Biological detection reagent, preparation method, depression evaluation kit and application of depression evaluation kit in curative effect evaluation of Chinese and western medicines

Abstract

The invention provides a biological detection reagent, a preparation method, a depression evaluation kit and application of the depression evaluation kit in curative effect evaluation of Chinese and western medicine, the biological detection reagent comprises NH2-Fe3O4 and a labeled aptamer which are connected, the labeled aptamer comprises Apt and fluorescein which are connected, and the Apt is connected with the NH2-Fe3O4; the biological detection reagent can generate changes of a colorimetric signal, a fluorescence signal and an electrochemical signal in the presence of ALD, and can detect ALD concentration from multiple dimensions and perform cross validation and calibration based on the three signals, so that the accuracy and reliability of ALD concentration detection are greatly improved; meanwhile, the accuracy and the reliability of the biological detection reagent in the evaluation of the anti-depression effect of the Chinese and western medicines are also improved.

Description

Technical Field

[0001] This application relates to the field of biological detection reagent technology, and in particular to a biological detection reagent, a preparation method, a depression assessment kit, and its application in the evaluation of the efficacy of traditional Chinese and Western medicines. Background Technology

[0002] Aldosterone is a mineralocorticoid associated with hypothalamic-pituitary-adrenal axis dysfunction and has been identified as a potential biomarker for depression. Aldosterone can be detected in various bodily fluids, including serum, saliva, and urine. However, aldosterone is usually present in very low concentrations, and existing detection technologies are often not sensitive enough to accurately detect the amount of aldosterone in bodily fluids. Summary of the Invention

[0003] In view of this, the purpose of this application is to propose a biological detection reagent, a preparation method, a depression assessment kit, and its application in the evaluation of the efficacy of traditional Chinese and Western medicines, so as to solve the problem that the sensitivity of existing detection technologies is insufficient and it is difficult to accurately detect the content of aldosterone in biological fluids.

[0004] To achieve the above objectives, this application provides a biological detection reagent, comprising: NH2-Fe3O4 and a labeled aptamer connected together, wherein the labeled aptamer comprises Apt and fluorescein connected together, wherein the Apt is connected to the NH2-Fe3O4.

[0005] Optionally, the serial number of the tagged aptamer is 5'-6-FAM-CTC TCG GGA CGA CAG ATA GTTGTT CTT AGC GAT GTT CAG CGT TGT CGT CCC-3'.

[0006] Based on the same inventive concept, this disclosure also provides a method for preparing a biological detection reagent, comprising: Prepare an NH2-Fe3O4 solution by placing NH2-Fe3O4 in HEPES buffer containing NaCl; The labeled aptamer solution was added to NH2-Fe3O4 solution, mixed, and incubated to obtain the bioassay reagent.

[0007] Optionally, the NH2-Fe3O4 includes the following preparation steps: NaOAc·3H2O, dopamine hydrochloride and FeCl3·6H2O were dissolved in ethylene glycol, stirred and heated under pressure to obtain crude NH2-Fe3O4 product; The crude NH2-Fe3O4 product was washed and dried to obtain NH2-Fe3O4.

[0008] Based on the same inventive concept, this disclosure also provides a depression assessment kit, including the above-mentioned biological detection reagents.

[0009] Based on the same inventive concept, this disclosure also provides an application of a reagent kit in the evaluation of the efficacy of traditional Chinese and Western medicines.

[0010] As described above, the biological detection reagent provided in this application includes: NH2-Fe3O4 and a labeled aptamer connected together. The labeled aptamer includes Apt and fluorescein connected together, wherein Apt is connected to NH2-Fe3O4. NH2-Fe3O4 is formed by introducing amino groups onto the surface of Fe3O4, and the amino groups on its surface provide binding sites for the labeled aptamer. The labeled aptamer is a bifunctional molecule formed by chemical coupling of Apt (aptamer) and fluorescein (label). Apt binds to the surface of NH2-Fe3O4 through electrostatic adsorption. NH2-Fe3O4 has peroxidase-like catalytic activity. When it binds to Apt, the catalytic activity of NH2-Fe3O4 is significantly enhanced. At the same time, Apt is a non-conductive biomolecule. When NH2-Fe3O4 binds to Apt, a non-conductive component is introduced, leading to an increase in the charge transfer resistance and a decrease in the potential on the surface of NH2-Fe3O4. Fluorescein is attached to Apt to label Apt. Due to the fluorescence quenching effect of NH2-Fe3O4, the fluorescence signal of fluorescein attached to Apt is suppressed by NH2-Fe3O4.

