ENZYMATIC CONVERSION-BASED COLORIMETRIC BIOSENSOR PLATFORM, DIAGNOSTIC SYSTEM, PRODUCTION AND ANALYSIS METHOD

Abstract

The invention is a biosensor platform, diagnostic system, fabrication method, and analytical method for the determination of target analytes in biological samples via enzymatic conversion and colorimetric detection. The platform includes sample application, enzymatic conversion, colorimetric reaction, reference and / or calibration, and optional digital analysis components. In the preferred application, the target analyte is Indoxyl-β-D-Glucuronide (IDG), which is converted to indoxyl via β-glucuronidase, the resulting indoxyl is converted into colored reaction products via oxidation, and the resulting colorimetric signal is evaluated visually and / or digitally. The platform can be implemented in test strip, lateral flow, microfluidic cartridge, microplate, and other suitable analytical formats.

Description

1 TARIFF ENZYMATIC CONVERSION-BASED COLORIMETRIC BIOSENSOR PLATFORM, DIAGNOSTIC SYSTEM, PRODUCTION AND ANALYSIS METHOD Technical Area The invention describes the colorimetric measurement of the level of Indoxyl-β-D-Glucuronide (IDG) in a urine sample. It involves a test strip to determine its status. The invention also describes the level of Indoxyl-β-D-Glucuronide (IDG) found in a urine sample. Production method and use of a test strip for colorimetric determination. It is related to. The invention also involves electronic imaging of the colorimetric signal occurring on the test strip, 10 digital image processing, calibration, computational evaluation, data transfer, and software. electronic analysis system and method for evaluation through analysis based on analysis It is related. State of the Art Chronic kidney disease (CKD) is a condition in which kidney function deteriorates over time. It is characterized by a decrease in the amount of blood flow, and may not present with noticeable clinical signs in the early stages. Therefore, various laboratory analyses are used in its diagnosis. Nowadays in the evaluation of kidney function on serum and urine samples The biochemical analyses performed are based on the measurement of various biomarkers. It is widely used. In this context, changes in kidney function 20 In addition to traditional biomarkers, gut microbiota is also used in its assessment. for the evaluation of metabolites derived from the source and their levels in urine. Approaches are also being investigated to obtain urine samples non-invasively. The sampling process should be easy to implement and repeatable when necessary. Because it allows for sampling and is suitable for serial monitoring, it is used in biological analyses. 25 It constitutes an advantageous type of example. However, the importance of these analyses... Since some of the work is carried out in a laboratory environment, suitable analytical equipment is required. It may require laboratory infrastructure and trained personnel. For example sample preparation, transfer to the appropriate instrument for measurement, and measurement results. Sequential processes such as evaluation can increase the analysis time; device usage 30 Related analysis methods include applications outside the laboratory or in field conditions. 2 This can be limiting, especially due to the need for non-portable analytical devices. The fact that the analysis process may require multiple steps means that current methods are not suitable for the field. These limitations can make its implementation operationally difficult under these conditions. biological samples can be collected quickly and practically outside the laboratory or at the patient's bedside. 5 The need for analytical approaches that can enable evaluation It increases. Furthermore, obtaining the results may require a certain amount of analysis time, and This situation demonstrates the applicability of the analyses in different use cases. It can have an impact. Accordingly, the rapid and practical analysis of analytes found in biological samples 10 enzymatic and colorimetric detection principles for its identification Utilizing this, test systems and biosensor-based analysis approaches have been developed. Enzymatic methods for determining various analytes found in biological samples. Analytical methods based on reactions have been known for many years. In these methods... color resulting from the reaction of specific enzymes with substrates Colorimetric analysis by evaluating changes and differences in color intensity 15 This is being carried out. In addition, various tests are being used for the analysis of biological samples. Test strips, colorimetric analysis systems, and biosensor structures have been developed. The sample and reagents come into contact on a specific carrier structure, visually or orally. While they can be used as analysis platforms based on generating a measurable signal, Biosensor systems consist of 20 biological recognition or reaction elements. with a detection component for signal detection and evaluation It may include. However, signal detection in current biosensor systems and the need for additional reading devices for evaluation, the reagent used and the cost may increase depending on the detection systems, and the analysis time may also increase. the possibility of variation and the sensitivity to different analyte concentrations 25 Technical limitations such as these may exist. This type of enzymatic and colorimetric detection... The principles are based on the enzymatic reaction of a specific analyte into a product that can produce color. transformation and the resulting color change in determining the analyte level It also forms the basis for analytical approaches that rely on the use of this principle. Accordingly, an easily applicable test structure for analytes in biological samples 30 determination via and visual and / or digital analysis of the colorimetric results obtained development of analytical systems that can enable such evaluation These systems are needed. However, these systems require optical, electrochemical, and colorimetric analysis. Designed according to different detection principles, such as those mentioned above, its intended use and analysis Accuracy, analytical sensitivity, selectivity, analysis depending on the method and application conditions 35 3 differs in terms of technical performance parameters such as duration and repeatability. It can demonstrate. Chromogenic substrates used in enzymatic analysis and their reactions. Various technical solutions regarding their mechanisms are also included in the known state of the art. In addition, 5 different biomarkers found in biological samples analytical systems for determining and their laboratory and field use. Various suitable applications have been developed. Analyses are performed in a laboratory environment. While it can be carried out depending on specific analytical equipment and infrastructure, in field applications portability, the ability to perform analysis with fewer processing steps, and quick results. It is becoming increasingly important that it can be evaluated within a short period of time. However, general chromogenic 10 analytical approaches, Indoxyl-β-D-Glucuronide (IDG) found in urine samples its level via enzymatic conversion and the associated colorimetric signal It may not directly meet the specific analytical requirements for its identification. However, current technical solutions for the analysis of biological samples alternative analysis that can be developed and respond to different application needs 15 Work is ongoing to develop these approaches. In this context, specifically, analysis aimed at determining the level of IDG found in urine samples. Developing these approaches is important. Therefore, in the technical field, applications that can be used in the analysis of biological samples are needed. 20 that can be more easily adapted to different conditions and meet different usage needs The need for alternative analysis systems continues. As a result of the research conducted on this subject, it was determined that sample number EP2981619A1 "In a sample Detection and / or enumeration of enterobacteria requires at least one chromogenic and / or fluorogenic marker. An application titled "Use of phosphatase substrate" was found. The application concerns food and the environment. and the detection and / or quantification of Enterobacteria in biological samples 25 in which at least one chromogenic and / or fluorogenic phosphatase substrate is used The microbiological analysis system is explained. However, current technical solutions do not measure Indoxyl-β-D-Glucuronide (IDG) levels in urine. rapid on-site determination from the sample and the resulting colorimetric result all the needs for visual and / or digital evaluation together 30 It does not meet the requirements. 4 In conclusion, due to the negative aspects described above and the current solutions being the subject of discussion... Due to its shortcomings, an improvement is needed in the relevant technical field. It has been made. Purpose of the Invention The invention was created by drawing inspiration from existing situations and addressing the aforementioned drawbacks. 