An assessment method for depression biomarkers and its application

Abstract

This invention discloses a method for assessing a biomarker for depression and its application. A biosensor BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE is obtained through layer-by-layer assembly of BSA, AbGRα, Trp-AuNPs@IL-rGO, and GCE. This biosensor is then applied to the detection of the depression biomarker GRα protein. The linear regression equation for GRα in the ranges of 0.001–50 ng / mL and 50–500 ng / mL is ΔI(μA) = 1.907*lg[GRα] + 6.592(R). 2 =0.9907) and ΔI(μA)=14.07*lg[GRα]‑14.63(R 2 =0.9904), the limit of detection (LOD) was 0.283 pg / mL, and the reproducibility, long-term stability and specificity were all good.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to an assessment method for biomarkers of depression and its application. Background Technology

[0002] Currently, there are numerous methods available in the market and laboratories for detecting GRα protein, which mainly include:

[0003] 1. Gene Level. ① Southern blot / Northern blot method: The Southern blot / Northern blot method, which uses probe hybridization technology, can detect GR levels or its gene polymorphisms at the DNA or mRNA level. ② In situ hybridization method: The mRNA of the gene to be detected is hybridized with a corresponding fluorescently labeled nucleotide probe for detection. ③ PCR technology: Including reverse transcription PCR (RT-PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, which are suitable for detecting GR gene polymorphisms.

[0004] 2. Protein Levels. ① Radionuclide-labeled glucocorticoid binding assay: suitable for detecting biologically active glucocorticoids (GR) in the cytoplasm; competition assays can also be used for new drug development. ② Western blotting: a classic method for protein detection. SA Weaver et al. used this method to detect the content of glucocorticoid receptors in intact pig brains and pituitary glands. ③ Immunohistochemistry: allows for relatively accurate localization of GR. ④ ELISA: a sandwich-type antigen-antibody reaction. ⑤ Flow cytometry: allows for multi-parameter analysis of specimens; for example, in blood specimens, it can accurately select a specific cell population to analyze its GR expression level.

[0005] However, there are no reports on the application of biosensing technology to GR. Currently, the content of GRα protein is detected by RT-qPCR, Western blotting, and ELISA. However, when faced with the challenges of rapid detection and increasing complexity of biological samples, the above detection methods have drawbacks such as low sensitivity and complex operation. Biosensors with high simplicity and sensitivity can perform trace detection of complex samples in a shorter time, and such biosensors are urgently needed for development. Summary of the Invention

[0006] The purpose of this invention is to address the problems of time-consuming and labor-intensive detection of GRα protein and poor detection sensitivity in the existing technology, and to provide a GRα protein biosensor.

[0007] Another objective of this invention is to provide applications for GRα protein biosensors.

[0008] The technical solution adopted to achieve the purpose of this invention is:

[0009] A GRα protein biosensor is prepared by the following steps:

[0010] Step 1: Drop-coat the Trp-AuNPs@IL-rGO suspension onto a clean glassy carbon electrode GCE, and after drying, obtain Trp-AuNPs@IL-rGO / GCE. The Trp-AuNPs@IL-rGO includes amino-ion graphene and gold nanoparticles that are reduced and encapsulated by tryptophan.

[0011] Step 2: Activate Trp-AuNPs@IL-rGO / GCE using EDC / NHS solution, then immerse Trp-AuNPs@IL-rGO / GCE in ethylenediamine solution to block the carboxyl groups. Modify Trp-AuNPs@IL-rGO / GCE with antibody. During the modification process, the carboxyl groups at the Fc base and Fab shoulder of antibody AbGRα are immobilized onto the amino groups of Trp-AuNPs@IL-rGO / GCE. Use BSA to remove non-specific binding sites to block the electrode surface. Wash the electrode with PBS buffer solution to remove unbound BSA from the electrode surface, obtaining the biosensor BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE.

[0012] In the above technical solution, the cleaning method in step 1 is to polish the glassy carbon electrode on the surface of suede with dispersed alumina powder, then wash it alternately with water and anhydrous ethanol, and dry it in a nitrogen atmosphere to obtain the cleaned glassy carbon electrode GCE.