[0011] In the presence of ALD, due to the higher specific binding affinity of ALD to Apt than the interaction between Apt and NH2-Fe3O4, ALD competitively binds to Apt, causing Apt to dissociate from the NH2-Fe3O4 surface. This process triggers a series of changes: in catalytic color development, the detachment of Apt reduces the peroxidase-like activity of Fe3O4, thereby altering its catalytic ability towards the chromogenic substrate; ALD concentration can be detected by measuring changes in the color development intensity of the substrate. Electrochemically, after Apt dissociation, the number of non-conductive biomolecules on the NH2-Fe3O4 surface decreases, reducing charge transfer resistance and increasing current; ALD concentration can be detected based on current changes. Simultaneously, Apt detached from NH2-Fe3O4 is no longer affected by its fluorescence quenching effect; the fluorescence signal of the luciferin attached to Apt gradually recovers with increasing ALD concentration; ALD concentration can be detected by measuring changes in fluorescence signal intensity. Finally, through mutual calibration of these three detection methods, the concentration of ALD can be accurately determined. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in this application or related technologies, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 A and Figure 1 C is the SEM image of NH2-Fe3O4; Figure 1 B and Figure 1 D is a TEM image of NH2-Fe3O4; Figure 1 E is the particle size analysis diagram of NH2-Fe3O4; Figure 1 F is the XRD pattern of NH2-Fe3O4; Figure 1 G is the XPS spectrum of NH2-Fe3O4; Figure 1 H represents the N1s spectrum of NH2-Fe3O4; Figure 1 IL is an elemental mapping image of Fe, O, C and N in NH2-Fe3O4; Figure 1 M is a TEM image of NH2-Fe3O4@Apt; Figure 1 NR is the elemental mapping image of Fe, O, C, N and P in NH2-Fe3O4@Apt; Figure 1 S is the EDX spectrum of NH2-Fe3O4; Figure 1 T is the EDX spectrum of NH2-Fe3O4@Apt; Figure 2 A is a graph showing the change of absorbance at 650 nm over time; Figure 2 B is a Lineweaver-Burk diagram; Figure 2 C represents the UV-Vis spectrum used for feasibility verification in colorimetric mode; Figure 2 D is the fluorescence emission spectrum for feasibility verification in fluorescence mode; Figure 2 E is Fe3O4 / MGCE at 10 mV s -1 Up to 100 mV s -1 CV response plots at different scan rates; Figure 2F is a graph showing the relationship between the redox peak current and the square root of the scan rate; Figure 2 G is the EIS spectrum used for feasibility verification in electrochemical mode; Figure 2 H is the DPV response diagram used for feasibility verification in the electrochemical mode; Figure 2 I represents the Zeta potential analysis diagram.

[0014] Figure 3 A is the optimal concentration diagram of Fe3O4; Figure 3 B is the Apt concentration optimization graph; Figure 3 C represents the optimal concentration of NaCl; Figure 3 D is the TMB concentration optimization plot; Figure 3 E represents the optimized concentration of H2O2; Figure 3 F is the pH optimization diagram for the TMB colorimetric reaction; Figure 4 A represents the results of using the obtained bioassay reagent on 0 pg mL -1 -20 ng mL -1 Colorimetric detection graphs of different ALD concentrations within the sample; Figure 4 B is 5 pg / mL -1 -20 ng mL -1 Colorimetric mode calibration curves for different ALD concentrations; Figure 4 C shows photographs of test solutions with different ALD concentrations under sunlight; Figure 4 D represents the ImageJ grayscale value and 5 pg / mL. -1 -20 ng mL -1 Calibration curves between different ALD concentrations within the cell; Figure 4 E represents the result of using the obtained bioassay reagent on 0 pg mL -1 -20 ng mL -1 Fluorescence detection images at different ALD concentrations; Figure 4 F is 3 pg mL -1 -10 ng mL -1 Fluorescence mode calibration curves for different ALD concentrations; Figure 4 G represents the result of using the obtained bioassay reagent on 0 pg mL -1 -20 ng mL -1Electrochemical detection graphs of different ALD concentrations within the cell; Figure 4 H is 3 pg / mL -1 -20 ng mL -1 Electrochemical mode calibration curves for different ALD concentrations; Figure 4 I is a specificity analysis diagram of the three-modal biological detection reagent; Figure 4 J is a reproducibility analysis graph of five sets of three-modal biological detection reagents independently constructed under the same conditions; Figure 4 K is a graph representing the storage stability analysis of the three-modal biological assay reagents, which was checked every week for one month. Figure 4 L is a repeatability analysis chromatogram of biological detection reagents in electrochemical mode; Figure 5 A is the flowchart of the rat experiment; Figure 5 B is a graph showing the experimental weight test of rats; Figure 5 C represents the rat sucrose preference test graph; Figure 5 D is a graph of the forced swimming test in rats; Figure 5 E is a diagram of the open field test in rat experiments; Figure 5 F is the signal intensity diagram of ALD in rat serum samples detected in colorimetric mode; Figure 5 G is the signal intensity map of ALD in rat serum samples detected in fluorescence mode; Figure 5 H represents the signal intensity diagram of ALD in rat serum samples detected in electrochemical mode; Figure 5 IK is a comparison chart of ALD concentrations in serum from different groups of rats detected under different detection modes; Figure 6 A is the signal intensity diagram of ALD in human serum samples detected in colorimetric mode; Figure 6 B is the signal intensity diagram of ALD in human serum samples detected in fluorescence mode; Figure 6 C is the signal intensity diagram of ALD in human serum samples detected in electrochemical mode; Figure 6 DF is a graph showing the difference in ALD concentration in the serum of healthy controls (n = 10) and patients with depression (n = 10) under different detection modes; Figure 6G is the ROC curve used to evaluate the diagnostic value of biological test reagents for patients with depression by the area under the curve (AUC). Figure 6 H represents the signal intensity map of a three-modal biological detection reagent used to detect ALD in human saliva samples; Figure 6 I represents the ALD concentration in the saliva of a healthy individual; Figure 6 J represents the signal intensity map of a three-modal biological detection reagent used to detect ALD in human urine samples; Figure 6 K represents the ALD concentration in the urine of healthy individuals; Figure 6 L represents the total ALD content in the urine of a healthy individual over 24 hours. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0016] It should be noted that, unless otherwise defined, the technical or scientific terms used in the embodiments of this application should have the ordinary meaning understood by one of ordinary skill in the art to which this application pertains. The terms "first," "second," and similar terms used in the embodiments of this application do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are only used to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0017] As mentioned in the background, depression is a common mental illness characterized by persistent and significant low mood. Depression has four major high-risk characteristics: high prevalence, high relapse rate, high self-harm rate, and high suicide rate. Data from the World Health Organization shows that more than 280 million people worldwide suffer from depression. Suicide due to depression is the fourth leading cause of death among people aged 15 to 29, a critical period for academic, professional, and social development. Depression places a heavy burden on global public health and socioeconomic systems. Therefore, early and accurate diagnosis of depression is crucial for timely intervention and effective treatment. However, current diagnostic methods rely heavily on subjective clinical interviews and questionnaire-based assessments, which are prone to bias and lack objectivity.