5 It aims to solve the problem. The main objective of the invention is the enzymatic conversion of target analytes found in biological samples. and enables its rapid and practical determination via colorimetric reaction. The goal is to develop a biosensor platform. The target analyte in a preferred application. Indoxyl-β-D-Glucuronide (IDG) is the platform in question, and the colorimetric level of IDG is 10⁻⁶. to be determined as such and the result obtained visually and / or digitally This allows for evaluation. Another objective of the invention is to appropriately identify target analytes found in different biological samples. using enzymatic conversion elements and colorimetric reaction systems The aim is to create a modular analysis platform that allows for the determination of 15 problems. Another aim of the invention is to visually identify biomarkers found in biological samples. The goal is to establish an analytical system that allows for evaluation. Another aim of the invention is in the evaluation of chronic kidney disease. The goal is to develop a usable and practically applicable analysis system. Another aim of the invention is to create an easy-to-use 20-inch device that can be used in the analysis of biological samples. an analysis system that contributes to obtaining feasible and reliable results to place. Another objective of the invention is to separate the colorimetric signal occurring on the test strip. imaging, image preprocessing, region identification, color analysis, numerical evaluation, processing through results generation and reporting components, reference and 25 normalization with calibration data, evaluation of reaction kinetics, and The results obtained can be used via portable reader, mobile application, user interface, wireless a modular system that enables delivery through communication and data management infrastructures The goal is to establish an analysis system. The invention is used to achieve the purposes described above; it is based on the detection of substances found in urine samples. A method for colorimetric determination of indoxyl-β-D-glucuronide (IDG) levels. test strip; at least one supporting structure and the arrangement on said supporting structure It must contain at least one reactive site, and the said reactive site must be at least one β-glucuronidase. It contains at least one oxidizing agent along with its enzyme. 5 The invention also involves determining the level of Indoxyl-β-D-Glucuronide (IDG) found in a urine sample. This is a method for producing a test strip for colorimetric determination. Production The method involves a reactive system containing at least one oxidizing agent and the β-glucuronidase enzyme. preparation and placement of the prepared reactive system on at least one carrier structure, at least 10 It involves applying it to a small, reactive area in the following steps. The invention also involves measuring the level of Indoxyl-β-D-Glucuronide (IDG) in a urine sample during testing. the conversion of the colorimetric signal generated in the strip into digital image data and the word the subject is the processing of image data together with reference and / or calibration data. electronically via software running on an electronic device 15 It is an electronic analysis system for determining this. The electronic analysis system;  An imaging system displays the colorimetric signal generated on the test strip. an image acquisition module that records the image data as digital image data by capturing it from the medium,  digital image data recorded by the image acquisition module preliminary 20 aimed at reducing variability resulting from imaging conditions An image preprocessing module that creates processed image data by applying processing techniques.  on the processed image data generated by the image preprocessing module By determining the reaction region, the area of ​​analysis where the colorimetric signal occurs. Module for identifying the reaction region that generates data,  25 on the processed image data generated by the image preprocessing module In normalizing and / or calibrating reaction zone data Generating reference region data by defining the reference area to be used. Reference region determination module,  Color parameters from reaction region data and / or reference region data The color analysis module, which generates digital color data by extracting optical density data, is 30.  Reference area for numerical color data generated by the color analysis module. normalize with data and / or calibration data and perform mathematical, statistical, by applying image processing and / or machine learning-based assessment 6 Numerical data that generates a quantitative assessment of the IDG level. evaluation module,  Numerical evaluation generated by the numerical evaluation module generating results that transform data into analytical output regarding IDG level module, 5  The analysis output generated by the results generation module is displayed to the user. converting into a transmissible digital, graphical and / or classified output format It includes a reporting module. The invention also involves converting a colorimetric signal generated on a biosensor platform into an electronic signal. a 10 for the numerical analysis of imaging and data processing. It is a method. The method;  Fixed display of the analysis platform under standard lighting conditions viewing under its distance and angle,  The image should be obtained in high-resolution RAW and / or JPEG format, and recorded with a timestamp, 15 • White balance, noise reduction, background correction, contrast adjustments are applied to the image. related to adjustment, brightness normalization, geometric and / or perspective correction. regional selection implementation,  Automatic designation of test area, control area, reference area and calibration area or semi-automatic identification and segmentation, 20  RGB, HSV, HSL, CIELAB, XYZ, YUV, CMYK, YCbCr from the specified regions and / or grayscale parameters, optical density and color channel ratios removal,  Reference region of the test signal, calibration region, internal reference dye and / or Normalization using the internal normalization region, 25  Ratio method for normalized data, calibration curve, linear regression, nonlinear model, mathematical model, statistical model and / or Converting to analyte concentration using a machine learning model,  Obtained at fixed time intervals from the start of the reaction images, time-lapse viewing, continuous viewing and / or video 30 Monitoring time-dependent color change and reaction kinetics via recording. determination, 7  Image processing of color, optical density, feature and / or reaction kinetics data, statistical classification, artificial intelligence, machine learning and / or deep learning evaluation using algorithms,  The accuracy, repeatability, signal-to-noise ratio, and detection limit of the analysis result, 5 with determination limit, recovery, sensitivity and valid / invalid result decision criteria. undergoing quality control,  Analysis regarding analyte concentration and / or evaluation result the creation of the result,  The analysis results in the form of numerical, graphical and / or risk classification. reporting, 10  Saving the analysis result with the date, time, analysis number, and user information. and archiving,  Analysis results can be accessed via mobile application, laboratory information management system, and wireless network. data communication and / or transfer via cloud server,  Analysis data recorded in offline conditions will be available in the cloud when a connection is established. synchronization with the server and / or other data management systems,  Signals obtained from multiple reaction regions of the same sample multiple analysis outputs are evaluated within a common digital analysis framework. creation It includes the steps of the process. 20 The structural and characteristic features and all the advantages of the invention are given in the figures below. This becomes clearer thanks to the detailed explanation written with references to these figures. This will be understood, and therefore the evaluation should also take these forms and detailed explanations into consideration. It needs to be done by taking precautions. 25 Figures that will help understand the invention. Figure 1 shows the general operation of the enzymatic conversion-based colorimetric biosensor platform. It is a schematic representation of the principle. Figure 2 shows the relationship between target analyte concentration and colorimetric signal. This is an example / schematic calibration illustration. 30 Figure 3 illustrates the application of colorimetric signals in digital imaging, image processing, color analysis, and This is a schematic representation of how the result is converted into a numerical analysis result through calibration. 8 Figure 4 shows a sample of the modular biosensor platform based on enzymatic conversion. Applications include enzymatic conversion, colorimetric reactions, reference and calibration, and digital processes. It illustrates the overall system architecture between the analysis and the output result. Figure 5 shows the biosensor platform's reagent preparation, the transfer of reagents to the carrier matrix. Application, drying, stabilization, lamination and assembly, test strip format 5 It shows the stages of cutting, quality control, packaging, labeling, and storage. Figure 6 is a representative flowchart of the method described in the invention. REFERENCE NUMBERS 1. Sample application module 10 2. Enzymatic conversion module 3. Colorimetric reaction modulus 4. Reference / calibration module 5. Digital analysis module 6. Analysis result 15 7. Sample pad 8. Enzymatic reaction site 9. Colorimetric reaction area 10. Control area 11. Reference / calibration zone 20 12. Absorbent pad 13. Carrier layer 1001. Fixed viewing distance of the analysis platform under standard lighting conditions. and viewed under the angle 25 1002. The image should be obtained in high-resolution RAW and / or JPEG format, and recorded with a timestamp 1003. White balance, noise reduction, background correction, contrast adjustment to the image. Area of ​​interest with adjustment, brightness normalization, geometric and / or perspective correction. The implementation of the election 30 1004. Automatic designation of test zone, control zone, reference zone and calibration zone. or semi-automatic identification and segmentation 9 1005. RGB, HSV, HSL, CIELAB, XYZ, YUV, CMYK, YCbCr from the specified regions. and / or extraction of optical density and color channel ratios using grayscale parameters. 