Depression Early Diagnosis Kit for Salivary Cortisol Concentration and Quantitative Analysis Using Magnetic Beads

Abstract

The present invention relates to an in vitro diagnostic kit capable of early diagnosis of depression by concentrating cortisol in saliva using magnetic beads and performing quantitative analysis. By modifying the surface of magnetic beads (Fe₃O₄) to bind a cortisol-specific antibody (Anti-Cortisol Ab) with HRP (Horseradish Peroxidase), the device enables the effective capture of cortisol and signal amplification. The magnetic beads bound to cortisol are concentrated using a magnetic field to remove unnecessary components, and the signal is detected by reacting with a TMB solution to induce a color change. The strip sensor is designed to optimize the reaction while the sample travels to the absorption pad using capillary action, thereby enhancing signal uniformity and improving sensitivity. By applying smartphone-based RGB analysis technology, the color changes of the T-Line and R-Line are quantitatively analyzed, allowing for real-time measurement of cortisol concentration. The present invention enables signal amplification of more than 30 times compared to the existing ELISA method and can be used quickly and easily without specialized equipment, so it can be utilized as an in vitro diagnostic system for the early diagnosis of depression and stress monitoring.

Description

Technology Field

[0001] This invention belongs to the field of Point-of-Care Testing (POCT) and biosensor technology, and relates to a technology for concentrating and quantitatively analyzing cortisol in saliva using magnetic beads. In particular, a strip sensor capable of detecting cortisol with high sensitivity by utilizing magnetic concentration and HRP signal amplification is developed, and a system capable of real-time quantitative analysis is provided by applying smartphone-based RGB analysis. Through this, an early diagnostic kit capable of effectively monitoring indicators of depression and stress is realized. Background Technology

[0003] Cortisol is a major hormone associated with the human stress response, and its concentration in the body is used as an important biomarker reflecting health conditions such as depression, chronic stress, and adrenal dysfunction. Conventional methods for measuring cortisol primarily utilize enzyme-linked immunosorbent assays (ELISA) or chromatography-based analysis (LC-MS) using blood, urine, or saliva samples; however, these methods have disadvantages, such as being time-consuming, requiring specialized equipment and skilled personnel, and making real-time monitoring difficult.

[0004] Recently, with the advancement of Point-of-Care Testing (POCT) technology, the development of biosensors utilizing strip sensors is actively underway to enable rapid and convenient on-site diagnosis. In particular, saliva-based analysis methods are attracting attention as a more user-friendly approach than blood analysis because they are non-invasive, easy to collect samples from, and allow for repetitive monitoring. However, existing saliva-based cortisol sensors have limitations, such as low sensitivity, making it difficult to detect trace amounts of cortisol and challenging to perform quantitative analysis.

[0005] Accordingly, the present invention aims to develop a strip sensor capable of detecting cortisol with high sensitivity by utilizing magnetic bead-based magnetic concentration technology and HRP (Horseradish Peroxidase) signal amplification, and to establish a quantitative analysis system using smartphone RGB tracking to provide a faster and simpler early diagnostic kit compared to the existing ELISA method. The problem to be solved

[0007] Existing saliva-based cortisol analysis methods have limitations, such as low sensitivity making it difficult to detect trace amounts of cortisol, restricted quantitative analysis, and long analysis times that hinder real-time diagnosis. Additionally, they require specialized equipment or skilled personnel, making them difficult to utilize at home or in the field. The present invention aims to develop an early diagnosis kit for depression that significantly improves cortisol detection sensitivity by applying magnetic bead-based magnetic concentration and HRP signal amplification technologies, and enables real-time quantitative analysis through smartphone RGB analysis. Through this, the invention seeks to provide a non-invasive, rapid, and user-friendly in vitro diagnostic system. means of solving the problem