[0018] Aldosterone (ALD), a mineralocorticoid associated with hypothalamic-pituitary-adrenal axis dysfunction, has been identified as a potential biomarker for depression. Elevated ALD levels have been observed in patients with major depressive disorder, suggesting its value in objective diagnosis. Importantly, ALD can be detected in various bodily fluids, including serum, saliva, and urine, enabling minimally invasive and non-invasive sampling methods. However, ALD is typically present at extremely low concentrations (pg / mL), making accurate quantification highly challenging. Traditional methods such as radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay have been used for ALD detection, but they often suffer from insufficient sensitivity, especially when non-invasively analyzing trace amounts of ALD. Furthermore, these techniques are costly, time-consuming, and dependent on skilled personnel. These limitations underscore the urgent need to develop innovative bioassays that are not only highly sensitive and accurate but also rapid, cost-effective, and suitable for clinical and home applications.

[0019] The following is in conjunction with the appendix Figure 1-6 The embodiments of this application will be described in detail below.

[0020] Example 1: Components of a biological detection reagent A biological detection reagent includes: NH2-Fe3O4 and a labeled aptamer connected together, wherein the labeled aptamer includes Apt and fluorescein connected together, wherein the Apt is connected to the NH2-Fe3O4.

[0021] Additionally, the serial number of the tagged aptamer is 5'-6-FAM-CTC TCG GGA CGA CAG ATA GTT GTTCTT AGC GAT GTT CAG CGT TGT CGT CCC-3'.

[0022] Specifically, NH2-Fe3O4 is formed by introducing amino groups onto the Fe3O4 surface, which provide binding sites for the labeled aptamer. The labeled aptamer is a bifunctional molecule formed by chemical coupling of Apt (aptamer) and luciferin (label). Apt binds to the NH2-Fe3O4 surface via electrostatic adsorption. NH2-Fe3O4 exhibits peroxidase-like catalytic activity; when it binds to Apt, its catalytic activity is significantly enhanced. Simultaneously, Apt is a non-conductive biomolecule; when NH2-Fe3O4 binds to Apt, it introduces a non-conductive component, leading to an increase in charge transfer resistance and a decrease in potential on the NH2-Fe3O4 surface. Luciferin (6-carboxyluciferin (6-FAM)) is attached to Apt to label it. Due to the fluorescence quenching effect of NH2-Fe3O4, the fluorescence signal of luciferin attached to Apt is suppressed by NH2-Fe3O4.

[0023] In the presence of ALD, due to the higher specific binding affinity of ALD to Apt than the interaction between Apt and NH2-Fe3O4, ALD competitively binds to Apt, causing Apt to dissociate from the NH2-Fe3O4 surface. This process triggers a series of changes: in catalytic color development, the detachment of Apt reduces the peroxidase-like activity of Fe3O4, thereby altering its catalytic ability towards the chromogenic substrate; ALD concentration can be detected by measuring changes in the color development intensity of the substrate. Electrochemically, after Apt dissociation, the number of non-conductive biomolecules on the NH2-Fe3O4 surface decreases, reducing charge transfer resistance and increasing current; ALD concentration can be detected based on current changes. Simultaneously, Apt detached from NH2-Fe3O4 is no longer affected by its fluorescence quenching effect; the fluorescence signal of the luciferin attached to Apt gradually recovers with increasing ALD concentration; ALD concentration can be detected by measuring changes in fluorescence signal intensity. Finally, through mutual calibration of these three detection methods, the concentration of ALD can be accurately determined.

[0024] Example 2: Preparation method of biological detection reagent A method for preparing a biological detection reagent, comprising: S100: Prepare an NH2-Fe3O4 solution by placing NH2-Fe3O4 in HEPES buffer containing NaCl; S200: Add the labeled aptamer solution to the NH2-Fe3O4 solution, mix, and incubate to obtain the bioassay reagent.

[0025] For example, NH2-Fe3O4 was dissolved in HEPES buffer containing NaCl to prepare a solution with a concentration of 200 μg / mL. -1The NH2-Fe3O4 solution was prepared. Then, 5 μM labeled aptamer solution (a buffer solution prepared by placing the labeled aptamer in TE buffer) was added to 150 μL of NH2-Fe3O4 solution, mixed thoroughly, and incubated at 37°C in the dark for 20 minutes to obtain the bioassay reagent.

[0026] Example 3 Preparation of NH2-Fe3O4 The NH2-Fe3O4 is prepared by the following steps: NaOAc·3H2O, dopamine hydrochloride and FeCl3·6H2O were dissolved in ethylene glycol, stirred and heated under pressure to obtain crude NH2-Fe3O4 product; The crude NH2-Fe3O4 product was washed and dried to obtain NH2-Fe3O4.

[0027] Specifically, 6.53 g NaOAc·3H2O, 0.114 g dopamine hydrochloride, and 3.24 g FeCl3·6H2O were dissolved in 60 mL ethylene glycol. After vigorous stirring for 30 minutes, the resulting solution was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 200 °C for 12 hours to obtain a black crude NH2-Fe3O4 product. The black crude NH2-Fe3O4 product was thoroughly washed with deionized water and ethanol, and then vacuum dried at 60 °C to obtain the NH2-Fe3O4.