1006. Test signal reference region, calibration region, internal reference dye and / or normalization using the internal normalization region 1007. Ratio method of normalized data, calibration curve, linear regression, 5 nonlinear model, mathematical model, statistical model and / or machine conversion to analyte concentration using a learning model 1008. Obtained at fixed time intervals from the start of the reaction. images, time-lapse viewing, continuous viewing and / or video recording Monitoring time-dependent color change and determining reaction kinetics 10 1009. Image processing of color, optical density, feature and / or reaction kinetics data, statistical classification, artificial intelligence, machine learning and / or deep learning evaluation using algorithms 1010. Accuracy, repeatability, signal-to-noise ratio, detection limit, determination of the analysis result. Quality 15 with limit, recovery, sensitivity and valid / invalid result decision criteria. to be checked 1011. Analysis relating to analyte concentration and / or evaluation result. the creation of the result 1012. Analysis results in numerical, graphical and / or risk classification form. reporting 20 1013. Recording the analysis result with date, time, analysis number and user information. and archiving 1014. Analysis results via mobile application, laboratory information management system, wireless data communication and / or transmission via cloud server 1015. Analysis data recorded in offline conditions will be available in the cloud when a connection is established. synchronization with the server and / or other data management systems 1016. Common signals obtained from multiple reaction regions of the same sample Generating multiple analysis outputs by evaluating them within a digital analysis framework. Detailed Description of the Invention 30 This detailed explanation describes the enzymatic conversion-based colorimetric model that is the subject of the invention. biosensor platform, the structure and working principle of the platform, production method, methods of use, preferred test lane and other platform configurations, and electronics. and digital analytics structures, solely for the purpose of better understanding the invention. It is explained. The invention describes a suitable enzymatic conversion of a target analyte present in a biological sample. conversion of the element into a reaction product suitable for color formation and The product in question has a measurable 5 through oxidation and / or colorimetric reaction. It is based on the principle of signal generation. It is formed as a result of enzymatic transformation. The reaction product is formed into a colored product with a suitable oxidizing agent and / or reaction system. can be converted, and the resulting colorimetric signal can be visually and / or digitally displayed. This can be evaluated using image analysis. The target in a preferred application. The analyte is Indoxyl-β-D-Glucuronide (IDG), and the glucuronide bond in the IDG structure is β-10. It is hydrolyzed by the glucuronidase enzyme to form indoxyl and D-glucuronic acid. is brought in; the resulting indoxyl then undergoes oxidation to form indigo and / or indigoid structures. This creates and produces a blue and / or purple colorimetric signal. Thus Enzymatic conversion, oxidation, and color formation are successive technical steps. This can be done as follows. 15 The biological and metabolic basis upon which the working principle of the invention described above is based. This is explained below. Metabolism of dietary tryptophan by the gut microbiota As a result, indole is formed in the intestine. The formed indole is then processed in the liver. As a result of the metabolic transformations that take place, it is converted to indoxyl; indoxyl is then 20 Conjugation with sulfate or glucuronide results in indoxyl sulfate and indoxyl-β-, respectively. It forms indoxyl derivatives such as D-glucuronide (IDG). Among these indoxyl derivatives... The analyte directly targeted within the scope of this invention is Indoxyl-β-D-Glucuronide (IDG). Indoxyl derivatives, in question, can be eliminated from the body via the kidneys. If renal elimination by the kidneys is reduced, the biological 25 of these metabolites... Their levels in fluids can vary, and these metabolites are related to the microbiome and the kidneys. It can be used to evaluate the relationship between functions. IDG in urine The presence of a measurable indoxyl derivative in the sample and changes in its level are renal It can contribute to the evaluation of changes in elimination, The use of metabolites as target biomarkers in evaluating this relationship is 30. This provides the opportunity. The metabolite directly targeted for analysis within the scope of the invention is the one mentioned. The subject is indoxyl-β-D-glucuronide (IDG), an indoxyl derivative, found in urine samples. IDG levels are determined colorimetrically. 11 In a preferred application, the invention is a disposable test strip. It is being structured. In another application, the invention involves a biosensor, a detection interface, a display or sensing device, a calculation module and a processing module It is implemented as an integrated diagnostic system. This system is biological. 5 microbiome-derived metabolites in the sample and their association with kidney function Detection of biomarkers, conversion of selected metabolites into color-producing products. enzymatic conversion, visualization of the resulting signals and / or digitization and evaluation of kidney function using the obtained data. It ensures that the process is carried out for that purpose. In applications where the invention is implemented as an integrated system, 10 is obtained from the test strip. The obtained colorimetric data are used for digital imaging, signal quantification, and computational analysis. It may be subjected to data processing and / or artificial intelligence-based analysis processes. The test strip, which is the subject of this invention, essentially consists of a carrier structure and a structure mounted on that carrier. It consists of reaction zones placed within it. The carrier structure is mechanically attached to the test strip. In addition to providing support, the reaction zones are determined on the test strip at 15 the organization of locations and the protection of those locations during use It functions as a structural element that provides support and testing. In this respect, the load-bearing structure acts as a test element. It ensures the preservation of the structural arrangement of the strip. Testing in the preferred application. the strip allows the user to securely hold the test strip in contact with the sample. It includes a holding region (handle) that provides a secure grip. The holding region is 20 degrees away from the reactive regions. It is physically separated and does not come into direct contact with the sample. This arrangement, by reducing the likelihood of the user directly contacting reactive areas, user-generated This contributes to reducing the risk of contamination. Therefore, the holding area, the test This contributes to the use of the strip without direct contact with the sample and reactive areas. It ensures the mechanical integrity of the test strip in the continuation of the holding area. 25 There is a support layer that provides protection. The support layer allows for the use of the test strip. bending, deformation, or reduction in mechanical strength during this process It prevents this. In addition, the support layer protects the test strip from moisture changes. limiting potential dimensional changes and the dimensional accuracy of the test strip It contributes to maintaining its stability. In this respect, the support layer, test 30 It serves to maintain the mechanical and dimensional stability of the strip. Support At least one reagent pad is placed on the sample sheet. The reagent pad is placed on the urine sample. Enables the enzymatic conversion of Indoxyl-β-D-Glucuronide (IDG) found there. It contains the reagents. The reagent pad ensures the transport of these reagents. 12 In addition, the sample comes into contact with the reagents and undergoes enzymatic conversion of IDG into it. It creates a reaction microenvironment in which a dependent colorimetric reaction takes place. Therefore, the reactive pad is intended for the enzymatic and colorimetric determination of IDG. It forms the main functional region where reactions take place. In the preferred application, the reactive pad consists of a porous cellulose-based structure. 5 The porous cellulose structure allows the urine sample to be absorbed into the reactive zone by capillary action. suitable for the transport and distribution of β-glucuronidase enzyme on the reactive pad The matrix provides the β-glucuronidase enzyme to work in this porous cellulose matrix. It is immobilized onto the surface. This immobilization involves the β-glucuronidase enzyme. The reagent pad should be held stably on the surface and the reaction should occur during contact with the sample. This helps to limit its movement away from the region. Thus, enzyme test It is fixed on the strip and the reaction is controlled when it comes into contact with the urine sample. This ensures that the process takes place within this structure, in the reactive region of the sample. It supports the transport and retention of the immobilized enzyme in the reaction site. Beneath the porous cellulose layer lies a polymer binder layer. 15 Polymer binder layer is preferred in applications using polyvinyl alcohol (PVA), chitosan or It consists of combinations of these. PVA and chitosan are used together. In applications, these polymers act as film-forming agents and carriers for reactive components. The properties that contribute to its retention on the structure can be utilized together. The polymer binder layer ensures the enzyme is held on the carrier structure and the reactive system is 20 It ensures the preservation of its stability. In addition, the polymer binder layer is reactive. maintaining the even distribution of components on the reactive zone and contact with the sample. This can help to keep the reactive system contained in that region. Here, the stability of the reactive system depends on the activity of the β-glucuronidase enzyme and the reactive system. This refers to contributing to the preservation of the functionality of the components during storage. 