Biosynthetic hydroxyapatite-based protein kinase B electrochemical sensor and skin cell senescence application research

Abstract

The invention designs a biosynthetic hydroxyapatite-based protein kinase B (Akt1) electrochemical sensor which is applied to Akt1 activity analysis in a skin cell aging process. Hydroxyapatite is biologically synthesized through bacillus subtilis and is uniformly deposited on the surface of a glassy carbon electrode by utilizing a potentiometric deposition method. The surface of the material contains rich phosphate groups and hydroxyl groups, can be combined with lanthanum ions, and provides a good interface for subsequent sensor detection. In order to detect the activity of Akt1, a substrate polypeptide containing three phosphorylation sites is designed. The Akt1 is subjected to phosphorylation of a substrate polypeptide, negative charges are increased, combination of the Akt1 and lanthanum ions on the surface of the electrode is enhanced, the impedance on the surface of the electrode is increased, and the activity of the Akt1 is reflected. And the unphosphorylated substrate polypeptide cannot be effectively attached and shows a relatively small impedance value. The accuracy of the sensor is verified by adopting an Akt1 inhibitor MK-2206. Human skin fibroblasts (GNHu49) are taken as a model, and research finds that the activity of Akt1 in senescent cells is remarkably reduced, so that an experimental basis is provided for researching the activity change of Akt1 and the change of cell functions in the skin senescence process. The invention provides a new technical means for skin aging research, anti-aging drug evaluation and cosmetic technology.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing an electrochemical sensor and its application in skin cell aging research, in particular, by Bacillus subtilis biosynthesis of hydroxyapatite, combined with the specific recognition of lanthanide ions and phosphate ions, an electrochemical sensor for the activity analysis and detection of protein kinase B (Akt1) is developed. This technology can be used to study the changes of Akt1 activity in the process of skin aging, and provides a new detection tool for skin aging research. The technology involved belongs to the field of biosynthetic materials and analytical sensing technology. BACKGROUND

[0002] Skin aging is one of the most significant and intuitive manifestations of the aging process in the human body, mainly manifested as decreased skin elasticity, wrinkle formation, dark and dry skin, and other phenomena. With age, the structure and function of the skin change, and this process is usually influenced by genetic factors, lifestyle, and environmental factors such as ultraviolet radiation and pollution. Skin aging is not only closely related to the natural aging process in the body, but also significantly affected by external adverse factors. The core mechanisms of skin aging mainly include the degradation of collagen and elastin, the weakening of epidermal cell proliferation and repair capacity, and the decline of skin barrier function. Over time, the skin's self-repairing function gradually weakens, antioxidant capacity decreases, and cell renewal and regeneration capacity decreases, thus accelerating the aging process. This aging process is usually manifested in the form of thinning of the epidermis, pigmentation, dryness, and loss of skin elasticity. In modern society, with the increasing trend of population aging, skin aging-induced health problems have gradually become an important factor affecting people's quality of life. Skin aging not only affects the appearance, but also is closely related to skin diseases, immune function decline, and other health problems. Although there are many anti-aging products on the market, most of them rely on external drugs or surgical intervention, with limited effect and possible side effects. Therefore, developing new early detection methods and effective intervention strategies has become a key issue in current skin aging research, and has important social significance and application value.

[0003] With the in-depth study of the mechanism of skin aging, more and more studies have begun to focus on the role of intracellular signaling pathways in the aging process. As an important serine / threonine kinase, Akt1 plays a key role in the process of skin aging. Akt1 is a core member of the PI3K / Akt / mTOR signaling pathway and is widely involved in cell proliferation, survival, metabolism, antioxidant response, and damage repair. Through the regulation of multiple downstream targets, Akt1 can promote the growth and repair of skin cells, thereby delaying the aging process. In the process of skin aging, the activity of Akt1 is directly related to the self-repair ability of skin cells. Studies have shown that the dysregulation of the Akt1 signaling pathway can lead to decreased proliferation, increased apoptosis, and weakened repair function of skin cells, thereby accelerating the aging process. On the contrary, the enhancement of Akt1 activity helps to promote the regeneration and repair of skin cells, effectively inhibiting the aging process. In addition, Akt1 is also involved in the regulation of skin antioxidant function, which can reduce the damage of oxidative stress to skin cells, thereby further delaying aging. Therefore, as a key regulatory factor in the process of skin aging, Akt1 has become an important target in the study of skin aging, with wide research value and application prospect. Although there are some studies on the detection of PKA (protein kinase A) activity, there is still a large gap in the efficient detection method of Akt1 activity. Studies have shown that Akt1 activity is closely related to skin aging and related diseases. Therefore, the development of more accurate and sensitive Akt1 activity detection methods is of great significance for the early diagnosis, intervention and treatment of skin aging.