[0013] In the above technical solution, the Trp-AuNPs@IL-rGO is prepared through the following steps:

[0014] Step s1: Disperse GO in double-distilled water, add amino ionic liquid, sonicate and stir continuously, then add alkaline substance and sonicate again, heat and stir the resulting mixed solution under reflux, centrifuge after the reaction is complete, wash the obtained solid with ethanol and double-distilled water alternately to obtain amino ionic graphene IL-rGO.

[0015] Step s2: After heating the aqueous solution of HAuCl4 to boiling, amino acid solution is added dropwise. After the reaction is completed, the resulting reaction system is quenched in an ultrasonic ice bath. After quenching, the system is centrifuged and the obtained solid particles are washed with double-distilled water to obtain amino acid-reduced and encapsulated gold nanoparticles AA-Au NPs.

[0016] Step s3: The IL-rGO and AA-Au NPs are uniformly dispersed in double-distilled water and then mixed and stirred. The mass ratio of IL-rGO and AA-Au NPs is 5:1 to 1:1. After the reaction is completed, the mixture is centrifuged to obtain AA-Au NPs@IL-rGO.

[0017] In the above technical solution, the Trp-AuNPs@IL-rGO suspension in step 1 is an aqueous solution of Trp-AuNPs@IL-rGO with a concentration of 0.5-1 μg / mL and a volume of 5-20 μL.

[0018] In the above technical solution, the EDC / NHS solution in step 2 is a mixed solution of EDC aqueous solution and NHS aqueous solution, the concentration of EDC aqueous solution is 50-300mM, the concentration of NHS aqueous solution is 50-300mM, the volume ratio of EDC aqueous solution to NHS aqueous solution is 1:1, the activation time is 20-50 minutes, and the concentration of ethylenediamine solution is 50-300mM.

[0019] In the above technical solution, in step 2, 0.5-1% BSA is used to remove non-specific binding sites, the concentration of the PBS buffer solution is 8-12 mM, and the pH of the PBS buffer solution is 7.2-7.6.

[0020] In the above technical solution, the concentration of antibody AbGRα in step 2 is 0.5-1 μg / mL, and the incubation time of AbGRα is 60-120 minutes.

[0021] In another aspect of the present invention, the biosensor is used in detecting GRα protein content.

[0022] In another aspect of the present invention, the method for detecting GRα protein using a biosensor, i.e., an assessment method for depression biomarkers, includes the following steps:

[0023] Step S1: Incubate BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE in GRα protein standard solutions of different concentrations. After incubation, perform tests to establish a concentration-DPV standard curve.

[0024] Step S2: Immerse BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE in the GRα protein solution to be tested for incubation. After incubation, perform DPV test. Substitute the test results into the standard curve obtained in step S1 to calculate the concentration of the GRα protein solution to be tested.

[0025] In the above technical solution, the incubation time for immersing in GRα protein standard solutions of different concentrations in step S1 and the incubation time for immersing in the GRα protein solution to be tested in step S2 are both 90-150 minutes.

[0026] In the above technical solution, in step S1, when the concentration of the GRα protein standard solution is 0.001-50 ng / mL, the equation of the standard curve is ΔI(μA)=1.907*lg[GRα]+6.592(R 2 =0.9907), when the concentration of the GRα protein standard solution is 50 to 500 ng / mL, the equation of the standard curve is ΔI(μA) = 14.07lg[GRα] - 14.63(R) 2 =0.9904), where [GRα] is the concentration of GRα protein.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. This invention is the first to use GRα protein as a biomarker (a biomarker for depression) to develop an electrochemical biosensing platform. The sensitivity of the biosensor is improved by using amino acid-coated AuNPs and IL-rGO, thereby increasing the sensitivity of GRα analysis and reducing the detection difficulty and cost.

[0029] 2. The BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE biosensor exhibits excellent detection performance, with a detection range from 0.001 ng / mL to 500 ng / mL and a LOD value of 0.283 pg / mL. This biosensor can sensitively detect the GRα content in samples, demonstrating good stability and reproducibility. GRα levels in the hippocampus and blood cells can be measured using this sensor, and it shows good correlation with ELISA and Western blot analysis.