25 In this respect, the polymer binder layer holds the reactive components in the reactive region. an intermediate layer that contributes to the retention and maintenance of the stability of the reactive system. It serves this function. Beneath the polymer binder layer is the mechanical part of the test strip. It has a polyester-based backing film that provides its durability. Polyester-based The backing film provides mechanical resistance to the test strip, maintaining its shape during use and 30 It is preferred because it contributes to the preservation of structural integrity. This backing film, physical protection of the test strip during production, transportation, storage and use. It maintains its integrity. Thus, the layers forming the test strip are relative to each other. their positions are maintained and the reactive area is not bent or twisted during use. 13 This helps to limit the deformation of the test strip's structural integrity. It is supported that it works consistently. Therefore, polyester-based support The film is mechanically designed to preserve the physical integrity of the layered structure of the test strip. It provides support. At least one indicator area following the reactive pad. It is located there. The indicator region is the area where 5 of the products formed as a result of the enzymatic reaction are located. This is the region where the color change resulting from oxidation is observed. β- Indoxyl, formed as a result of hydrolysis carried out by the glucuronidase enzyme, is an oxidizing agent. It undergoes oxidation in the presence of the agent, and as a result of this oxidation, it becomes colored. Chromogenic products are formed, generating an observable colorimetric signal. In the preferred configuration, the color formation occurring in this area is blue and / or purple. 10 It is in shades of gray. The intensity of the resulting color depends on the IDG level in the urine sample. differences in color intensity are correlated and used in the assessment of IDG levels. This can be used. This evaluation involves comparing the resulting color to a reference color scale and visually. by comparing and / or digitally analyzing color intensity This can be achieved. Therefore, the indicator region contains enzymatic and oxidative 15 the colorimetric signal generated as a result of the reactions is observed and evaluated It constitutes the region. In a preferred configuration, the enzyme site and the indicator site are marked on the test strip. They are arranged separately. In another configuration, the test strip is used to measure the sample. a sample site where it is applied, followed by an enzyme site, and then a 20 It includes an indicator region. Thus, the sample moves in a specific flow direction. The sample reaches the reaction zones in sequence. The sample's movement between these zones... its progression is via capillary flow occurring through the porous structure of the test strip. This ensures that the sample reaches the enzyme site and then the indicator site, respectively. This allows for an alternative configuration where the enzyme site and the indicator site are the same. They are arranged together within the composite structure. Thus, via enzymatic reaction. The colorimetric reaction can be carried out within the same reaction matrix. The subject is composite structure, enzymatic and colorimetric reactions of components. integration of separate reaction sites within a single reaction matrix This allows for a reduction in the number of reactions. Using the same reaction matrix, 30 colorimetric reaction of the reaction product formed as a result of enzymatic transformation by allowing the components to come into contact in the same environment, thus enabling the reaction. reducing the need for physical transfer between steps and colorimetric It contributes to the generation of the signal in the same reaction environment. 14 The test strip also measures the capillary flow mechanism of the sample, in other words, the capillary action. There is a flow path that enables its transport by impact. Sample, application It is transported from this region to the reaction areas via capillary absorption mechanisms. The liquid front that forms during the advancement of the sample, porous carrier structure. by moving along it, the sample reaches the enzyme site and the indicator site 5 Capillary flow ensures controlled delivery of the sample to the reaction sites. ensuring that it reaches its destination and that its contact with the reactants occurs in a specific flow pattern. This contributes to the control of reaction time and the comparison of different measurements. This is important in terms of establishing repeatable analysis conditions. The test strip in this design is nitrocellulose-based. Thanks to its porous structure, nitrocellulose has 10 providing a suitable environment for the transport of the sample by capillary flow and its protein structure It is preferred because it offers a surface suitable for the retention of reactants on the carrier structure. It can be done. In alternative constructions, the load-bearing structure is polyvinylidene fluoride (PVDF) or It can also be structured as a cellulose-based material. The PVDF-based carrier structure is reactive. immobilization of components and transport of the sample within a porous structure 15 Nitrocellulose-based carriers are preferred in applications where a suitable membrane structure is chosen. It can be used as an alternative to the existing structure. The preferred configuration is the test strip. It is approximately 3-5 mm wide. The upper surface of the test strip is protective and waterproof. A support layer may be present. This protective and waterproof support... The layer provides mechanical protection of the test strip, as well as environmental protection of reactive zones. limiting direct contact of the reactive system with moisture and external factors. It can contribute to maintaining its stability. At the bottom, the sample... a spreading layer and / or hydrophobic barrier that supports controlled propagation It can receive it. The spreading layer ensures that the sample is evenly distributed on the test strip. 25 This provides a hydrophobic barrier that prevents the sample from reaching areas outside the designated flow path. by limiting the spread of fluid flow in the direction of reaction zones. It contributes to its guidance. In the application of the invention for performing multiple analyses, the biosensor acts as a support. The sample pad (7), conjugate pad, reaction membrane and 30 are arranged on the layer. in the form of a multilayer lateral flow test strip containing an absorbent pad This can be achieved. The lateral flow principle involves the porous layers of the sample. This allows the sample to be transported via passive capillary flow throughout the reaction. eliminating the need for an external pumping system to advance the material to its destinations. It removes. In this configuration, the sample pad (7) is placed on the test strip of the applied sample 35 to be taken up and to move in the direction of flow; conjugate pad, reagent used in analysis contact of the components with the sample; reaction membrane, analytes a reaction in which reactions are carried out and measurable signals are generated to carry the reaction areas; the absorbent pad absorbs the fluid passing through the reaction areas and It ensures the continuity of capillary flow. The support layer is mentioned in point 5. The subject is the structural integrity of the layers on the test strip and their relationship to each other. This contributes to the preservation of their positions. Within the scope of the invention, "multiple analysis" is the same. Multiple biomarkers present in the sample appear on different test strips located on the same test strip. It refers to the simultaneous evaluation through reaction zones. In this configuration, multiple reaction sites can be spatially separated from each other.10 and allows for the detection of different analytes on the same test strip. Reaction the regions are physically separated from each other and the sample flow follows a defined flow path. directing the reactants located in different reaction sites along the way limiting unwanted interactions arising from different analytes The colorimetric signals are spatially separated from each other, and each signal corresponds to its respective reaction. 15 It contributes to the independent evaluation of the region. The biomarkers to be detected in the multiple analysis application are of microbiome origin. metabolites, biomarkers associated with renal function, and additional biochemical These indicators may include biomarkers. The biomarkers mentioned here are examples only. Multiple analysis applications are not limited to these. Microbiome-derived metabolites 20 among them indoxyl sulfate and indoxyl glucuronide; related to kidney function Biomarkers may include creatinine, albumin, microalbumin, and phosphate. The combined evaluation of these biomarkers suggests microbiome-derived metabolic activity. changes in biochemical markers associated with different aspects of kidney function by evaluating them together on the same sample and thus a more comprehensive 25 It enables biochemical evaluation to be carried out. In an application, the conjugate pad is designed to interact with target biomarkers. It contains functionalized nanoparticles. The sample comes into contact with the conjugate pad. Upon this, the functionalized nanoparticles on the pad are mobilized. It participates in capillary flow along with the sample and moves towards the reaction membrane 30 These nanoparticles are transported. These nanoparticles include gold nanoparticles and latex nanoparticles. from silica nanoparticles, carbon nanoparticles or quantum dots These nanoparticles can be selected. In the analysis system, they can be optically and / or measurably detected. They must be able to exhibit characteristics suitable for generating a signal and their surfaces must be target 16 enabling their functionality with recognition elements specific to biomarkers They are preferred for this reason. Nanoparticles are antibodies, aptamers, enzyme-linked. They can be functionalized with binding proteins or molecularly imprinted polymers. These binding elements bind to other substances found in the target biomarker sample. Selectively recognizing components and establishing a link between the target biomarker and the nanoparticle is possible. It allows for the creation of a specific interaction. In one example