[0028] The morphology of NH2-Fe3O4 synthesized by the above method was characterized at different magnifications using scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0029] like Figure 1 A- Figure 1 As shown in Figure E, NH₂-Fe₃O₄ exhibits a classic, uniformly dispersed spherical morphology with an average particle size of approximately 411.89 nm. The crystal structure of NH₂-Fe₃O₄ was further investigated using X-ray diffraction (XRD). Figure 1As shown in Figure F, the XRD pattern shows that the characteristic diffraction peaks of NH2-Fe3O4 are located at 2θ values ​​of 18.22° (111), 30.08° (220), 35.44° (311), 43.06° (400), 53.48° (422), 57.00° (511), and 62.52° (440), which can be attributed to the cubic spinel structure of magnetite. Furthermore, the surface chemical composition of NH2-Fe3O4 was analyzed using X-ray photoelectron spectroscopy (XPS). Figure 1 C 1s and O 1s nuclear level peaks were observed at 285.08 eV and 532.08 eV, respectively (G). Figure 1 The inset G shows a double peak in the Fe 2p region, with binding energies of 711.18 eV and 725.08 eV, corresponding to Fe 2p... 3 / 2 and Fe 2p 1 / 2 To further confirm amino functionalization, high-resolution XPS analysis was performed on the N 1s region. Figure 1 As shown in Figure H, two significant peaks were observed at 399.68 eV and 401.78 eV, which are attributed to the free amine group (-NH2) and the protonated amine group (-NH3), respectively. + This confirms the successful synthesis of NH2-Fe3O4.

[0030] Furthermore, elemental composition analysis was performed using elemental mapping images and energy-dispersive X-ray (EDX) spectroscopy. Figure 1 As shown in the image, Fe, O, C, and N elements are uniformly distributed within the NH₂-Fe₃O₄ nanoparticles. The presence of N indicates the successful introduction of amino groups.

[0031] Subsequently, Apt was anchored to the surface of NH2-Fe3O4 using the method of Example 2, forming NH2-Fe3O4@Apt. This NH2-Fe3O4@Apt is simply a substance formed by Apt attaching to the NH2-Fe3O4 surface and does not contain fluorescein. TEM images of NH2-Fe3O4@Apt show that its morphology remains almost unchanged. Figure 1 M). Furthermore, Fe, O, C, N, and P elements were detected in NH2-Fe3O4@Apt, and their spatial distribution was visualized based on their relative abundance using element mapping. Figure 1 N- Figure 1 R). Compared with NH2-Fe3O4, the increase in N content and the presence of P element in NH2-Fe3O4@Apt confirm that Apt has been successfully adsorbed on the surface of nanoparticles (R). Figure 1 S and Figure 1The T;Cu signal originates from the copper mesh used to support the sample. For clarity and subsequent results analysis, NH2-Fe3O4 will be abbreviated as Fe3O4 in the following text.

[0032] Example 4 Feasibility Analysis Test of Biological Detection Reagents Example 4.1 To evaluate the feasibility of a colorimetric model (a method for quantitative analysis of ALD based on the catalytic activity and regulatory role of Fe3O4 and Apt by detecting colorimetric changes in the substrate), the catalytic kinetics of Fe3O4 and Fe3O4+Apt were first investigated using TMB (3,3',5,5'-Tetramethylbenzidine, 3,3',5,5'-Tetramethylbenzidine) as the substrate in the presence of a constant concentration of H2O2. Figure 2 As shown in Figure A, Fe3O4 coated with Apt exhibits higher catalytic activity compared to bare Fe3O4. Furthermore, the Michaelis constant (Km) calculated from the Lineweaver-Burk plot... m The concentrations were 0.328 mM (Fe3O4) and 0.152 mM (Fe3O4+Apt), respectively. Figure 2 B). K after Apt adsorption m The lower value indicates a higher affinity for the TMB substrate, thus ensuring reliable catalysis-based colorimetric analysis of ALD. The improved catalytic activity is likely due to the hydrogen bonding and π-π stacking interactions between the DNA bases of Apt and the amino and benzene rings on the TMB substrate, respectively, enhancing the substrate affinity and catalytic performance of Fe3O4. Subsequently, the feasibility of ALD detection in colorimetric mode was verified. Figure 2 As shown in Figure C, the Fe3O4 nanozyme only exhibits peroxidase-like activity in the presence of both TMB and H2O2. Functionalization with Apt significantly enhances the catalytic activity of Fe3O4, consistent with the catalytic kinetics results. The presence of ALD induces competitive dissociation of Apt from the Fe3O4 surface through specific binding, leading to a significant decrease in catalytic activity, confirming the sensitive response of this bioassay reagent to ALD in colorimetric mode.

[0033] Example 4.2 To evaluate the feasibility of a fluorescence model (a method for quantitative analysis of ALD based on the fluorescence quenching effect of Fe3O4 on 6-FAM-labeled Apt, using changes in fluorescence intensity for analysis), fluorescence analysis of ALD was performed using fluorescently labeled Apt. Figure 2As shown in Figure D, Apt labeled with 6-FAM alone exhibited the highest fluorescence intensity. When Apt anchored to the Fe3O4 surface, the fluorescence intensity decreased significantly due to a quenching effect. The introduction of ALD induced the release of Apt from Fe3O4, leading to fluorescence recovery, thus demonstrating the feasibility of this bioassay reagent for detecting ALD in fluorescence mode.