[0004] The present application designs a protein kinase B electrochemical sensor based on biosynthesis of hydroxyapatite, and applies it to the analysis of Akt1 activity in the process of skin cell aging. The method first uses Bacillus subtilis to biosynthesize hydroxyapatite, and then deposits the biosynthesized hydroxyapatite material on the surface of a glassy carbon electrode by potential deposition. Because the material surface contains rich phosphate groups and hydroxyl groups, it can form specific coordination with lanthanum ions (La 3+) binding, thereby providing a good interface for subsequent sensing detection. In order to realize the detection of Akt1 activity, the present application further designs a substrate polypeptide containing three phosphorylation sites. In this system, Akt1 phosphorylates the substrate polypeptide, increasing the negative charge of the polypeptide, which can more effectively be adsorbed to the electrode surface by lanthanide ions. When the substrate polypeptide is phosphorylated by Akt1, the impedance value of the electrode surface increases, thereby realizing the detection of Akt1 activity through impedance changes. If the substrate polypeptide is not phosphorylated, it cannot be effectively attached to the electrode surface, showing a smaller impedance value. In addition, MK-2206 is used as an inhibitor of Akt1 to study its inhibitory effect on Akt1 activity, further verifying the accuracy and reliability of the sensor in detecting Akt1 activity. Finally, human skin fibroblasts (GNHu49) are used as a model to explore the difference in Akt1 activity between normal cells and senescent cells. The study found that the activity of Akt1 in senescent cells was significantly reduced, which provided strong experimental evidence for the subsequent study of the correlation between the change in Akt1 activity and the change in cell function in the process of skin aging. The method of the present application provides a new idea and technical means for exploring the changes in cell signaling in the process of skin aging, evaluating the effect of anti-aging drugs, and developing skin anti-aging and cosmetic technology. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a protein kinase B electrochemical sensor with good specificity, high sensitivity, fast detection speed, and accurate and reliable results, and its application research in the field of skin cell aging.

[0006] The technical solution adopted by the present application to solve the above technical problems is as follows: a biosynthetic hydroxyapatite-based protein kinase B electrochemical sensor and skin cell aging application research, the specific steps are as follows:

[0007] (1) Biosynthesis of hydroxyapatite (BHAP)

[0008] Collect 100-200 grams of soil sample, put the collected soil sample into 50 mL of sterile normal saline (0.85% NaCl), shake and mix thoroughly, remove the supernatant and centrifuge. Repeat this process 2-3 times to ensure that the excess bacteria are removed. Use LB medium (Luria-Bertani medium) for separation, inoculate the treated soil sample into the LB medium, vortex mix, and culture at 37℃, 180rpm on a shaker for 24 hours. After serial dilution of the culture solution by 1000 times, plate coating is performed on the LB agar plate. After 24-48 hours of culture, typical single colonies are selected for purification. The selected colonies are inoculated again into the LB medium after purification, and the growth characteristics are confirmed to be Bacillus subtilis.

[0009] The purified B. subtilis was inoculated into 100 mL LB medium with 1% (v / v) inoculation amount. The bacteria were cultured for 48 hours at 37°C, 150 rpm in a constant temperature shaker to promote the growth of bacteria and the phosphorization reaction. After 48 hours of bacterial culture, 2 mL of 1 M sodium glycerophosphate and 2 mL of 1 M calcium chloride (CaCl2) solution were added. The constant temperature shaker was continued to be used for culture, with the condition set as 37°C, 150 rpm, for 48 hours, during which the bacteria secreted organic acids to promote the precipitation of phosphorus and calcium, and finally form the hydroxyapatite (BHAP). After the end of the culture, the precipitate was collected by centrifugation at 6000 rpm for 10 minutes. The precipitate was washed with deionized water for 5 times to remove the soluble impurities. After centrifugation, the precipitate was continuously washed with deionized water to ensure the purity of the precipitate. Finally, the washed precipitate was transferred to an oven, with the temperature set as 50°C, and dried for 24 hours until the precipitate was completely dried, for storage.