application, the reaction membrane has a pore size of 8–15 µm. It consists of nitrocellulose; the reaction sites are 3–8 mm apart from each other. They are separated. The distance between the reaction zones affects the capillary flow of the sample. While enabling it to move between regions in this direction, different reactions 10 in a way that will allow the regions to be spatially separated from each other This spatial distinction can be determined. This spatial separation occurs in different reaction regions. unwanted interaction between reactants and the resulting signals limiting and separately analyzing the signal obtained from each reaction region. It contributes to the evaluation. The pore size of the reaction membrane is 15 the capillary flow rate of the sample within the membrane and, consequently, the sample It can affect the contact time with reactants in the reaction areas. Therefore... The selection of pore size determines a flow pattern suitable for the reaction zones of the sample. in terms of ensuring that it reaches the inside and that sufficient contact time is provided for the reaction. It is important. The reaction membrane allows for reactions related to target biomarkers. In addition to carrying the reaction sites where it takes place, through its porous structure the sample moves through the reaction zones by capillary flow and the fluid flow It forms a fundamental structure that contributes to its regulation. Gold nanoparticles If used, the diameter of the nanoparticles is 10–100 nm, and in a preferred application... The nanoparticle size can be in the range of 20–60 nm, and the total number of nanoparticles per test strip is 5–25. It can be found in the 500 µg range. The reagent system on the test strip contains enzymatic indoxyl-β-D-glucuronide (IDG). will enable the transformation and the colorimetric monitoring of the reaction that occurs. It is prepared in this way. The reactive system in question involves the enzymatic conversion of IDG. The process involves obtaining a colorimetric signal from the resulting product after the transformation. the components necessary for its formation and maintaining the reaction conditions together It includes. The reactive system basically consists of; at least one β-glucuronidase enzyme, at least one oxidizing agent, at least one buffer solution, at least one stabilizer, and at least one moisture content. It contains a regulator. β-glucuronidase enzyme is immobilized on the reactive pad. 17 and the use of a stabilizer within the reagent system, the enzyme on the test strip contributes to the preservation of its activity and its maintenance under storage and usage conditions. It provides. In the preferred configuration, the amount of β-glucuronidase per test strip is 0.2- It is between 2 U. The amount of enzyme in question is within the range of the sample applied to the test strip. Suitable enzyme 5 is required to carry out the enzymatic conversion of the IDG found. ensuring its activity and the analysis conditions of the enzymatic reaction test strip. It is determined by considering whether it can be carried out within it. As an oxidizing agent. A ferric / ferrocyanide system, peroxidase, MnO₂ and / or oxygen are used. The subject is oxidizing agents, which can be used individually or in combination as alternatives to each other. They can be used in combinations, and their combined use is mandatory. It is not. The oxidizing agent is enzymatic hydrolysis carried out via β-glucuronidase. by contributing to the oxidation of indoxylin formed as a result, the colored product and related It enables the generation of a colorimetric signal. In the selection of the oxidizing agent, Suitability for carrying out indoxylin oxidation, colorimetric signal Contribution to its formation, stability within the reactive system and suitability under analytical conditions 15 Technical characteristics such as reaction behavior can be taken into consideration. Preferred In this formulation, the amount of oxidizing agent per test strip is between 0.1 and 2 mg. The buffer system regulates the pH of the reaction medium. Preferred In this formulation, the pH value of the buffer solution is in the range of 5.5-6.5. This pH range refers to: Supporting the activity of the β-glucuronidase enzyme and the enzymatic 20 of IDG providing a suitable reaction environment in order for the transformation to take place It is preferred for this purpose. The amount of buffer per test strip is 1-3 µg. is applied. The amount of buffer in question depends on the contact of the sample with the reactive system. during this process, the pH of the reaction medium must be kept within the appropriate range, and the pH It is determined in a way that will contribute to maintaining its stability. Reactive system 25 It also contains trehalose, albumin, or similar stabilizers. In addition to contributing to the storage stability of the reactive system, stabilizers also provide β- Contributes to preserving the activity of the glucuronidase enzyme during storage and use. It provides. In the preferred setup, the amount of stabilizer is 5-20 µg per test strip. It is among them. The amount of stabilizer in question affects β-glucuronidase 30 during storage. limiting the potential loss in enzyme activity and the reactive system This is determined by considering the preservation of its functionality. In the reactive system, there is also... at least one hum regulator that ensures the maintenance of moisture balance in the reaction environment. It is located there. This humidity regulator controls the amount of moisture in the reactive zone and By contributing to the maintenance of moisture balance, it protects reactive components from moisture changes. 18 help to limit the physical and / or functional changes that may result. The preferred setup involves an amount of moisture regulator of 2-10 mg per test strip. The reactive system ensures the preservation of enzyme activity throughout the reaction, the color formation should be repeatable and the test strip should remain stable during storage. It is prepared in a way that will ensure it remains. Here, the color formation is 5 Repeatability, using different test strips of the same or comparable samples. Similar colorimetric responses when evaluated under the same analytical conditions. It means that it can be obtained. The reactive system described above involves the enzymatic conversion of IDG and its associated components. Based on the generation of a colorimetric signal, and described in detail below, 10 It ensures the implementation of the working principle. The operating principle of the platform described in the invention is based on the enzymatic processing of the target analyte. conversion and the colorimetric analysis of the resulting product. It is based on evaluation via reaction pathway. In the preferred IDG application. Analysis process; application of the biological sample to the sample application area, IDG β-15 hydrolysis via the glucuronidase enzyme, resulting in indoxylin. oxidation, formation of colored product and the resulting colorimetric signal visual evaluation, comparison with a reference color scale and / or digital sequential steps such as evaluation using image analysis It consists of. In other applications of the platform, the target analyte and the selected enzymatic 20 Similar conversion and colorimetric detection steps depending on the conversion system. It is applicable. The reaction that occurred is described as follows. Indoxyl-β-D-Glucuronide + H₂O → Indoxyl + D-Glucuronic Acid The indoxyl formed as a result of this reaction is present in the subsequent oxidation on the test strip. by participating in the intermediate step, it forms the basis for the generation of the colorimetric signal. This product is formed by the hydrolysis of D-glucuronic acid via β-glucuronidase. It is the other reaction product that results. Indoxyl, formed as a result of hydrolysis, is an oxidizing agent present in the reactive system. and / or undergoes oxidation in the presence of atmospheric oxygen. Oxidation 30 As a result, indigo and / or indigoid structures are formed, and consequently 19 A visible blue and / or purple color is formed. Here, indigo and indigoid structures, This can occur as a result of indoxylin oxidation and is observed on the test strip. Color reaction products that contribute to the formation of the colorimetric signal. In this reaction chain, the oxidizing agent is indoxylin, which is formed as a result of hydrolysis. by enabling its oxidation, it transforms into indigo and / or indigoid colored products, and this is called 5 This contributes to the generation of the associated colorimetric signal. The resulting color is tested. It occurs in the indicator area located on the strip. The indicator area, It is not only an area where the resulting colorimetric signal is observed, but also indoxylin. colored products are formed as a result of oxidation and the resulting colorimetric It forms the reaction region where the signal is generated and evaluated. Reaction 10 The resulting color intensity is due to the presence of Indoxyl-β-D-Glucuronide in the urine sample. It is related to the amount. Blue and / or purple color occurring in colorimetric evaluation. While hue can be used to visually determine the reaction, color intensity... Changes in color are taken into account when evaluating the IDG level in the sample. The relationship between the intensity and the amount of IDG is quantitatively 15% of the colorimetric signal. It forms the basis for the evaluation and the reference values ​​and / or color intensity by comparing the numerical values ​​obtained as a result of digital image analysis This allows for the determination of the IDG level in the sample. Thus, the test... The resulting color is compared to the reference color scale to determine the IDG (Individual Difference) in the sample. It is possible to assess the level. The reference color scale is 20 on the test strip. used in the comparative evaluation of the color change that occurs It can be configured as an external assessment tool. In an application, the reaction consists of two successive reaction steps. The first In the reaction step, hydrolysis of IDG occurs through the