[0034] Example 4.3 To verify the feasibility of an electrochemical mode (a method for quantitative analysis of ALD based on potential changes generated by the connection or dissociation of Fe3O4 and Apt) for detecting ALD, 10 mM K3[Fe(CN)6] was used as a redox probe in 0.1 M KCl solution at a range of 10–100 mV s. -1 Cyclic voltammetry (CV) curves of Fe3O4 / MGCE (magnetic glassy carbon electrode) were recorded within the scan rate range. Figure 2 As shown in Figure E, the redox current increases with increasing scan rate. There is a strong linear relationship between the peak current and the square root of the scan rate, with the linear equation being: I pa = 8.552v 1 / 2 + 9.177 (R1 2 =0.996) and I pc = -8.576 v 1 / 2 -8.014 (R2 2 = 0.994) Figure 2 F). These results indicate that the redox reaction at the Fe3O4 / MGCE interface follows a diffusion-controlled process.

[0035] In containing 10 mM K 3 [Fe(CN) 6 Electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) measurements were performed in a 0.1 M KCl solution. Figure 2 As shown in G, the diameter of the semicircle in the Nyquist plot of EIS corresponds to the charge transfer resistance (Rct) of the modified electrode. Compared with the bare MGCE (magnetic glassy carbon electrode), the Rct of Fe3O4 / MGCE is significantly lower due to the good conductivity of Fe3O4. ct The reduction occurs when Apt adsorbs onto the Fe3O4 surface, introducing non-conductive biomolecules that lead to a decrease in R. ct Significantly increased. In the presence of ALD, Apt dissociates from the Fe3O4 surface, leading to Rct The current response in the DPV analysis decreased. As expected, the current response in the DPV analysis exhibited a similar pattern to R. ct The opposite trend ( Figure 2 These results confirm the feasibility of using this bioassay reagent to detect ALD in electrochemical mode.

[0036] Furthermore, zeta potential analysis was used to further verify the feasibility of ALD detection. Figure 2 As shown in Figure I, the zeta potential of Fe3O4 decreased from -10.05 mV to -42.15 mV after the introduction of Apt, indicating that the negatively charged nucleic acid Apt successfully modified Fe3O4. Notably, this shift to a more negative potential may help attract the oppositely charged TMB substrate to the nanoparticle surface, thereby enhancing substrate affinity and catalytic activity. When ALD competitively replaces Apt, the zeta potential shifts back to a more positive value (-27.11 mV). These zeta potential results are consistent with the trends observed in colorimetric, fluorescence, and electrochemical detection modes, collectively confirming the feasibility of Apt-modified Fe3O4 for ALD detection in all three analytical modes.

[0037] Example 5: Optimization Experiment of Preparation Conditions for Biological Detection Reagents To obtain optimal performance of the bioassay reagent, various reaction conditions were optimized for the preparation method of the bioassay reagent in Example 2. First, the absorbance (ΔA) before and after Apt adsorption onto Fe3O4 under different concentrations of Fe3O4, Apt, and NaCl was studied. 652 =A Fe3O4 + Apt – A Fe3O4 ) and fluorescence intensity (ΔF 518 = F Apt – F Fe3O4 + Apt The changes in ) . For example Figure 3 As shown in Figure A, when the Fe3O4 solution concentration is 200 μg / mL -1 At a certain point, ΔA reached its maximum value, indicating that Fe3O4 and Apt were sufficiently bound. However, with further increases in the Fe3O4 solution concentration, ΔA decreased, possibly due to increased background absorbance caused by excess Fe3O4. Conversely, ΔF continuously increased with increasing Fe3O4 concentration, indicating that increased Fe3O4 content exacerbated fluorescence quenching. Considering both absorbance and fluorescence changes, a 200 μg / mL solution was selected. -1 Fe3O4 was used for subsequent experiments. A certain concentration of Apt was mixed with Fe3O4 solution to prepare a bioassay reagent, such as... Figure 3As shown in Figure B, ΔA increases with increasing Apt concentration, peaking at 5 μM and decreasing beyond that concentration. This decrease is likely due to excess Apt saturating and completely coating the Fe3O4 surface. Similarly, ΔF tends to stabilize at 5 μM Apt, indicating that fluorescence quenching has reached saturation. Therefore, 5 μM was determined to be the optimal concentration of Apt. Furthermore, the concentration of NaCl in the HEPES buffer was optimized to eliminate the electrostatic repulsion between Fe3O4 and Apt, thereby promoting rapid adsorption. Figure 3 As shown in Figure C, both ΔA and ΔF increase with increasing NaCl concentration, reaching an inflection point at 150 mM, indicating that Apt can be effectively adsorbed onto Fe3O4 at this ionic strength. Therefore, 150 mM NaCl was chosen as the buffer system.

[0038] Subsequently, the concentrations of TMB and H2O2, as well as the pH value of the buffer system, were optimized for the TMB colorimetric reaction in the colorimetric mode of Example 4.1. Figure 3 As shown in Figures D and E, the reaction rate gradually increases with increasing TMB and H2O2 concentrations. When the TMB and H2O2 concentrations reach 3 mM and 40 mM, respectively, the absorbance of the reaction system reaches its maximum and tends to stabilize. Therefore, 3 mM TMB and 40 mM H2O2 were selected as the optimal concentrations. Furthermore, the pH value of the buffer system has a significant impact on the TMB oxidation reaction. Figure 3 As shown in Figure F, the reaction is most favorable at pH 4.0; excessively acidic or alkaline conditions will weaken the catalytic activity. Therefore, pH 4.0 was determined to be the optimal pH for the catalytic buffer system.