[0009] The present invention relates to an in vitro diagnostic kit capable of early diagnosis of depression by concentrating cortisol in saliva using magnetic beads and performing quantitative analysis. By modifying the surface of magnetic beads (Fe₃O₄) to bind a cortisol-specific antibody (Anti-Cortisol Ab) with HRP (Horseradish Peroxidase), the device enables the effective capture of cortisol and signal amplification. The magnetic beads bound to cortisol are concentrated using a magnetic field to remove unnecessary components, and the signal is detected by reacting with a TMB solution to induce a color change. The strip sensor is designed to optimize the reaction while the sample travels to the absorption pad using capillary action, thereby enhancing signal uniformity and improving sensitivity. By applying smartphone-based RGB analysis technology, the color changes of the T-Line and R-Line are quantitatively analyzed, allowing for real-time measurement of cortisol concentration. The present invention enables signal amplification of more than 30 times compared to the existing ELISA method and can be used quickly and easily without specialized equipment, so it can be utilized as an in vitro diagnostic system for the early diagnosis of depression and stress monitoring.

[0010] An early diagnosis kit for detecting cortisol in saliva according to one embodiment of the present invention comprises: magnetic beads (Fe₃O₄) surface-modified with a cortisol-specific antibody (Anti-Cortisol Ab) and HRP (Horseradish Peroxidase); a reaction solution containing the magnetic beads that reacts with cortisol in a saliva sample; a magnetic concentration unit that concentrates the magnetic beads bound to cortisol using a magnetic field in the reaction solution containing the magnetic beads; a chromogenic unit that induces a color change by reacting with a TMB (3,3',5,5'-Tetramethylbenzidine) solution after washing the concentrated magnetic beads; and a strip sensor composed of a sample pad, a test line (T-Line), a reference line (R-Line), and an absorbent pad to confirm the presence and concentration of cortisol, and quantitatively analyzes the cortisol concentration by analyzing the color change of the strip sensor.

[0011] A method for quantitatively analyzing cortisol in saliva according to one embodiment of the present invention comprises the steps of: modifying the surface of a magnetic bead with an amine group (-NH₂) or a carboxyl group (-COOH) and binding a cortisol-specific antibody (Anti-Cortisol Ab) and HRP thereto; mixing the modified magnetic bead with a saliva sample to capture cortisol; concentrating the magnetic bead bound with cortisol using a magnetic field and removing unnecessary components; reacting the washed magnetic bead with a TMB (3,3',5,5'-Tetramethylbenzidine) solution to induce color development; and quantitatively analyzing the cortisol concentration through a color change of a strip sensor in a strip sensor composed of a sample pad, a test line (T-Line), a reference line (R-Line), and an absorbent pad to confirm the presence and concentration of cortisol. Effects of the invention

[0013] This invention utilizes magnetic bead-based magnetic concentration technology to effectively capture cortisol in saliva and applies HRP signal amplification to detect cortisol with a sensitivity up to 30 times higher than conventional ELISA methods. Furthermore, by optimizing fluid flow within the strip sensor to enhance signal uniformity and minimizing background signals, it enables precise analysis, thereby providing higher reliability than existing cortisol sensors. In addition, by applying smartphone RGB analysis technology, cortisol concentration can be quantitatively analyzed without the need for separate, expensive equipment, allowing users to monitor depression and stress levels in real-time on-site. Unlike conventional blood-based analysis methods, this invention enhances user convenience by employing a non-invasive saliva detection method. Since results can be verified quickly with simple operations, it is easy to utilize in Point-of-Care Testing (POCT) environments. Moreover, the analysis of cortisol concentration enables the early diagnosis of stress and depression, and continuous monitoring contributes to personalized mental health management and the establishment of smart healthcare systems. Brief explanation of the drawing

[0015] Figure 1 illustrates the structure and operating principle of an early depression diagnosis kit that concentrates and quantitatively analyzes cortisol in saliva using magnetic beads according to the present invention. Various embodiments are now described with reference to the drawings, and throughout the drawings, similar reference numerals are used to denote similar elements. For illustrative purposes, various descriptions are provided in this specification to facilitate an understanding of the invention. However, it is evident that these embodiments can be practiced without such specific descriptions. In other examples, known structures and devices are presented in the form of block diagrams to facilitate the description of the embodiments. Specific details for implementing the invention

[0016] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. As the present invention is susceptible to various modifications and may take various forms, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to the specific disclosed forms, and it should be understood that the invention includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the invention. Similar reference numerals have been used for similar components in the description of each drawing.