[0010] (2) Preparation method of BHAP modified electrode

[0011] 10 mg of dried BHAP precipitate was taken and added to 10 mL of deionized water, and dispersed using an ultrasonic cleaning instrument, with the ultrasonic treatment time set as 20 minutes and the frequency set as 50 kHz, to ensure that the particles were completely dispersed and formed a uniform suspension, and finally a BHAP solution with a concentration of 1 mg / mL was obtained. A glassy carbon electrode was selected as the working electrode (labeled as GCE), an Ag / AgCl electrode was selected as the reference electrode, and a platinum electrode was selected as the counter electrode. The constant potential method was set, with the potential set as 0.4 V (vs. Ag / AgCl) and kept stable. 1 mL of the prepared BHAP solution was added to the electrolytic cell for electrochemical deposition, with the deposition time set as 30 minutes, and the deposition process was carried out at room temperature. After the deposition was completed, the electrode was taken out and gently washed with deionized water to remove the suspended particles that were not deposited on the surface, to ensure that the electrode surface was clean, and was labeled as BHAP / GCE.

[0012] (3) La 3+ @Preparation method of BHAP modified electrode

[0013] The electrode modified with BHAP was directly immersed in 0.05 M LaCl3 (lanthanum chloride solution) to ensure that the electrode was completely immersed in the solution. The immersion time was 30 minutes, and the process was carried out at room temperature. After the completion of the compounding process, the electrode surface was gently washed with deionized water to remove the unabsorbed lanthanum element ions, to ensure the stability of the electrode surface compound, and was labeled as La 3+ @BHAP / GCE

[0014] (4) Peptide / La 3+ @Preparation method of BHAP modified electrode

[0015] A 10 μL Akt1 phosphorylation reaction system was constructed according to the following procedure: 1 μL of 1000 nM Akt1 solution was added to achieve a final Akt1 concentration of 100 nM; 1 μL of 10 μM substrate polypeptide (Ac-Gly-Ser-Pro-Glu-Thr-Asp-Ser-Ala-NH2) solution was added to achieve a final substrate polypeptide concentration of 1 μM; 1 μL of 1 mM ATP solution was added to achieve a final ATP concentration of 100 μM; 1 μL of 10 mM MgCl2solution was added to achieve a final magnesium ion concentration of 1 mM; 1 μL of 1 mM DTT solution was added to achieve a final DTT concentration of 0.1 mM; 0.5 μL of 0.1 mg / mL BSA solution was added to achieve a final BSA concentration of 0.05 mg / mL; and the rest was filled up to 10 μL with 50 mM Tris-HCl (pH 7.5) buffer. The reaction system was reacted at a constant temperature of 30 °C, and the reaction time was 60 minutes. After the reaction was completed, the reaction solution was diluted 5-fold with deionized water, and then 10 μL of the diluted solution was added dropwise to the electrode surface and maintained for 10 minutes, which was marked as Peptide / La 3+ @BHAP / GCE, and then the electrode was placed in a 5 mM potassium ferricyanide redox solution to complete the electrochemical impedance (EIS) test.

[0016] Analysis and detection of different concentrations of Akt1: In the above step (step 4), the concentration of Akt1 was changed, and the final concentration was controlled to be: 0 nM (control group), 0.1 nM, 0.5 nM, 1 nM, 3 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 150 nM, 200 nM. Other conditions remained unchanged. By collecting the electrochemical EIS response signals of Akt1 at different concentrations, quantitative analysis of Akt1 activity can be achieved.

[0017] Analysis of different concentrations of MK-2206: In the above experimental procedure (step 4), first, 1 μL of MK-2206 was pre-incubated with Akt1 (final concentration: 100 nM) for 5 minutes to ensure the binding of MK-2206 to Akt1 and exert the inhibitory effect. Then, follow the original experimental procedure for subsequent operations, keeping other conditions unchanged. By this pre-incubation method, the inhibitory effect of MK-2206 on Akt1 can be effectively evaluated, providing important basic data for the detection of Akt1 and analysis and treatment of related diseases (such as skin aging, etc.). In order to further evaluate the efficacy of MK-2206, especially its inhibitory effect on Aktl, different concentrations of MK-2206 were used in the experiment, with the final concentration controlled at: 0 μM (control group), 0.01 μM, 0.05 μM, 0.08 μM, 1 μM, 10 μM, 20 μM, 30 μM, 50 μM, 70 μM, 100 μM, 150 μM, 200 μM. By using these different concentrations of MK-2206, the inhibitory effect of MK-2206 on Akt1 can be more comprehensively evaluated, and a concentration-response curve can be further drawn. By analyzing these data, the IC 50 value of MK-2206 can be calculated, thereby realizing the quantitative evaluation of its inhibitory effect on Akt1 activity.