[0039] Example 6 Performance Analysis Experiment of Biological Detection Reagent Under optimal conditions, ALD was used as a model analyte for trimodal detection. Specifically, NH2-Fe3O4 was dissolved in HEPES buffer (10 mM, pH 7.0) containing 150 mM NaCl to prepare a solution with a concentration of 200 μg / mL. -1 The NH2-Fe3O4 solution was prepared. Then, 3 μL of labeled aptamer solution (5 μM) was added to 150 μL of NH2-Fe3O4 solution, mixed thoroughly, and incubated at 37°C in the dark for 20 minutes. Subsequently, 3 μL of ALD solution of different concentrations was added, and the mixture was incubated at 37°C in the dark for another 20 minutes. After incubation, colorimetric, fluorescence, and electrochemical signals were recorded.

[0040] Colorimetric method: Transfer 50 μL of the final reaction mixture to a 96-well plate, then add 60 μL of TMB (3 mM) and H2O2 (40 mM), both prepared with acetate buffer (10 mM, pH 4.0). Incubate at 37°C in the dark for 15 minutes, then terminate the reaction with 100 μL of stop solution. Record absorbance using a microplate reader for UV-Vis spectroscopy analysis. Additionally, capture images using a smartphone within 15 minutes and perform quantitative analysis using ImageJ software.

[0041] Specifically, as the concentration of ALD increases, the absorbance peak in the ultraviolet-visible (UV-vis) spectrum gradually weakens. This is because the detachment of Apt from the Fe3O4 surface leads to a decrease in its peroxidase-like activity. Figure 4 A). For example Figure 4 As shown in B, at 5 pg mL -1 Up to 20 ng mL -1 Within the range, the absorbance value at 652 nm shows a good linear relationship with the logarithm of ALD concentration, and the linear regression equation is y = -0.068 × lg C ALD + 0.522 (R) 2 =0.996). Limit of detection (LOD) according to 3S D / m criterion calculation (where S) D The standard deviation of 10 blank determinations (where m is the slope of the calibration curve) was 1.528 pg / mL. -1 Furthermore, in colorimetric mode, ALD detection can also be achieved through color recognition via a smartphone. Figure 4 C). For example Figure 4 As shown in D, at 5 pg mL -1 Up to 20 ng mL -1 Within the specified range, the grayscale values ​​obtained using ImageJ show a good linear relationship with the logarithm of ALD concentration, and the regression equation is y = 13.697 × lgC. ALD + 107.064 (R) 2 = 0.992), LOD is 1.643 pg mL -1 .

[0042] Fluorescence mode: 50 μL of the final reaction solution was transferred to a 96-well plate, and the fluorescence emission spectrum was recorded using a microplate reader excited at 494 nm.

[0043] Specifically, in fluorescence mode, as the ALD concentration increases, aptamer dissociation alleviates the fluorescence quenching effect, leading to a gradual increase in the fluorescence signal under 494 nm excitation. Figure 4 E). For example Figure 4 As shown in F, at 3 pg mL -1 Up to 10 ng mL -1 Within this range, the fluorescence intensity at 518 nm exhibits a linear relationship with the logarithm of the ALD concentration, with the regression equation being y = 154.523 × lg C. ALD +125.886 (R) 2 = 0.994), its LOD is 1.016 pg mL -1 .

[0044] Electrochemical procedure: A magnetic glassy carbon electrode was polished for 2 minutes on a chamois surface dispersed with alumina powder (0.1 and 0.05 μm particle sizes), then alternately cleaned with water and anhydrous ethanol for 3 minutes each, and finally dried under nitrogen. The cleaned magnetic glassy carbon electrode was then immersed in a reaction solution (labeled aptamer and NH₂-Fe₃O₄ reaction solution) for 30 seconds to modify the electrode surface. The modified electrode was then dried at room temperature, and electrochemical measurements were performed. The detailed electrochemical procedure is as follows: All electrochemical measurements were performed in a standard three-electrode system, consisting of a working electrode (magnetic glassy carbon electrode), a reference electrode (Ag / AgCl electrode saturated with KCl), and a counter electrode (a 1 mm diameter Pt wire). CV and DPV experiments were performed at a scan rate of 50 mV / s, with a potential range of -0.2 to +0.6 V. EIS measurements were performed under open-circuit conditions, with a frequency range of 100 kHz to 0.1 Hz and an amplitude signal of 5 mV. All electrochemical experiments were conducted at room temperature in a 0.1 M KCl solution containing 10 mM K₃[Fe(CN)₆].

[0045] In electrochemical mode, as the ALD concentration increases, the reduction of non-conductive biomolecules on the electrode surface leads to a gradual increase in peak current. Figure 4 G). For example Figure 4 As shown in H, at 3 pg mL -1 Up to 20 ng mL -1 Within the range, ΔI (ΔI = I–I0, where I is the current response at a specific ALD concentration, and I0 is 0 pg / mL) -1 The current at that time showed a good linear relationship with the logarithm of ALD concentration, and the regression equation was y = 9.498 × lg C. ALD + 1.201 (R) 2 = 0.995), and the LOD in electrochemical mode was 1.137 pg / mL. -1 .