action of the β-glucuronidase enzyme. And as a result of this hydrolysis, indoxyl is formed. Indoxyl is present in the reaction chain at 25 It is an unstable intermediate product that undergoes oxidation to form colored products. It forms the starting material for the subsequent reaction step. Second reaction step, the continuation of the indoxyl formed in the first reaction step It involves the transformation of indoxyl into indigo through oxidation. It forms. Indigo and / or indigoid 30 formed in the second reaction step. colored structures, the blue and / or purple colorimetric signal observed on the test strip It forms the basic reaction products that enable its formation. Two consecutive reactions Performed in steps, the enzymatic conversion of IDG and the colorimetric signal successive separate reaction stages of the oxidation step leading to its formation ...to be carried out as and the necessary reaction conditions for each step separately 35 It allows for regulation. During the reaction The oxidizing system used includes ferric / ferrocyanide, peroxidase, MnO₂ and / or atmospheric It can be composed of oxygen. The β-glucuronidase enzyme is immobilized on the reagent pad. By immobilizing the enzyme, the enzyme is kept on the test strip, 5 the reaction takes place in a controlled manner and enzyme activity during storage This ensures the enzyme's protection during sample flow. Immobilization also prevents the enzyme from being immobilized during sample flow. limiting the movement away from the reactive site and thus the enzymatic reaction Contributing to maintaining the positional stability of the area where it occurred on the test strip. This ensures that the enzyme is retained in a specific area on the reactive pad and its activity is 10. preservation involves creating similar reaction conditions on different test strips and... Contribution to the intertest repeatability of colorimetric responses obtained depending on the test. This is the preferred application method where the enzyme is deposited onto a porous cellulose matrix. the enzyme is immobilized, and the polymer binder layer holds the enzyme onto the carrier structure. maintaining its position and moving away from the reactive zone during sample flow 15 by contributing to its limitation, the enzyme remains mechanically and positionally stable. It ensures retention. The polymer binder layer also acts as a reactive pad in the production process. maintaining structural integrity between the load-bearing structure and the test strip contributes to the stable maintenance of the relative positions of the layers. It can provide. 20 Measurement and evaluation of results performed after the reaction is complete. During this stage, the color change that occurs on the test strip is a result of the reaction. This varies depending on the amount of indigo and / or indigoid structures formed. The subject is color change and color intensity, which can be visually evaluated and / or It can be analyzed numerically using digital image analysis. The preferred 25 In practice, a reference color scale is provided along with the test strip. Reference color The scale is based on specific reference levels of the color tone obtained from the test result. This allows for a comparison of the resulting colorimetric signal with the reference signal. It allows for semi-quantitative evaluation according to their levels and visual Differences in opinions that may arise among users during the evaluation process are 30%. It contributes to its reduction. In an application, the reference color scale includes colorless, light blue, medium blue, and dark blue. It includes different color levels. The color levels in question are for illustrative purposes only. 21 However, the reference color scale is not limited to these. In different applications, the colors to be analyzed Depending on the IDG concentration ranges and the assessment approach used. color scales containing different numbers and / or different color intensities of reference levels can be used. These reference color levels are known as IDG. Colorimetric responses obtained from reference samples with concentrations are based on 5 It can be created by taking and different color levels are predetermined IDG This can be correlated with concentration ranges. Thus, what occurs in the test sample... IDG by comparing the resulting color change with the relevant reference color level. This allows for a semi-quantitative assessment of the level. In one example application, reference color levels are used for 10 cases of chronic kidney disease. It is used in the evaluation. Accordingly, different color intensities have different IDG values. It is associated with levels, and this association is known as IDG. Calibration data including colorimetric responses obtained against concentrations This can be achieved based on the following principles. The results obtained show that kidney functions... It can be used in the evaluation. In this context, 15 is obtained from color evaluation. The result obtained is not a diagnosis of chronic kidney disease by itself, but rather a urine sample. based on determining the IDG level in the sample and assessing kidney function. It is used as a biomarker indicator to support the assessment. In an application, a reference color scale is evaluated visually. In the evaluation, the color change occurring on the test strip corresponds to the reference color scale 20. Comparison under the same lighting and observation conditions as possible, evaluation that may result from environmental conditions and user feedback It contributes to reducing differences. In alternative applications, testing The image of the strip is captured by digital imaging systems and the color The density is analyzed numerically. In the digital evaluation, the test strip 25 RGB color channel values ​​and / or words in the image obtained from the indicator region The subject is the analysis of equivalent numerical color intensity data derived from color values. It can be used as a parameter. Data obtained as a result of digital analysis (6), It is processed using computational evaluation methods and clinical decision support. It is used in the processes. 30 In one application of the invention, the test strip, the preparation of the reactive system, and the reactive system... application to the load-bearing structure, drying, and quality control processes obtained through a production method that includes the stages of production and packaging. 22 These stages are carried out sequentially in the production process. Each production stage is described in detail below. The first stage of the production method... In this stage, the reactive system is prepared. In the preferred application, β-glucuronidase is used. The enzyme is first dissolved in a phosphate buffer. The phosphate buffer contains the enzyme. creating a suitable pH environment and preparing the reactive system during β-5 In order to contribute to the preservation of the activity of the glucuronidase enzyme It is used. In one application, the buffer system has a pH of approximately 5.5-6.5. is being prepared. The pH range in question affects the activity of the β-glucuronidase enzyme. suitable for supporting and enabling the enzymatic conversion of IDG. It is preferred in order to ensure the reaction conditions are met. 10 Following this, the oxidizing agent is prepared in a separate solution phase. The prepared... The enzyme solution and the oxidizing agent solution are combined in a controlled manner. Preparing the enzyme and oxidizing agent initially in separate solution phases, the reagent This helps to preserve the functionality of the components before assembly; If the subject is the controlled combination of solutions, then the components are reactive system 15 It supports a regular and homogeneous distribution within the reactive system. A stabilizer is then added. At this stage, the stabilizer acts as a reactive agent. Adding the reactive mixture to the system increases the activity of the β-glucuronidase enzyme. preparation and subsequent preservation during storage and the reactive system It contributes to maintaining its stability. Reagent 20 with the addition of stabilizer. The system is homogenized. The homogenization process involves enzymes, oxidizing agents, the stabilizer and other reactive components are homogeneous in the reactive mixture ensuring its distribution and when the mixture is applied to the carrier strip To support the even distribution of reactive components along the test strip. This is carried out as follows. The reactive system also maintains the moisture balance during storage at 25%. It contains at least one moisture regulator that contributes to its preservation. The prepared reagent The mixture is stabilized for 24 hours at a temperature of approximately 4°C in the preferred application. This stabilization process involves applying the reactive system to the carrier strip. before application, the components must reach a stable state in the mixture and 30 in order to contribute to making the reactive system suitable for application. This is being carried out. Within this scope, the preparation and stabilization of the reactive system is performed. The pH, temperature, and processing time applied during the process are taken into account in controlling the production process. These are among the basic process parameters taken. These parameters... Maintaining the activity of the β-glucuronidase enzyme within the specified processing conditions and preserving the functionality of reactive components and ensuring a controlled production process 35 23 It contributes to the realization of the stabilization process. Next, the carrier strips are transferred to the reagent application stage. In one application... The carrier structure consists of nitrocellulose-based strips. The nitrocellulose-based carrier structure... Thanks to its porous structure, the sample moves along the test strip by capillary flow. providing a suitable environment and the reaction zone of the applied reactive components 5 It is preferred because it contributes to retention. In alternative applications The carrier structure can also be made of PVDF or cellulose-based strips. PVDF or Cellulose-based carrier structures; targeted capillary flow properties, with reactive components. compatibility, retention of reagents on the carrier structure and use of the test strip Considering the physical requirements related to the conditions, a nitrocellulose-based carrier structure was used. Alternatively, it can be preferred. The prepared reactive system involves carrier strips and reactive strips. The strips are applied by immersing them in a bath. The immersion process, the carrier strip surface makes controlled contact with the reactive system and the reactive to contribute to the homogeneous and regular distribution of components throughout the carrier structure This is carried out for this purpose. 