[0030] 3. Compared with existing methods for detecting GRα protein (ELISA and Western blot), this method requires less time, is simpler to operate, and has lower detection costs. The sensor of this invention can accurately assess the GRα level of the sample without the need for a large number of samples and secondary antibody incubation. The detection method of this invention is novel and simple, which is helpful for the research and development of antidepressant drugs. Attached Figure Description

[0031] Figure 1 The image shows the antibody immobilization ability of tryptophan-reduced gold nanoparticles evaluated using absorbance.

[0032] Figure 2 The diagram shows the fabrication process of the GR-α immunobiosensor.

[0033] Figure 3 The results of cyclic voltammetry (CV) tests are shown for glassy carbon electrodes modified with different materials (rGO, IL-rGO, AuNPs@IL-rGO, Trp-AuNPs@IL-rGO).

[0034] Figure 4 The figure shown is a differential pulse voltammetry (DPV) test result after layer-by-layer self-assembled glassy carbon electrodes.

[0035] Figure 5 The figure shown is the electrochemical impedance spectroscopy (EIS) test results after the layer-by-layer self-assembled glassy carbon electrode.

[0036] Figure 6 The diagram shows the material quantities and antibody incubation time for the GRα protein detection method of the present invention. An optimization diagram of antibody incubation amount and antigen incubation time is also shown.

[0037] Figure 7 The results of BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE for detecting GRα from 1 pg / mL to 500 ng / mL are shown, where A is the raw DPV data and B is the standard curve.

[0038] Figure 8 The diagram shows the verification of the anti-interference ability, stability, and repeatability of the constructed sensing platform.

[0039] Figure 9 The diagram shows the detection of GRα in blood cells and hippocampus of rats using commercial ELISA, WB, and the biosensor of the present invention in control group (Con), model group (Mod), and fluoxetine group (Flu). A is a schematic diagram of animal grouping (n=6), B is the detection of GRα by enzyme-linked immunosorbent assay (ELISA), C is the detection of GRα by Western blot assay, D is the detection of GRα in hippocampus using the present invention, and E is the detection of GRα in blood cells using the present invention. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0041] Example 1 1.1

[0043] The preparation method of Trp-AuNPs@IL-rGO includes the following steps:

[0044] Step 1: Synthesis of amino-ionic graphene (IL-rGO)

[0045] Dispersing GO in 25 mL of double-distilled water yields 0.5 mg / mL. -1 A homogeneous GO solution was prepared, and 5g of an amino-ionic viscous liquid (IL-NH2, 1-aminopropyl-3-methylimidazolium chloride (purchased from Shanghai Chengjie Chemical Co., Ltd.)) was added. The resulting mixture was continuously stirred and sonicated for 30 minutes, followed by the addition of 25mg KOH. The mixture was then sonicated for another 50 minutes. After sonication, the turbid mixture transformed into a homogeneous solution (a uniformly dispersed gray solution). This homogeneous solution was heated under reflux at 80°C and vigorously stirred for 24 hours to obtain a reaction solution. The reaction solution was centrifuged, and the precipitate was IL-rGO. The precipitate was washed alternately with ethanol and double-distilled water, and then dispersed in double-distilled water (1mg / mL). It was stored at 4°C for subsequent experiments.

[0046] Step 2: Synthesize tryptophan-reduced and encapsulated gold nanoparticles (Trp-AuNPs)

[0047] In this system, Trp is used as a reducing agent to fully utilize its reducing properties. In addition, Trp also acts as a capping agent and a bridging agent in this system. When it acts as a capping agent, it can improve the stability of nanomaterials and make them less prone to aggregation. When it acts as a bridging agent, the free amino and carboxyl groups can effectively fix antibodies, while the free carboxyl groups can fix ionic amino acids.