[0046] To investigate the specificity, reproducibility, stability, and repeatability of the bioassay reagents, based on the above research, several non-target steroid compounds were used to test the bioassay reagents, including those with a concentration of 1 ng / mL. -1 Cortisol (COR), corticosterone (CORT), testosterone (TES), progesterone (P4), and estradiol (E2). For example... Figure 4 As shown in I, with 100 pg mL -1 The signals obtained by ALD were significantly different, and the responses produced by these interfering substances were negligible in all detection modes, indicating extremely low cross-reactivity and high specificity. Reproducibility was examined by preparing five independent batches of the bioassay reagent. The differences in colorimetric, fluorescence, and electrochemical signals were small, with relative standard deviations (RSDs) of 2.15%, 2.46%, and 2.23%, respectively, indicating consistent manufacturing processes and reliable reproducibility. Figure 4 J). To assess storage stability, the bioassay reagent was stored at 4°C and tested weekly for four weeks. Figure 4 As shown in K, the signal strength decreased slightly over time, but more than 87% of the original response was retained in all modes, indicating acceptable long-term stability. The repeatability under electrochemical conditions was verified by testing the same bioassay reagent in 10 consecutive runs. The RSD of the results was 1.77%, reflecting stable output and excellent repeatability. Figure 4 In summary, these results highlight the stability of this bioassay reagent and its great potential for accurate quantification of ALD in clinical settings.

[0047] Example 7: Application of biological detection reagents in experiments evaluating the efficacy of traditional Chinese and Western medicines To evaluate the practical application value of biological testing reagents in the evaluation of the efficacy of traditional Chinese and Western medicines, a classic depression model—Chronic unpredictable mild stress (CUMS)—was established.

[0048] Thirty-six male Sprague-Dawley rats were housed in a controlled environment at a constant temperature of 22 ± 1°C and a relative humidity of 55 ± 5%, with free access to food and water. After a 1-week environmental adaptation period, the formal experiment began. All animals were cared for in accordance with the guidelines in the Guide for the Care and Use of Laboratory Animals, and the rat experimental protocol was approved by the Animal Ethics and Welfare Committee, with the approval number IRM / 2-IACUC-2402-015.

[0049] Thirty-six male Sprague-Dawley rats were randomly divided into 6 groups: healthy control group (Con), depression model group (CUMS), paroxetine treatment group (Par), high-dose group of traditional Chinese medicine Sini Powder (SNPH), medium-dose group (SNPM), and low-dose group (SNPL) ( Figure 5 A). Among them, the rats in the healthy control group were housed under standard conditions, with free access to food and water, and a 12-hour day-night cycle. For the rats in other groups, a chronic unpredictable mild stress (CUMS) model was used, and a series of mild and unpredictable stressors were randomly applied every day for four consecutive weeks to induce depressive-like behaviors in the rats. These stressors included 24-hour food and water deprivation, 24-hour day-night reversal, 24-hour wet bedding, 4-hour physical restraint, 4-hour ultrasonic stimulation, 24-hour cage tilt, 1-minute tail clamping, 10-minute forced swimming, and 24-hour overcrowding. One week after stress induction, gavage administration began in each group and continued for three weeks while stress was ongoing. The rats in the Con group and the CUMS group were gavaged with normal saline; the rats in the Par group were gavaged with paroxetine (2 mg / kg); the rats in the SNPH group, SNPM group, and SNPL group were gavaged with the decoction of Sini Powder at doses of 6 g / kg, 3 g / kg, and 1.5 g / kg, respectively. During the experiment, the rats were evaluated behaviorally using body weight measurement, sucrose preference test (SPT), forced swimming test (FST), and open field test (OFT). In the SPT, the intakes of pure water and 1% sucrose solution were recorded to evaluate anhedonia. Sucrose preference was calculated as the ratio of the sucrose solution intake to the total liquid intake (sucrose + water). The FST was performed for 5 minutes to measure the immobility time and the time of attempting to escape. The OFT was used to evaluate spontaneous locomotor and exploratory behaviors, and the number of grid crossings, standing, and grooming behaviors within 5 minutes was recorded.

[0050] As Figure 5 shown in B, the baseline body weights of the rats in each group were comparable, but the body weight of the rats in the CUMS group decreased significantly compared with that in the Con group after modeling. Similarly, the SPT results of the rats in the CUMS group were significantly lower than those in the Con group ( Figure 5 C). FST results showed that the active escape time and resting time of rats in the CUMS group were shortened (C). Figure 5 D). In OFT, the number of grid crossings, standing, and grooming behaviors of rats in the CUMS group were also significantly reduced ( Figure 5 E). These results collectively confirm the successful establishment of the CUMS depression model. Conversely, rats in the Par group showed significant improvement in body weight and reduced depressive symptoms in SPT, FST, and OFT after treatment with paroxetine. Furthermore, treatment with Sini Powder (SNP) also improved depressive-like behaviors in a dose-dependent manner, with higher doses resulting in more significant improvements in body weight and behavioral indicators. Notably, the efficacy of the high-dose SNP group was comparable to that of the paroxetine group. These behavioral tests preliminarily validate the effectiveness of the drug intervention.

[0051] Following behavioral testing, all rats were anesthetized with chloral hydrate, and blood samples were collected from the abdominal aorta. After standing for 1 hour, the samples were centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was collected and stored at -80°C for later use. Subsequently, the serum ALD levels of rats in different experimental groups were detected using this bioassay reagent, aiming to reflect the efficacy of antidepressant treatment through quantitative ALD analysis. Signal changes were recorded to estimate and compare the relative concentrations of ALD in the serum of rats in each group. Figure 5 F- Figure 5 As shown in Figure H, the colorimetric, fluorescence, and electrochemical signal responses are highly consistent. This bioassay reagent can effectively distinguish subtle differences in ALD levels in the serum of different groups of rats, and its detection results are highly consistent with those of commercially available ELISA methods, indicating that this bioassay reagent has excellent analytical accuracy. Figure 5 I). like Figure 5 As shown in Figure J, ALD levels were significantly elevated in the CUMS group rats compared to the control group. ALD concentrations were significantly reduced in the Par group rats after paroxetine treatment. Similarly, SNP treatment also led to a decrease in ALD levels in rats, showing a clear dose-dependent trend. Figure 5 As shown in K, the mean serum ALD concentration in different groups of rats showed a correlation with... Figure 5 The trend observed in J was consistent with that in the SNPH group rats, and the ALD level in the SNPH group rats was comparable to that in the Par group rats. These findings are consistent with the behavioral test results, further confirming the applicability and reliability of this bioassay reagent in evaluating the efficacy of traditional Chinese and Western medicines, highlighting its potential in supporting new drug development.