[0018] Akt1 and its small molecule inhibitor MK-2206 were detected by electrochemical impedance spectroscopy (EIS) method. In the experiment, the alternating frequency range was set to 10 -2 ~ 10 5 Hz, the initial voltage was 0.245 V, the amplitude was 5 mV, and the solution was 5 mM [Fe(CN)6] 3- / 4- By measuring the electrochemical impedance values corresponding to different concentrations of Akt1, a quantitative relationship between electrochemical impedance and Akt1 concentration was established. According to this relationship, the Akt1 content in the sample to be tested can be accurately determined. In addition, using this method to monitor the activity of Akt1 in skin cells helps to reveal the relationship between epigenetics and skin aging and its development rules.

[0019] Invention principle: The present application designs an electrochemical sensor based on biosynthetic hydroxyapatite for detecting Akt1 activity. The sensor forms an interface rich in phosphate groups by uniformly depositing biosynthetic hydroxyapatite on the surface of a glassy carbon electrode. Subsequently, La 3+The specific binding and charge attraction of phosphate ions promote the adsorption of Akt1 phosphorylation substrate polypeptides. When the negative charge on the substrate polypeptide increases, it can be effectively attached to the electrode surface, resulting in an increase in electrode impedance, thereby realizing the detection of Akt1 activity. The accuracy of the sensor is verified by using Akt1 inhibitor MK-2206. The research using skin fibroblasts as a model shows that the Akt1 activity in senescent cells is significantly reduced, providing experimental basis for subsequent anti-aging research. The present application provides a new technical means for signal monitoring and anti-aging treatment in the process of skin aging.

[0020] Compared with the prior art, the present application has the following advantages: first, by using biosynthetic hydroxyapatite material, an environmentally friendly and efficient electrochemical sensor interface is provided, which has good stability and repeatability; second, the substrate polypeptide designed to contain multiple phosphorylation sites can effectively improve the sensitivity and specificity of Akt1 activity detection; in addition, the sensor can monitor the activity change of Akt1 in real time and quantitatively, and the accuracy thereof is verified by MK-2206, ensuring the reliability of the detection results; finally, the technology of the present application is not only suitable for Akt1 activity detection, but also can provide important data support in skin aging and anti-aging research, providing a new idea and technical means for early diagnosis and treatment of related diseases. The advantages thereof are:

[0021] (1) Electrochemical interface innovation. Traditional hydroxyapatite synthesis is mostly through chemical methods (such as hydrothermal method, precipitation method, etc.), while biosynthesis by Bacillus subtilis and other microorganisms not only is environmentally friendly and avoids the use of harmful chemical reagents, but also can be produced on a large scale at a lower cost without harsh conditions such as high temperature and high pressure. The crystal structure and particle size of hydroxyapatite can be precisely controlled by Bacillus subtilis biosynthesis, thereby improving its surface properties such as the distribution of phosphate groups. Biosynthetic hydroxyapatite has good biocompatibility and is suitable for biomedical and biosensor applications.

[0022] (2) Modification mechanism innovation. La 3+ The affinity of phosphate groups provides a new interface construction strategy for electrochemical sensors. La 3+ The combination of phosphate groups through electrostatic interaction forms a stable interface on the electrode surface, which is crucial for subsequent sensor signal detection (such as Akt1 phosphorylation reaction). La 3+ The combination of phosphate groups is relatively rare in the field of electrochemical sensors, especially in the detection of Akt1 activity, and such interface design is novel.

[0023] (3) The target is novel. Although the detection of PKA is relatively mature, the detection of Akt1 is still relatively less, especially in the direct monitoring of Akt1 activity by electrochemical method. The electrochemical sensor developed in the application can realize high sensitive detection of Akt1, and a linear equation has been obtained: the impedance response is linearly related to the concentration of Akt1 (R 2 = 0.991), and the detection limit is 0.053 nM, which proves its high sensitivity in low concentration Akt1 detection.

[0024] (4) High specificity. The sensor has high specificity for Akt1, and other control substances such as alkaline phosphatase (ALP), terminal transferase (TdT), glucose oxidase (GOx), uricase (UOx) and lysozyme (LZM) do not interfere with the system, ensuring accurate detection of Akt1 activity.

[0025] (5) Inhibitor screening. The electrochemical sensor of the application can be used for MK-2206 inhibitor screening, and the relationship between MK-2206 and Akt1 inhibition is analyzed by electrochemical response, which further verifies the applicability of the sensor.