[0048] The specific procedure is as follows: heat 100 mL of HAuCl4 (0.1 mM) aqueous solution to boiling, and add 3.0 mL of 100 mM tryptophan solution dropwise at a constant rate of 0.2 mL / min. -1 After 10-15 minutes, a red colloid was observed to form. The synthesis was completed after 30 minutes. To terminate the reaction, the resulting solution was quenched in an ultrasonic ice bath for 15 minutes to ensure good dispersion of the nanoparticles. Then, the solution was centrifuged at 9000 rpm, and the supernatant was discarded to obtain tryptophan-reduced and encapsulated gold nanoparticles (Trp-AuNPs). These nanoparticles were washed 2-3 times with double-distilled water, dispersed in double-distilled water (1 mg / mL), and stored at 4°C until use.

[0049] Step 3: Load amino acid-encapsulated gold nanoparticles onto amino-ionized graphene to obtain Trp-AuNPs@IL-rGO

[0050] The aqueous solution of IL-rGO obtained in step 1 and the aqueous solution of Trp-AuNPs obtained in step 2 were mixed in equal volumes at 37°C for 30 minutes. The two were bound by the amino groups on the surface of IL-rGO and the carboxyl groups exposed on the surface of Trp-Au NPs. The loading of Trp-Au NPs onto the surface of IL-rGO was achieved by physical adsorption. 1.2

[0052] The antibody immobilization and antigen detection capabilities of tryptophan-reduced gold nanoparticles were investigated. First, different materials, such as Trp-AuNPs@IL-rGO, were adsorbed into 96-well plates, with 20 μL of material added to each well. The plates were incubated at 37°C for two hours, and the unbound fraction was discarded. The plates were then washed three times with PBS. Next, 10 μL of EDC / NHS (0.1 mM, 1:1 v / v) was added to activate the materials, and the plates were activated at room temperature for 30 min. The supernatant was discarded, and the plates were washed three more times with PBS. Finally, 3.75 μL of mL of [unspecified solution] was added to each well. -1 The antibody AbGRα was incubated with the material at 4°C for 90 min, followed by the same washing procedure. 10 μL of 1:500 HRP-labeled secondary antibody was added to each well and incubated for 1 hour, followed by the same washing procedure. Then, 150 μL of TMB was used for color development at 37°C for 30 min, and finally, 50 μL of 2 mM sulfuric acid was used for termination. The absorbance was measured at 650 nm. Figure 1 As shown in the results, Trp-AuNPs@IL-rGO exhibits superior performance in antibody immobilization compared to AuNPs@IL-rGO. 1.3

[0054] A method for preparing a GRα protein sensor includes the following steps:

[0055] Step 1, as follows Figure 2 As shown, GCE was polished on the surface of suede with dispersed alumina powder, then washed with water and anhydrous ethanol alternately for 2 minutes, and finally dried in nitrogen. A Trp-AuNPs@IL-rGO suspension with a concentration of 1 mg / mL and a volume of 10 μL was drop-coated onto the cleaned glassy carbon electrode and air-dried at room temperature to obtain Trp-AuNPs@IL-rGO / GCE. The Trp-AuNPs@IL-rGO includes amino-ion graphene and gold nanoparticles reduced and encapsulated by tryptophan on it.

[0056] Step 2: Trp-AuNPs@IL-rGO / GCE was activated for 30 minutes using a 1:1 volume ratio of 100mM EDC aqueous solution and NHS aqueous solution. Then, Trp-AuNPs@IL-rGO / GCE was immersed in a 100mM ethylenediamine solution to block the carboxyl groups. Utilizing the carboxyl groups at the bottom of the antibody Fc and the shoulder of the Fab, a concentration of 0.75 μg / mL was added. -1Antibody AbGRα solution was immobilized onto the amino group of Trp-AuNPs@IL-rGO / GCE (specifically, the immobilization method was as follows: antibody AbGRα solution was dropped onto the electrode interface of Trp-AuNPs@IL-rGO / GCE, where antibody AbGRα solution was a mixture of antibody AbGRα and 10mM PBS), and incubated for 90 minutes. Then, non-specific binding sites on AbGRα-Trp-AuNPs@IL-rGO / GCE were removed with 1% BSA solution to block the electrode surface (to prevent non-specific adsorption of immunoassay reagents and to prevent the analyte GRα from adhering to sites other than antibody AbGRα). The electrode was then gradually washed with PBS (10mM, pH=7.4) buffer solution to remove BSA adhering to the electrode surface, resulting in BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE.