[0052] Example 8: Application of biological detection reagents in clinical trials To assess the clinical applicability of this bioassay reagent, we analyzed real samples from minimally invasive (serum) and non-invasive (saliva and urine) sources.

[0053] Human serum samples (samples from healthy individuals (n = 10) and samples from patients with depression (n = 10)) were obtained from Tianjin Chest Hospital (Tianjin, China). Collected blood was allowed to stand for 1 hour, then centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was collected and stored at -80°C for later use. Saliva was collected using a sterile saliva collector and centrifuged at 3000 rpm for 20 minutes. The supernatant was separated, transferred, and stored at -80°C until use. Urine samples were collected as a 24-hour composite sample. All urine samples were collected in sterile beakers, thoroughly mixed, and centrifuged at 3000 rpm for 20 minutes. The supernatant was separated and stored at -80°C for later use. All samples (including serum, saliva, and urine) were analyzed using the developed bioassay reagents according to the established assay protocol.

[0054] For minimally invasive testing, this bioassay reagent was used to quantify ALD concentrations in serum samples from healthy individuals (n = 10) and patients with depression (n = 10). Figure 6 As shown in AC, there were significant differences in colorimetric, fluorescence, and electrochemical signal responses between the two groups of subjects. Furthermore, this bioassay successfully distinguished between serum ALD levels in healthy subjects and patients, and the results were consistent with those obtained using commercially available ELISA methods. Figure 6 D). For example Figure 6 E and Figure 6 As shown in Figure F, serum ALD concentrations in patients with depression were significantly higher than those in healthy subjects (P<0.001). Furthermore, receiver operating characteristic (ROC) curve analysis indicated that this bioassay reagent had a high diagnostic accuracy for depression (AUC = 0.92) and could effectively distinguish between healthy subjects and patients with depression. Figure 6 G). These results indicate that this bioassay reagent has great potential for the clinical diagnosis of depression based on serum samples.

[0055] It is worth noting that this bioassay reagent not only enables minimally invasive testing but also non-invasive analysis, thus expanding its application to home testing scenarios. This bioassay reagent was used to determine ALD levels in the saliva and urine of eight healthy individuals. Figure 6 H and Figure 6 As shown in Figure J, the signal intensity trends are consistent across colorimetric, fluorescence, and electrochemical modes. Figure 6 I and Figure 6As shown in K, the ALD concentrations measured in saliva and urine ranged from 25.37 to 91.14 pg / mL. -1 and 872.82 to 1586.43 pg mL -1 Furthermore, the detection results of this method are highly consistent with those of commercially available ELISA methods, demonstrating the reliable analytical accuracy of this biological detection reagent. In addition, the total excretion of ALD in urine over 24 hours ranges from 0.92 to 2.20 μg (…). Figure 6 These findings demonstrate that bioassay reagents serve as a powerful platform for non-invasive testing, offering significant potential for point-of-care testing (POCT) and home applications.

[0056] Based on the same inventive concept, this embodiment also discloses a depression assessment kit, including the aforementioned biological detection reagents. This depression assessment kit is suitable for serum, saliva, and urine samples, is easy to operate, has a rapid response, and possesses good clinical adaptability and potential for home use.

[0057] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of this application is limited to these examples; under the concept of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of the embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0058] Although this application has been described in conjunction with specific embodiments thereof, many substitutions, modifications, and variations of these embodiments will be apparent to those skilled in the art from the foregoing description. The embodiments of this application are intended to cover all such substitutions, modifications, and variations falling within the broad scope of the claims of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the embodiments of this application should be included within the protection scope of this application.

Claims

1. A biological detection reagent, characterized in that, include: The NH2-Fe3O4 and the labeled aptamer are connected, the labeled aptamer comprising connected Apt and fluorescein, wherein the Apt is connected to the NH2-Fe3O4.

2. The biological detection reagent according to claim 1, characterized in that, The serial number of the tagged aptamer is shown as 5'-6-FAM-SEQ ID NO:1-3'.

3. A method for preparing a biological detection reagent, characterized in that, include: Prepare an NH2-Fe3O4 solution by placing NH2-Fe3O4 in HEPES buffer containing NaCl; The labeled aptamer solution was added to NH2-Fe3O4 solution, mixed, and incubated to obtain the bioassay reagent.

4. The method for preparing a biological detection reagent according to claim 3, characterized in that, The NH2-Fe3O4 is prepared by the following steps: NaOAc·3H2O, dopamine hydrochloride and FeCl3·6H2O were dissolved in ethylene glycol, stirred and heated under pressure to obtain crude NH2-Fe3O4 product; The crude NH2-Fe3O4 product was washed and dried to obtain NH2-Fe3O4.

5. A depression assessment kit, characterized in that, Includes the biological detection reagents described in claims 1-2.

6. The application of the kit according to claim 5 in the evaluation of the efficacy of traditional Chinese and Western medicines.

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