15 during the immersion process, it is absorbed into the carrier structure. Reactive system quantity; immersion time; removal of carrier strips from the reactive bath. application parameters such as speed and carrier structure's reactive system absorption capacity This can be controlled by taking into account the homogeneity of the reagent bath and the application process. to maintain homogeneous distribution of reactive components within the solution throughout the process This can be sustained by ensuring suitable mixing conditions. Thus, different carriers 20 the strips should come into contact with the reactive system under similar conditions and the reactive components This helps maintain the consistency of the distribution on the strips. Excess reagent remaining on the strip surface after application of the reagent system. is removed. Removal of excess reactant allows the reactive components to be removed from the carrier structure. to support a more homogeneous and controlled distribution on the surface and reaction 25 This helps to limit the accumulation of unwanted reactive agents outside of designated areas. During the application of the reactive system onto the carrier strip, β-glucuronidase The enzyme is immobilized on a porous cellulose matrix. In the application, β-glucuronidase enzyme is immobilized onto a porous cellulose matrix. This is done. In order to hold the enzyme on the carrier structure, PVA, chitosan or 30 A polymer binder system containing combinations of these is used. Polymer The binding system contributes to the retention of the β-glucuronidase enzyme on the carrier structure. In addition to providing this, it also creates a protective effect on the carrier surface caused by the reactive components. preserving the physical integrity of the coating and its continuity over the reactive region 24 It contributes. After the reagent application process is completed, the strips It is then taken to the drying stage. In the preferred method, the drying process is carried out under vacuum. Vacuum Drying underneath allows for the controlled removal of the liquid phase within the reactive system. and to contribute to the fixation of reactive components on the carrier structure 5 is preferred. In addition, controlled application of drying conditions, β- It contributes to maintaining the functionality of the glucuronidase enzyme. The strips are approximately The reactive system is dried under vacuum for 30-60 minutes on the carrier structure. This ensures that the excess moisture in the carrier structure is fixed. The drying time in question involves removing the excess moisture from the carrier structure. and with sufficient removal of the liquid phase, the reactive coating is applied to the carrier structure 10 drying that will contribute to the preservation of physical integrity and stability This is determined by taking into account the conditions for achieving the drying process. Following completion, test strips are processed for final product quality before the packaging stage. They are subjected to control procedures. As part of quality control, test strips are examined first. Reaction performance is being evaluated. For this purpose, 15 reactions occurring in the test strips are assessed. The rate of color development is observed. Negative control studies are then conducted. This is being carried out. Negative control measures include those that do not contain Indoxyl-β-D-Glucuronide. The response of the test strip is evaluated using samples. Negative control. Studies show that unwanted substances are detected in the test lane under conditions where the target analyte, IDG, is absent. 20 for the purpose of evaluating whether a colorimetric signal occurs is being carried out. In the continuation of quality control processes, Indoxyl-β-D-Glucuronide is applied at specific concentrations. Using validation samples containing the results of test strips to different concentrations The response is being evaluated. IDG is used in validation samples. The concentrations will represent the predicted operating range of the test strip, and the word 25 the colorimetric response to different IDG levels within the range in question They can be selected in a way that will allow for evaluation. Verification samples the behavior of the test strip in generating a colorimetric response to samples containing IDG and the color change that occurs in response to different IDG concentrations It is used for evaluation purposes. Testing is also carried out after production. 30 The storage stability of the strips is being evaluated. In this context, different temperatures and Stability tests are conducted under humid conditions. These stability tests are performed during the testing process. in addition to evaluating the physical integrity of the strips under storage conditions in turn, the activity of the β-glucuronidase enzyme, the functionality of the reactive components and consequently the performance of generating the associated colorimetric response is maintained throughout the storage period. This is carried out to assess whether it is protected. Storage In evaluating its stability, the preservation of β-glucuronidase enzyme activity, The ability of the test strip to change color against the sample containing IDG, the resulting Performance criteria such as colorimetric signal intensity and color development time are taken into consideration. 5 It can be obtained. As a result of stability tests, the activity of the β-glucuronidase enzyme and Whether the analytical performance of the reactive system is maintained during storage; test The ability of the strip to develop color against the sample containing IDG, resulting in Based on performance criteria such as colorimetric signal intensity and color development time. are evaluated. Quality control processes carried out in this way involve testing 10 test strips. the ability to generate and store the expected colorimetric response to the target analyte to evaluate the preservation of reactive system performance throughout the period This is being carried out. Test strips that successfully complete quality control processes. They are packaged in a way that protects them from moisture. In the preferred application, each test strip... The moisture-proof foil is placed individually inside the packaging. This foil contains 15... In addition to providing physical protection to the test strip, the packaging also protects against reactive environmental moisture. by limiting the access of the β-glucuronidase enzyme and other reactive components to the region. reducing exposure to moisture-induced changes and storage of the reactive system It contributes to maintaining its functionality throughout this period. In one application, a desiccant material is also included in the packaging. It can be placed inside. The desiccant material may be found inside the packaging or by helping to retain moisture that may reach the packaging during storage. the moisture level inside the packaging must be kept under control and the reactive system must be protected from moisture. It supports protection against changes. Packaged test strips are single. It is provided to the user for single use. The test strip is for single use only (25). its configuration, cross-contamination that may occur between different samples to reduce the risk and / or sample contamination that may result from previous use This contributes to preventing reactive residues from affecting the subsequent analysis result (6). This type of packaging ensures the physical integrity of the test strip. the integrity and reactive system's resistance to environmental humidity-induced changes during storage 30 It contributes to protection against [the virus]. Below is a urine test strip produced and packaged as described above. The method of use in the analysis of the sample is explained. 26 The test strip that is the subject of this invention detects Indoxyl-β-D-Glucuronide (IDG) in urine samples. It is used for the rapid and colorimetric determination of blood levels. During the testing process, the first step is to collect the urine sample to be analyzed. The sample is collected in a suitable container. Following sample collection, the test strip is applied. It is being removed from its packaging, and care is being taken to ensure that the reactive areas are not contaminated. 