[0026] (6) Strong practicability. The sensor has the advantages of low material consumption, fast operation and high sensitivity, and can realize efficient screening of Akt1 and small molecule inhibitors. At the same time, through intracellular Akt1 activity analysis, the relationship between Akt1 and skin aging is revealed, which has significant social, economic and scientific value.

[0027] In summary, the application provides a protein kinase B electrochemical sensor based on biosynthetic hydroxyapatite, which is successfully applied to skin cell aging research. The sensor has high sensitivity, good selectivity, simple operation, fast analysis and other advantages, and can be widely used in low concentration Akt1 detection and small molecule inhibitor screening, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the comparison chart of electrochemical EIS response of different modified electrodes;

[0029] Figure 2 is the linear relationship between the EIS response of the sensor to different concentrations of Akt1 and the concentration;

[0030] Figure 3 is the nonlinear fitting curve of the EIS response of the sensor to different concentrations of MK-2206 and the concentration;

[0031] Figure 4 is the selectivity experiment chart of the sensor;

[0032] Figure 5Figure for anti-interference experiment of the sensor;

[0033] Figure 6 Figure for detection experiment of the sensor on the content of Akt1 in cells under different H2O2 concentrations.

[0034] Figure 7 Figure for detection experiment of the sensor on the content of Akt1 in cells under different incubation times. DETAILED DESCRIPTION

[0035] The application will be further described in detail below with reference to the following examples.

[0036] Example 1 Preparation of electrochemical EIS sensor

[0037] (1) Biosynthesis of hydroxyapatite (BHAP)

[0038] A 100-200 gram soil sample was collected, and the collected soil sample was placed in 50 mL of sterile normal saline (0.85% NaCl) and mixed thoroughly by shaking. The supernatant was removed and centrifuged. This process was repeated 2-3 times to ensure that the excess bacteria were removed. The treated soil sample was inoculated into LB medium (Luria-Bertani medium) and mixed by vortexing, and then cultured at 37°C and 180 rpm for 24 hours. After the culture was diluted 1000 times, it was plated on LB agar plates. After 24-48 hours of culture, a single colony with a typical morphology was selected for purification. The selected colony was again inoculated into LB medium after purification, and its growth characteristics were confirmed.

[0039] The purified Bacillus subtilis was inoculated into 100 mL of LB medium at a volume ratio of 1%. The culture was incubated at 37°C and 150 rpm for 48 hours to promote bacterial growth and phosphorization. After 48 hours of bacterial culture, 2 mL of 1M sodium glycerophosphate and 2 mL of 1M calcium chloride (CaCl2) solution were added. The culture was continued on a constant temperature shaker at 37°C and 150 rpm for 48 hours. During this period, the bacteria secreted organic acids, which promoted the precipitation of phosphorus and calcium, and finally formed hydroxyapatite (BHAP). After the culture was completed, the precipitate was collected by centrifugation at 6000 rpm for 10 minutes. The precipitate was washed with deionized water 5 times to remove soluble impurities. After centrifugation, the precipitate was washed with deionized water to ensure the purity of the precipitate. Finally, the washed precipitate was transferred to an oven and dried at 50°C for 24 hours until the precipitate was completely dried and stored for future use.

[0040] (2) Preparation method of BHAP modified electrode

[0041] Take 10 mg of dry BHAP precipitate, add 10 mL of deionized water, disperse using an ultrasonic cleaner, ultrasonic treatment time is 20 minutes, frequency is set to 50 kHz, ensure that the particles are completely dispersed and form a uniform suspension, finally obtain a BHAP solution with a concentration of 1 mg / mL. Select a glassy carbon electrode as the working electrode (labeled GCE), an Ag / AgCl electrode as the reference electrode, and a platinum electrode as the counter electrode. Set the constant potential method, the potential is 0.4 V (vs. Ag / AgCl) and keep stable. Add 1 mL of prepared BHAP solution to the electrolytic cell for electrochemical deposition, the deposition time is 30 minutes, and the deposition process is carried out at room temperature. After deposition, remove the electrode and rinse it gently with deionized water to remove suspended particles that have not been deposited on the surface, ensuring that the electrode surface is clean, labeled as BHAP / GCE.