[0017] The terms used in this application are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as "comprising" or "having" are intended to indicate the presence of the features, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, steps, actions, components, parts, or combinations thereof.

[0018] Figure 1 illustrates the structure and operating principle of an early depression diagnosis kit that concentrates and quantitatively analyzes cortisol in saliva using magnetic beads according to the present invention.

[0019] First, when a saliva sample is injected into the kit, it is mixed with a solution containing magnetic beads, causing cortisol to bind to cortisol-specific antibodies on the surface of the beads. Subsequently, the magnetic beads are concentrated using a magnetic field, and after removing unnecessary components through a washing process, a TMB solution is added to react with HRP and induce a color change.

[0020] The strip sensor consists of a sample pad, a test line (T-Line), a reference line (R-Line), and an absorbent pad, and a fluid flow is formed by capillary action as the sample moves along the strip.

[0021] Furthermore, HRP reacts with TMB to induce a color change, and signals are detected at the test and reference lines. RGB values ​​can be measured and quantitatively analyzed using a smartphone camera. By linking a smartphone app with the strip sensor, cortisol concentration can be quantitatively calculated based on the analyzed RGB values, and the results can be visually displayed.

[0022] The present invention provides a Point-of-Care Testing (POCT) kit capable of early diagnosis of depression by concentrating cortisol in saliva with magnetic beads and quantitatively analyzing it using HRP signal amplification. To this end, rapid and accurate detection of cortisol is made possible by combining magnetic bead-based magnetic concentration technology, optimization of fluid flow within a strip sensor, a colorimetric reaction using a TMB solution, and quantitative analysis technology through smartphone RGB tracking.

[0023] The present invention comprises a process for capturing and concentrating cortisol using magnetic beads. To this end, the surface of Fe₃O₄ magnetic beads is modified with amine (-NH₂) or carboxyl (-COOH) groups to bind a cortisol-specific antibody (Anti-Cortisol Ab) with HRP (Horseradish Peroxidase). Through this process, cortisol in saliva samples is effectively captured by the magnetic beads, and signal sensitivity can be maximized by concentrating the beads using a magnetic field. HRP reacts with a TMB (3,3',5,5'-Tetramethylbenzidine) solution to produce color, thereby enabling the quantitative analysis of cortisol concentration. In this invention, at least 1,000 HRP molecules are bound to the magnetic beads to induce signal amplification, thereby providing a signal amplification effect of up to 30 times or more compared to the conventional ELISA method.

[0024] The strip sensor consists of a sample pad, a test line (T-Line), a reference line (R-Line), and an absorbent pad, and is designed to form a constant fluid flow through capillary action as a solution mixed with saliva samples and magnetic beads moves along the strip sensor. To optimize the fluid flow, Finite Element Analysis (FEA)-based simulations are performed to adjust the viscosity and surface tension of the TMB solution and maintain an optimal travel speed. Additionally, the TMB solution within the strip sensor performs the dual functions of washing and color development to reduce background signals and increase detection reliability.

[0025] The present invention includes a technology for quantitatively analyzing color change signals by applying smartphone-based RGB tracking technology. It measures color changes between a test line (T-Line) and a reference line (R-Line) using a smartphone camera and quantitatively calculates cortisol concentration by comparing them. It calculates the signal change rate through an RGB analysis algorithm and links the measured data with a smartphone app to allow real-time verification of results. Furthermore, the UI / UX is improved to allow users to intuitively view data, thereby enabling accurate cortisol analysis without the need for separate, expensive equipment.

[0026] The core technology of the present invention is quantitative analysis utilizing magnetic bead-based magnetic concentration, HRP signal amplification, fluid flow optimization, and smartphone RGB tracking, thereby providing a detection speed more than 10 times faster than existing methods, as well as high sensitivity and accuracy. While existing ELISA methods require specialized equipment in a laboratory setting, the present invention is developed as a Point-of-Care (POC) kit that can be used immediately in the field, offering high practicality and providing a simple in vitro diagnostic solution for monitoring depression and stress.