[0057] Example 2

[0058] The stepwise fabrication process of the BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE immunosensor was electrochemically characterized by CV, DPV, and EIS. All electrochemical measurements were performed in a standard three-electrode system, with different modified GCEs as the working electrode, an Ag / AgCl electrode containing saturated KCl as the reference electrode, and a 1 mm diameter platinum wire as the counter electrode. CV experiments were conducted in a solution containing 5.0 mmol / L Fe(CN)₆. 3- / 4- The experiments were conducted in a 0.1 mol / L KCl solution. CV experiments were performed at 100 mV / s within a potential range of -0.2 to +0.6 V. DPV experiments were performed in a solution containing 5.0 mmol / L Fe(CN)6. 3- / 4- PBS solution (0.1 mol / L) -1 The experiment was conducted at pH 7.4. EIS experiments were performed under open-circuit conditions with a frequency range of 6000 kHz to 1 Hz, an amplitude signal of 5 mV, and frequency intervals divided into 50 logarithmically spaced measurement points. All electrochemical experiments were conducted at room temperature.

[0059] The electroactivity of Trp-AuNPs@IL-rGO / GCE was tested at a scan rate of 100 mV / s using 5.0 mM [Fe(CN)6]. 3- / 4- The probe records the cyclic voltammetry curves of each electrode in a 0.1 M KCl aqueous solution (e.g., ...). Figure 3(As shown). Compared to the GCE electrode, the redox peak current of rGO / GCE is increased, the redox peak current of IL-rGO / GCE is further increased, the redox peak current of AuNPs@IL-rGO / GCE is significantly increased, and Trp-AuNPs@IL-rGO further promotes electron transfer on the GCE. The electroactive surface area is quantitatively estimated according to the Randles-Sevcikequation:

[0060] I pc =2.69×10 5 n 3 / 2 A eff D 1 / 2 v 1 / 2 C0 (1)

[0061] Where I pc Represents the cathode peak current, where n is the electron transfer number, A eff Corresponding electroactive surface area (cm²) 2 D is [Fe(CN)6] 3- / 4- The diffusion coefficient of the redox probe. (D = 6.70 ± 0.02 × 10⁻⁶) -6 cm 2 s -1 ), where v is the scan rate (0.1Vs) -1 C0 represents the concentration probe molecule (mmol / cm). -3 ).

[0062] The electroactive surface area was calculated using equation (1). The electroactive surface area of ​​Trp-AuNPs@IL-rGO / GCE was 5.93 × 10⁻⁶. -2 cm 2 The electrochemical surface area of ​​Trp-AuNPs@IL-rGO / GCE was 2.44, 1.95, 1.27, and 1.06 times that of GCE, rGO / GCE, IL-rGO / GCE, and AuNPs@IL-rGO / GCE (AuNPs are gold nanoparticles reduced by sodium citrate), respectively. Compared with AuNPs@IL-rGO / GCE, the electroactive surface area of ​​Trp-AuNPs@IL-rGO / GCE was not reduced. The good conductivity of AuNPs and the protonation and deprotonation of amino acids both contribute to accelerating electron transport. The significant enhancement of the electrochemical signal of Trp-AuNPs@IL-rGO compared with GCE, rGO / GCE, and IL-rGO / GCE is as follows: IL-rGO changed the irreversible aggregates of rGO on the electrode surface (after the formation of unfunctionalized rGO aggregates, they cannot be redispersed by ultrasound, etc.), increased the active surface area, and provided more active arch sites for AuNPs. Therefore, the electrode functionalized with Trp-AuNPs@IL-rGO exhibited the best electrochemical activity among them.

[0063] The stepwise construction process of a biosensor was measured using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). For example... Figure 4 As shown, due to the modification of non-conductive protein molecules hindering electron transfer, the current peak gradually decreased with the layer-by-layer immobilization of AbGRα, BSA, and GRα antibodies on AuNPs@IL-rGO / GCE. Furthermore, EIS also confirmed the successful construction of the proposed biosensor. Figure 5 The Nyquist plot shown uses the diameter of the semicircle to represent the Rct of the electrode, used to describe the immersion in [Fe(CN)6]. 3- / 4- The resistive behavior of different modified electrodes in solution. Compared with the GCE electrode, the resistance (Rct) of Trp-AuNPs@IL-rGO / GCE is significantly reduced, while Rct gradually increases with modification with AbGRα, BSA and GRα, indicating that the GRα biosensor was successfully constructed.