[0042] (3) La 3+ @Preparation method of BHAP modified electrode

[0043] The electrode modified with BHAP is directly immersed in 0.05M LaCl3 (lanthanum chloride solution), ensuring that the electrode is completely immersed in the solution. The soaking time is 30 minutes, and the process is carried out at room temperature. After the completion of the complexing process, the electrode surface is gently rinsed with deionized water to remove unabsorbed lanthanide element ions, ensuring the stability of the electrode surface complex, labeled as La 3+ @BHAP / GCE

[0044] (4) Peptide / La 3+ @Preparation method of BHAP modified electrode

[0045] Construct an Akt1 phosphorylation reaction system of 10 μL, the specific operation is as follows: add 1 μL of 1000 nM Akt1 solution to make the final concentration of Akt1 100 nM; add 1 μL of 10 μM substrate peptide (Ac-Gly-Ser-Pro-Glu-Thr-Asp-Ser-Ala-NH2) solution to make the final concentration of substrate peptide 1 μM; add 1 μL of 1 mM ATP solution to make the final concentration of ATP 100 μM; add 1 μL of 10 mM MgCl2 solution to make the final concentration of magnesium ions 1 mM; add 1 μL of 1 mM DTT solution to make the final concentration of DTT 0.1 mM; add 0.5 μL of 0.1 mg / mL BSA solution to make the final concentration of BSA 0.05 mg / mL; the rest is filled to 10 μL with 50 mM Tris-HCl (pH 7.5) buffer. The reaction system is reacted at a constant temperature of 30°C, and the reaction time is 60 minutes. After the reaction is completed, the reaction solution is diluted 5 times with deionized water, and then 10 μL of the diluted solution is added to the electrode surface and kept for 10 minutes, labeled as Peptide / La3+ @BHAP / GCE, and then the electrode was placed in 5 mM potassium ferricyanide redox solution to complete the electrochemical impedance test.

[0046] In 5 mM potassium ferricyanide redox solution ([Fe(CN)6] 3- / 4- ), the EIS response of each modified electrode was detected (as shown in Figure 1 ). With the modification of BHAP on the electrode surface, the impedance increased, which was mainly due to the presence of phosphate ions and hydroxyl groups on the surface of BHAP, which changed the charge distribution on the electrode surface and increased the impedance. Subsequently, with the further modification of La 3+ , the impedance decreased, indicating that the positive ion conduction of the La 3+ layer enhanced the conductivity of the electrode surface. When the polypeptide was adsorbed to the electrode surface, the impedance increased significantly, because the negative charge of the substrate polypeptide (Ac-Gly-Ser-Pro-Glu-Thr-Asp-Ser-Ala-NH2) increased after phosphorylation (phosphorylated peptide Ac-Gly-Ser(P)-Pro-Glu-Thr(P)-Asp-Ser(P)-Ala-NH2), and the negatively charged polypeptide interacted with the positively charged electrode surface, hindering the flow of electrons, resulting in an increase in impedance. These changes indicate the successful preparation of the sensor and its response characteristics under different modification conditions.

[0047] Example 2 Akt1 analysis detection

[0048] Based on step 4 of example 1, the concentration of Akt1 was changed to control its final concentration: 0 nM (control group), 0.1 nM, 0.5 nM, 1 nM, 3 nM, 5 nM, 10 nM, 20 nM, 50 nM, 100 nM, 150 nM, 200 nM. Other conditions remain unchanged. By collecting the electrochemical EIS response signals of Akt1 at different concentrations, quantitative analysis of Akt1 activity can be realized, and the results are shown in Figure 2 . The electrochemical EIS of the sensor has a good linear relationship with the concentration of Akt1, and the linear correlation equation of the electrochemical EIS response of the sensor to the log value of Akt1 concentration is y = 2029.02lgC Akt1 -49.63, R 2 = 0.991, the linear range is 0.1-100 nM, and the detection limit is 0.053 nM. It shows that the sensor realizes high sensitive detection of Akt1.

[0049] Example 3 MK-2206 analysis detection

[0050] In Step 4 of Example 1, 1 μL of MK-2206 was first pre-incubated with Akt1 (final concentration: 100 nM) for 5 minutes to ensure that MK-2206 could bind to Akt1 and effectively exert the inhibitory effect. Subsequently, the subsequent operations were continued according to the original experimental procedure, and other conditions were kept unchanged. This pre-incubation method helps to evaluate the inhibitory effect of MK-2206 on Akt1, providing important basic data for the detection of Akt1 and its analysis and treatment of related diseases (such as skin aging, etc.).