[0028] The implementation process of the present invention can be summarized as follows.

[0029] 1. Surface modification of magnetic beads and optimization of antibody (Anti-Cortisol Ab) and HRP binding

[0030] The binding of antibodies and HRP is optimized through surface modification of amine (-NH₂) or carboxyl (-COOH) groups of magnetic beads.

[0031] Using magnetic beads of 80 nm size, induce the binding of more than 1,000 HRPs.

[0032] We develop an antigen (Cortisol-Carrier) to enhance the binding affinity between magnetic beads and cortisol-specific antibodies.

[0033] 2. Cortisol capture and self-concentration processes in saliva

[0034] Cortisol in the saliva sample binds to cortisol-specific antibodies attached to magnetic beads.

[0035] It uses a magnetic field to concentrate magnetic beads and remove unnecessary components to maximize signal sensitivity.

[0036] 3. Induction of HRP reaction and color change using TMB solution

[0037] After the binding of the magnetic beads and cortisol is complete, a TMB (0.001% Tween 20) solution is added to induce the HRP reaction.

[0038] After fluid flow stops, TMB reacts with HRP to produce color, and signal differences occur depending on the cortisol concentration.

[0039] 4. Fluid Flow Optimization and Signal Detection Process within Strip Sensor

[0040] By controlling the fluid flow from the sample pad to the absorption pad, accurate signal detection is enabled on the test line (T-Line) and reference line (R-Line).

[0041] It ensures fluid flow uniformity and enhances washing functions to reduce background signals.

[0042] 5. Quantitative analysis using smartphone-based RGB tracking

[0043] Using a smartphone camera, color changes in the test line are analyzed, and cortisol concentration is quantified based on RGB values.

[0044] By applying a signal analysis algorithm, it is possible to calculate depression and stress indicators in real time.

[0046] The present invention provides a faster and simpler in vitro diagnostic system than existing methods by utilizing magnetic bead-based magnetic concentration and HRP signal amplification to detect cortisol in saliva with high sensitivity and to enable quantitative analysis through RGB analysis using a smartphone. This enables early diagnosis of depression and stress monitoring, and can be utilized as an in vitro diagnostic platform for real-time health management in conjunction with smart healthcare systems in the future.

[0047] Although the present invention has been described above with reference to preferred embodiments, those skilled in the art will understand that various modifications and changes can be made to the invention without departing from the spirit and scope of the invention as set forth in the following claims.

Claims

Claim 1 An early diagnosis kit for detecting cortisol in saliva, comprising: magnetic beads (Fe₃O₄) surface-modified with cortisol-specific antibodies (Anti-Cortisol Ab) and HRP (Horseradish Peroxidase); a reaction solution containing the magnetic beads that reacts with cortisol in a saliva sample; a magnetic concentration unit that concentrates the magnetic beads bound to cortisol using a magnetic field in the reaction solution containing the magnetic beads; a chromogenic unit that induces a color change by reacting with a TMB (3,3',5,5'-Tetramethylbenzidine) solution after washing the concentrated magnetic beads; and a strip sensor composed of a sample pad, a test line (T-Line), a reference line (R-Line), and an absorbent pad to confirm the presence and concentration of cortisol, and a quantitative analysis of cortisol concentration by analyzing the color change of the strip sensor. Claim 2 A method for quantitatively analyzing cortisol in saliva, comprising the steps of: modifying the surface of a magnetic bead with an amine group (-NH₂) or a carboxyl group (-COOH) and conjugating a cortisol-specific antibody (Anti-Cortisol Ab) and HRP thereto; mixing the modified magnetic bead with a saliva sample to capture cortisol; using a magnetic field to concentrate the cortisol-conjugated magnetic bead and remove unnecessary components; reacting the washed magnetic bead with a TMB (3,3',5,5'-Tetramethylbenzidine) solution to induce color development; and quantitatively analyzing the cortisol concentration through a color change of a strip sensor composed of a sample pad, a test line (T-Line), a reference line (R-Line), and an absorbent pad to confirm the presence and concentration of cortisol.

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