[0064] Example 3

[0065] A method for detecting the depression biomarker GRα protein using a biosensor prepared in Example 1 includes the following steps:

[0066] Step S1: Immerse BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE in GRα protein standard solutions of different concentrations and incubate for 90 min. After incubation, perform tests to establish a concentration-DPV standard curve.

[0067] Step S2: Immerse BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE in the GRα protein solution to be tested and incubate for 90 min. After incubation, perform DPV test and substitute the test results into the standard curve obtained in step S1 to calculate the concentration of the GRα protein solution to be tested.

[0068] Experimental variables can directly affect the performance of the GRα biosensor, such as Figure 6 As shown, a control experiment was conducted using the biosensor prepared by Trp-AuNPs@IL-rGO / GCE with 0 and 10.0 ng / mL GRα standards to optimize the performance of the GRα biosensor and its detection capability for GRα under different conditions.

[0069] To optimize detection performance, the volume of the Trp-AuNPs@IL-rGO suspension was investigated. Figure 6 (A), AbGRα incubation time ( Figure 6 (B) AbGRα concentration ( Figure 6 (C) and GRα incubation time ( Figure 6The influence of AbGRα on the performance of the GRα biosensor and its detection capability was investigated. Optimization criteria were established based on the generated blank-to-signal ratio (B / S). Optimal detection performance was achieved when the volume of Trp-AuNPs@IL-rGO (B / S 1.36) was 10 μL, the AbGRα incubation time was 90 min (B / S 1.09), the AbGRα concentration was 75 μg / mL (B / S 1.16), and the GRα incubation time was 120 min (B / S 1.09). Therefore, these conditions were used in subsequent experiments.

[0070] The analytical capabilities of the GRα biosensor obtained through DPV evaluation ( Figure 7 ΔI (the difference between the current measured in the presence and absence of the antigen) increases with increasing GRα concentration. The current response of DPV at different GRα concentrations is shown below. Figure 7 As shown in Figure A, ΔI showed two significant linear correlations with the logarithms of GRα concentrations from 0.001 to 50 ng / mL and from 50 to 500 ng / mL, respectively. The linear regression equation was ΔI(μA) = 1.907 × lg[GRα] + 6.592(R²). 2 =0.9907) and ΔI(μA)=14.07×lg[GRα]-14.63(R 2 =0.9904)(e.g. Figure 7 (As shown in B). Based on the 3sb / m standard, where sb is the standard deviation of 20 measurements performed without GRα, and m is the slope value of the calibration plot, the calculated limit of detection (LOD) is 0.283 pg / mL.

[0071] After the test is completed, the GRα protein is eluted with a regeneration solution, and the resulting biosensor can be recycled.

[0072] Example 4

[0073] Reproducibility, long-term stability, and specificity are key indicators for validating the efficiency and application value of biosensors. To determine total GRα protein in clinical samples such as blood cells and tissue extracts, IL-6, glucose oxidase (GOD), cholesterol esterase (ChOx), lactate dehydrogenase (LD), and ganglioside esterase (GE) (corresponding to...) were selected. Figure 8 In section A, numbers 2-6 on the horizontal axis are considered interfering substances. Characterization was performed by measuring GRα protein (0 or 10 ng / mL) in the serum of healthy individuals. Figure 8 As shown in Figure A, the B / S ratio changes were not significant or almost nonexistent among the different groups measuring GRα, indicating that the biosensor of the present invention can perform the determination of GRα protein in complex systems.