[0051] To further evaluate the inhibitory effect of MK-2206, especially its inhibitory effect on Akt1, different concentrations of MK-2206 were used in this experiment, with the final concentration set as: 0 μM (control group), 0.01 μM, 0.05 μM, 0.08 μM, 1 μM, 10 μM, 20 μM, 30 μM, 50 μM, 70 μM, 100 μM, 150 μM, 200 μM. Through these different concentrations of MK-2206, we can comprehensively evaluate its inhibitory effect on Akt1, and further draw the concentration-response curve. Through data analysis, the IC50 value of MK-2206 can be calculated, so as to quantitatively evaluate its inhibitory effect on Akt1 activity. As shown in Figure 3 , with the increase of the concentration of MK-2206, the EIS response value becomes smaller and smaller, indicating that the inhibitory effect of MK-2206 on Akt1 activity increases with the increase of the concentration. Through the test of the sensor, the half-inhibitory concentration (IC 50 ) of Akt1 is 23.9 μM.

[0052] Example 4 Specificity, Anti-interference analysis detection

[0053] In the selectivity and anti-interference experiment, the concentration of Akt1 (1) and other enzymes is 100 nM, and the abbreviations of other enzymes used are as follows: alkaline phosphatase (ALP, 2), terminal transferase (TdT, 3), glucose oxidase (GOx, 4), uricase (UOx, 5) and lysozyme (LZM, 6). In Step 4 of Example 1, sensors were prepared using the same concentration of other enzymes instead of Akt1. The results are shown in Figure 4 , compared with Akt1, the electrochemical EIS response of the sensor to other enzymes is smaller, indicating that the sensor has good selectivity for the detection of Akt1. In addition, as shown in Figure 5 , when Akt1 is present, even if other enzymes are superimposed, the measured impedance value is still similar, indicating that the presence of other enzymes basically does not affect the analysis and detection of Akt1.

[0054] Example 5 Intracellular Akt1 content analysis

[0055] To investigate the changes of intracellular Akt1 content when human skin fibroblasts (GNHu49) were induced into senescent state by hydrogen peroxide (H2O2). The effects of different concentrations of H2O2 and different time points of stimulation on Akt1 content were compared to explore the role of Akt1 in the aging process.

[0056] GNHu49 was inoculated into DMEM medium containing 10% fetal bovine serum and cultured in a cell incubator at 37°C and 5% CO2. Cells were seeded in 6-well plates or 96-well plates with an initial seeding density of 1×10 4 Cells / well and cultured to about 70-80% confluence. Two groups of experiments were set up: 1. Different concentrations of H2O2 were used to treat cells, with concentrations of 0 μM (control group), 50 μM, 100 μM and 200 μM; 2. After selecting H2O2 concentration of 100 μM, different time points were set: 0 hours, 24 hours, 48 hours and 72 hours. At the end of incubation, cells were gently washed twice with PBS to remove residual culture medium. Then lysis solution (such as RIPA buffer) was added for cell lysis, and incubated on ice for 20 minutes. Cells were scraped using a spatula and lysed by ultrasonic disruption. The lysed cell solution was centrifuged at 14,000 rpm for 10 minutes to separate cell membranes, nuclei and other organelles, and obtain supernatant containing dissolved proteins. The supernatant (i.e. cytoplasmic fraction) was used for subsequent analysis, including Akt1.

[0057] As shown in Figure 6 , the electrochemical EIS signals of cells treated with different concentrations of H2O2 (0 μM, 50 μM, 100 μM, 200 μM) for 24 hours were compared. The results showed that in the 0 μM, 50 μM, 100 μM treatment groups, the EIS signal increased slightly, indicating that the induction of H2O2 might cause a certain degree of oxidative stress response. Oxidative stress usually maintains cellular homeostasis by activating antioxidant defense mechanisms (such as Nrf2 pathway). However, when the concentration of H2O2 increased to 200 μM, the content of Akt1 decreased significantly, suggesting that high concentration of H2O2 might inhibit the expression of Akt1 by up-regulating other oxidative stress receptors or repair pathways, thereby exacerbating the process of cell aging.

[0058] As shown in Figure 7As shown, after treatment with 100 μM H2O2, different time points (0 h, 24 h, 48 h, 72 h) were set. After 24 h of incubation, the expression of Akt1 was up-regulated, which might be that the cells tried to resist oxidative damage through the Akt pathway, promoting cell repair and survival. At this time, the concentration of H2O2 might trigger stronger oxidative stress and activate the PI3K / Akt signaling pathway. However, as the oxidative damage continued (to 48 h and 72 h), the cells gradually entered the aging or apoptosis stage. As an important part of the anti-aging signaling pathway, the decrease in the expression of Akt1 might indicate that the cells entered the aging state, and the cell proliferation and repair ability decreased. These results showed that the changes in the expression of Akt1 could be used as a marker for the aging process of cells, and might be involved in the regulation of aging. By further analyzing these changes, we can better understand the role of Akt1 in cell aging and the changes in its signaling pathway, providing important experimental data for the study of aging mechanisms.

