colloid of graphene oxide, chloroauric acid and L-cysteine ​​in step 20) comprises the following steps: Step 201) 2 mL of 5 mg / mL graphene oxide solution and 1 mL of 6 mg / mL chloroauric acid solution are mixed, and magnetic stirring is used to mix them evenly; Step 202) adding 2 mL of 20 mg / mL L-cysteine ​​to the solution obtained in step 201), and then using magnetic stirring to mix them evenly to obtain a mixed solution of graphene oxide, chloroauric acid and L-cysteine ​​in step 20); Step 30) placing the flexible screen-printed electrode obtained in step 20) in a water bath at 70° C. for 12 h; Step 40) The flexible screen-printed electrode obtained in step 30) is placed in deionized water and soaked for 30 minutes, then taken out and blown dry with a small stream of nitrogen and then frozen in a -20°C refrigerator for 24 hours; Step 50) freeze-drying the flexible screen-printed electrode obtained in step 40) for 6 hours, and the transduction matrix is ​​thus prepared; Step 60) 10 μL of an aptamer solution that can specifically recognize cortisol is evenly dripped onto the working electrode area of ​​the flexible screen-printed electrode obtained in step 50), and then placed in a refrigerator at 0 to 4 degrees Celsius to dry overnight, thereby completing the preparation of a cortisol aptamer functionalized flexible electrode; the sequence of the aptamer that can specifically recognize cortisol in step 60) is 5′-SH(CH2)6-ATG GGC AAT GCG GGG TGG AGA ATG GTT GCC GCA CTT CGGC-MB-3′.

2. A cortisol aptamer functionalized flexible electrode, characterized in that: The method for preparing a cortisol aptamer functionalized flexible electrode according to claim 1 is used to prepare the electrode.

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