[0074] The stability of BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE was verified by the following method: five biosensors prepared using the same method were stored at 4°C, and 0 and 10 ng / mL GRα standard solutions were measured using the biosensors over 1–15 days. Results are as follows: Figure 8 As shown in Figure B, the B / S values ​​measured by the biosensor did not differ significantly over fifteen days, indicating good stability. Furthermore, the response reproducibility of the biosensor was evaluated by testing the performance of five biosensors prepared in the same manner over the same number of days. Figure 8 As shown in Figure C, consistent reproducibility can be observed with RSD values ​​of 1.1% and 1.3%.

[0075] Example 5

[0076] Eighteen 8-week-old male Sprague Dawley (SD) rats (220-250g; SCXK(JING)2017-0005) were purchased from the China National Institutes for Food and Drug Control (Beijing, China) and housed in a standard animal housing (room temperature: 20℃-24℃; relative humidity: 30%-40%). The rats were randomly divided into three groups: a control group (Con) and a model group (Mod). The control group was housed under normal conditions with sufficient water and light, with a 12-hour dark / light cycle. The model group (Mod) and the fluoxetine group (Flu) were used to induce chronic unpredictable mild stress (CUMS). The Con and Mod groups were administered physiological saline (10mL / kg) by gavage, while the model group was administered fluoxetine (10mg / kg) by gavage. Samples were collected after 28 days. After anesthesia with 10% chloral hydrate (0.03mL / kg), blood was collected from the abdominal aorta. After the rats were deemed dead, their hippocampi were harvested.

[0077] Chronic unpredictable mild stress (CUMS) was administered to rats for 4 weeks under the following conditions: dehydration (24 h), forced swimming (10 min), fasting (24 h), confinement in an empty water bottle (Wahaha, China) (4 h), crowded enclosure (24 h), tail clamping (1 min), 45° cage tilt (24 h), diurnal reversal (12 h / 12 ​​h), and noise (20 min). To ensure unpredictability, the above protocols were randomly assigned, with rats receiving one of them daily. Forced swimming (FST) was performed to assess depressive-like states in the rats. The rat experimental protocol was approved by the Animal Ethics and Welfare Committee, approval number IRM-DWLL-2020111.

[0078] Total protein was prepared from blood cells and hippocampal tissue using cell lysis buffer or RIPA buffer for further ELISA and WB analysis to determine GRα levels. ELISA and WB procedures were performed strictly according to the manufacturer's instructions. Briefly, the supernatant was boiled for 3 minutes and loaded for SDS / PAGE, followed immediately by transfer to a PVDF membrane. The desired membrane was blocked with 5% skim milk and incubated overnight with primary antibodies including AbGRα (32.8 kDa) and anti-β actin (41.6 kDa). After washing with PBST, the membrane was incubated with secondary antibody (1:2500) at room temperature for 1 hour. After thorough washing, the membrane was incubated with chemiluminescent HRP substrate and then exposed to a Tanon-5200 chemiluminescence instrument (Shanghai, China). The relative optical density of the bands was quantified using free software (NIH, Bethesda, MD, USA). Figure 9 As shown in Figures B, C, D, and E, the detection results of ELISA and Western blot demonstrate the consistency and reliability of the sensor detection results in this application.

[0079] To further demonstrate the practicality of the biosensor for detecting GRα protein in this invention, an analysis of GRα in clinical samples was conducted. In this study, GRα levels in the hippocampus and blood cells from different groups (control group (healthy), model (depressed), and fluoxetine (fluoxetine-induced depression)) were tested. Figure 9 As shown in Figures D and E, when using the biosensor, only a small amount of sample (less than 3 μL) with simple dilution is required. Simultaneously, GRα levels were detected using ELISA and Western blotting, and the results were consistent with those obtained by the biosensor of this invention, verifying the reliability of the biosensor's detection results. In this study, the construction of a depression model was confirmed, and compared with the control group, the GRα levels in the hippocampus and blood cells of the model group were significantly reduced. Figure 9 (B and C). In contrast, the inhibition of GRα expression was alleviated under fluoxetine treatment, indicating that the depression model system was successfully established.

[0080] To explore whether the obtained sensor could be used to detect GRα in animal samples, the current response was tested in samples collected from different groups. Figure 9 D, Figure 8 (See Tables 1 and 2). Biosensor responses in the healthy group were significantly higher than in the depressed group, while a significant increase was observed in the treatment group. These results are consistent with those obtained using Western blot and ELISA methods.