[0059] Of course, the above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the spirit and scope of the present application should also be within the scope of protection of the present application.

Claims

1. Research on the electrochemical sensor of biosynthesized hydroxyapatite-based protein kinase B and its application in skin cell aging, characterized in that... The mechanism is as follows: This invention designs an electrochemical sensor based on biosynthesized hydroxyapatite for detecting Akt1 activity. This sensor achieves this by uniformly depositing biosynthesized hydroxyapatite onto the surface of a glassy carbon electrode, forming an interface rich in phosphate groups. Based on this, lanthanum ions (La...)... 3+ By increasing the positive charge at the interface and specifically binding with phosphate ions, an ideal interface is provided for the detection of Akt1 activity. When Akt1 phosphorylates the substrate peptide, the negative charge on the substrate increases, allowing it to effectively adhere to the electrode surface, resulting in increased electrode impedance and thus enabling the electrochemical detection of Akt1 activity. The accuracy of the sensor was verified by using the Akt1 inhibitor MK-2206. Using skin fibroblasts as a model, the study showed that Akt1 activity is significantly reduced in senescent cells, providing experimental evidence for subsequent anti-aging research. This invention provides a new technical means for monitoring signal changes during skin aging and for anti-aging treatment.

2. The electrochemical sensor for biosynthesized hydroxyapatite-based protein kinase B and its application in skin cell aging research according to claim 1, characterized in that: Traditional hydroxyapatite synthesis typically employs chemical methods (such as hydrothermal synthesis and precipitation), while this invention utilizes microbial biosynthesis using Bacillus subtilis and other microorganisms. This method offers advantages such as being environmentally friendly, using harmless chemical reagents, and being low-cost, without requiring harsh conditions like high temperature and high pressure. Through biosynthesis, the crystal structure and particle size of hydroxyapatite can be precisely controlled, further optimizing its surface properties, such as the distribution of phosphate groups. This biosynthesized hydroxyapatite exhibits excellent biocompatibility and is suitable for applications in biomedicine and biosensors.

3. The electrochemical sensor for biosynthesized hydroxyapatite-based protein kinase B and its application in skin cell aging research according to claim 1, characterized in that: La 3+ The high affinity for phosphate groups provides an innovative interface construction strategy for the sensor. 3+ Through electrostatic and specific binding interactions, it binds to phosphate ions, forming a stable interface on the electrode surface, ensuring the accuracy of subsequent signal detection, and playing a particularly important role in the Akt1 phosphorylation reaction. 3+ The binding of phosphate groups has limited applications in the field of electrochemical sensors, and its application in Akt1 activity detection is still relatively novel.

4. The electrochemical sensor for biosynthesized hydroxyapatite-based protein kinase B and its application in skin cell aging research according to claim 1, characterized in that: Currently, the electrochemical detection of Akt1 remains a technological gap. The electrochemical sensor developed in this invention achieves high-sensitivity detection of Akt1, and a linear equation has been obtained: the impedance response is linearly correlated with the Akt1 concentration (R0). 2 =0.991), and the detection limit is 0.053 nM, indicating that the sensor has high sensitivity in the detection of low concentrations of Akt1.

5. The electrochemical sensor for biosynthesized hydroxyapatite-based protein kinase B and its application in skin cell aging research according to claim 1, characterized in that: Oxidative stress induced by H2O2 treatment led to changes in Akt1 activity. At low concentrations of H2O2 (≤100 μM), Akt1 expression was upregulated, suggesting that cells may resist oxidative stress by enhancing Akt1 activity. However, at high concentrations of H2O2 (≥200 μM), Akt1 expression significantly decreased, suggesting that excessive oxidative stress may exacerbate cellular senescence by inhibiting Akt1 activity. Furthermore, when H2O2 treatment lasted for more than 24 hours, Akt1 expression decreased significantly again, indicating that prolonged oxidative damage leads to cellular senescence or apoptosis. Changes in Akt1 expression at different concentrations and time points of H2O2 treatment can serve as a marker of cellular senescence, providing important experimental evidence for further research into the mechanisms of senescence.

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