[0081] Table 1. Detection of GRα content in rat blood cell and hippocampal extract samples according to the present invention.

[0082]

[0083]

[0084] A Mean ± ts / √n; n = 3; α = 0.05

[0085] Table 2. Validation of the recovery rate of this invention in rat blood cell and hippocampal extract samples.

[0086]

[0087] * Mean ± ts / √n; n = 3; α = 0.05

[0088] ** The value obtained by subtracting the content determined in Table 1 from the total found content.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A GRa protein biosensor, characterized by, Preparation by the following steps: Step 1, drop the Trp-AuNPs@IL-rGO suspension on the clean glassy carbon electrode GCE, and dry to obtain Trp-AuNPs@IL-rGO / GCE, wherein the Trp-AuNPs@IL-rGO comprises amino ionic graphene and gold nanoparticles reduced and wrapped thereon; The Trp-AuNPs@IL-rGO is prepared by the following steps: Step s1, disperse GO into double distilled water, add amino ionic liquid, ultrasonic treatment and continuous stirring, then add alkaline substance and ultrasonic treatment, heat and stir the obtained mixed solution to reflux, centrifuge after the reaction is completed, wash the obtained solid with ethanol and double distilled water alternately to obtain amino ionic graphene IL-rGO; Step s2, after the aqueous solution of HAuCl4 is heated to boiling, the amino acid solution is added dropwise, and after the reaction is completed, the obtained reaction system is quenched in an ultrasonic ice bath, and the obtained solid particles are washed with double distilled water to obtain amino acid reduced and wrapped gold nanoparticles Trp-Au NPs; Step s3, after the IL-rGO and Trp-Au NPs are uniformly dispersed in double distilled water respectively, they are mixed and stirred, wherein the mass ratio of IL-rGO to Trp-Au NPs is 5:1-1:1, and Trp-Au NPs@IL-rGO is obtained after centrifugal treatment after the reaction is completed; Step 2, activate Trp-AuNPs@IL-rGO / GCE with EDC / NHS solution, then immerse Trp-AuNPs@IL-rGO / GCE in ethylenediamine solution to block the carboxyl group, modify Trp-AuNPs@IL-rGO / GCE with antibody, in the modification process, the carboxyl groups at the Fc bottom and Fab shoulder of the antibody AbGRα are fixed to the amino groups of Trp-AuNPs@IL-rGO / GCE, use BSA to remove non-specific binding sites to block the electrode surface, wash the electrode with PBS buffer solution to remove the unconnected BSA on the electrode surface, and obtain the biosensor BSA-AbGRα-Trp-AuNPs@IL-rGO / GCE.

2. The biosensor of claim 1, wherein, The cleaning method in step 1 is to polish the glassy carbon electrode on the surface of suede dispersed with aluminum oxide powder, then wash with water and anhydrous ethanol alternately, and dry in a nitrogen atmosphere to obtain the cleaned glassy carbon electrode GCE.

3. The biosensor of claim 1, wherein, The Trp-AuNPs@IL-rGO suspension in step 1 is an aqueous solution of Trp-AuNPs@IL-rGO, and the concentration is 0.5-1 μg / mL, and the volume is 5-20 μL.

4. The biosensor of claim 1, wherein, The EDC / NHS solution in step 2 is a mixed solution of EDC aqueous solution and NHS aqueous solution, the concentration of EDC aqueous solution is 50-300 mM, the concentration of NHS aqueous solution is 50-300 mM, the volume ratio of EDC aqueous solution to NHS aqueous solution is 1:1, the activation time is 20-50 minutes, and the concentration of ethylenediamine solution is 50-300 mM.

5. The biosensor of claim 1, wherein, In step 2, 0.5-1% BSA is used to remove non-specific binding sites, the concentration of PBS buffer solution is 8-12 mM, and the pH of PBS buffer solution is 7.2-7.

6.

6. The biosensor of claim 1, wherein, The concentration of antibody AbGRα in step 2 is 0.5-1 μg / mL, and the incubation time of AbGRα is 60-120 minutes.

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