5 It is used by holding the test strip only during use. For this purpose, the test strip is only held during use. It is held by the area and there is direct contact between the reactive pad and the indicator area. This avoids user-generated contamination and reactive substances. contributing to limiting the impact of direct contact on affected regions The test strip is provided with the reactive areas in contact with the urine sample. The test strip is immersed in the sample. During the immersion process, the test strip absorbs the reagent. While ensuring that the areas make sufficient contact with the sample, the holding area Care is taken to ensure that it does not come into contact with the sample. Thus, the part that comes into contact with the sample... the limitation of the area and the immersion conditions in different test applications This contributes to its consistent application. The preferred application is test strip 15. It is held in the urine sample for approximately 1–2 seconds. This contact... The duration depends on the sufficient uptake of the urine sample by the reactive site and capillary action. This will allow the flow to be initiated and the sample to be transported to the reaction areas. This is determined by keeping the contact time controlled in different testing applications. Maintaining consistency in sampling conditions and consequently in analytical performance 20 It contributes to this. At the end of this period, the test strip is removed from the sample. The sample Following the procedure, the urine sample collected by the test strip is absorbed by capillary flow. It progresses to the reaction sites and is hydrolyzed via the IDG β-glucuronidase enzyme. indoxyl is formed as a result of this process, and as a result of the oxidation of the resulting indoxyl... Colorimetric color formation occurs. The colorimetric reaction takes 25 minutes. and the test strip is used to determine the formation of an evaluable color. It is allowed to sit for the duration of the reaction time. The resulting colorimetric signal, visually with the reference color scale after the reaction is complete. by comparison and / or using digital image analysis, and thus This allows for the determination of the IDG level in the sample. In this way, 30 The steps involved in the controlled application of the sample onto the test strip. application, the movement of the sample to the reaction sites via capillary flow, IDG determination as a result of enzymatic and colorimetric reactions and the resulting colorimetric This allows the signal to be used in the evaluation of the IDG level. 27 After the test strip is removed from the sample, a certain amount of time is allowed for the reaction to complete. It is expected that the waiting time in the preferred application will be approximately 30–120 seconds. It is among them. This waiting period allows enzymatic and oxidative reactions to progress. To allow the colorimetric signal to reach a suitable state for evaluation. It is applied for this purpose. Waiting time, enzymatic conversion and the following 5 the rate at which the oxidation reaction occurs, the characteristics of the sample, and the application Depending on the circumstances, this period may vary. The reaction is completed inside and the final color formation takes place. Waiting time. The color that appears on the test strip after the completion of the period is the reference color. It is evaluated by comparing it with the scale. Visual comparison is done with the test strip on a scale of 10. as closely as possible to the reference color scale and under appropriate lighting conditions This should be done in a way that can be observed, taking into account ambient light conditions and the user. to contribute to reducing differences in assessment that may arise from the interpretation. It provides. In one application, a reference color scale test strip is provided. An alternative 15 In practice, the reference color scale is included as a separate card within the kit. The reference color scale is provided with the test strip or included in the kit by the user. presenting the colorimetric signal resulting from the test in an easily accessible way After the waiting period is complete, directly with reference levels 20 It contributes. In the preferred application, the reference color scale uses different colors. It includes their color intensities. These color intensities refer to the different IDG colors in the sample. They are associated with reference levels representing concentration ranges, and Thus, the resulting colorimetric signal is half the range of IDG concentration. It allows for a quantitative evaluation. The resulting color tone is 25 The test is performed by determining the corresponding color on the reference color scale through visual comparison. The result can be interpreted; in alternative applications, color matching, test strip digitally capturing the image and analyzing numerical data regarding color intensity. This can be achieved through this method. In a sample application, different shades of Indoxyl-β-D-30 are used in the reference color scale. It is correlated with glucuronide levels. Accordingly, the change in color intensity, as an indicator that can be used in the evaluation of chronic kidney disease can be evaluated. In an application, the test result is directly evaluated by the user. It is evaluated visually. An alternative approach shows the image of the test strip. 28 The image is obtained via an optical reader. The resulting image shows the color intensity. It is analyzed for the purpose of determining it numerically. In another application, the test... The image of the strip is obtained with a mobile imaging device and processed through software. The image is processed. The color intensity determined as a result of image processing is compared with reference values. By comparing the results, a digital evaluation of the test outcome is provided. 5 In alternative applications, colorimetric data obtained from the test strip are used computationally. They are analyzed using evaluation methods and in clinical decision support systems. It is used. In one application, this assessment involves kidney function. in calculating an evaluation parameter created for monitoring It is used. The test strip is for single use only. One test strip is for single use only. Configuring it for reuse, eliminating residues that may result from a previous sample it could affect a subsequent analysis and what might happen between different samples By contributing to limiting the risk of cross-contamination, the analysis results... It supports the preservation of its credibility. In one application of the invention, the test strip can be used as a standalone diagnostic product. is presented. In another application, the test strip is used to detect Indoxyl- found in the urine sample. Included in a diagnostic kit for determining β-D-Glucuronide (IDG) levels. This diagnostic kit covers everything from sample collection to test administration. bringing together the essential components necessary for evaluating the resulting colorimetric outcome. It is structured to include at least one 20-minute urine sample for collection. sample collection container, at least one test strip for analysis of the collected sample, test strip on the sample At least one reference color scale for evaluating the resulting colorimetric signal. and at least one instruction manual for carrying out those operations It includes. The combined use of these kit components allows for the analysis of the urine sample. collection, application to the test strip, generation of the colorimetric signal dependent on IDG, and 25 The steps involved in evaluating the obtained result against a reference color scale are the same. This allows for sequential and controlled execution within the analysis system. This includes the instructions for use and the planned application of the testing process. The reference color scale supports the execution of the steps in accordance with the test. By comparing the colorimetric signal generated in the strip with the reference levels, 30 It functions as a component that helps in its evaluation. Thus, the word The subject is the supporting components, the implementation of the test, and the user's understanding of the results. This contributes to its consistent evaluation. 29 In one application, the diagnostic kit is used solely for visual assessment purposes. This The application directly compares the test result with a reference color scale. This allows for evaluation and external digital imaging or analysis. a quick and practical assessment without the need for a system It supports this. Therefore, this structure allows for non-laboratory use. 5 It can be used in conditions and applications requiring rapid assessment. In an alternative approach, the diagnostic kit allows for digital evaluation of the test result. It can be used with at least one optical reading system that provides this capability. The optical system in question... The reading system detects the color change occurring on the test strip, thus determining the color. It can convert density into numerical data. In alternative applications, test 10 Software-based analysis systems for evaluating the results. It can be used. The software system processes the image obtained from the test strip and determines the color. we can compare its density with reference values ​​and the evaluation result. It can transfer this to the user. In another application, the system can transfer colorimetric data obtained from the test strip to other clinical applications. It can be structured in a way that allows it to be evaluated together with the parameters. This Within this scope, the system can also include data on different biomarkers when necessary. It can be configured in a way that allows it to function. However, the basic identification process involves the test strip. It is based on the indoxyl-β-D-glucuronide reaction carried out on it. In alternative applications, the system includes 20 features for evaluating kidney function. It can work in conjunction with computational analysis modules. Computational analysis modules, By processing the colorimetric data obtained from the test strip, it provides the user with clinical data. It can provide information to support the evaluation. The system architecture is only as described above. not limited to the applications described, but also including colorimetric data obtained from the test strip. It can be used with different imaging, data processing and decision support systems. 25 It can be implemented in different ways to provide results. In one application, the colorimetric signals generated on the test strip can be captured by a smartphone camera or The image is displayed using an optical reader. The region of interest is shown on the resulting image. determination, color channel extraction, background normalization, and signal The calculation of the signal intensity can be performed. The calculated signal is 30. Their densities were converted to biomarker concentrations using calibration curves. It can be converted. Figure 2 shows the relationship between the target analyte concentration and the colorimetric signal. An example / schematic calibration representation is presented. Calibration relation is used. Colorimetric signal values ​​obtained from the biosensor platform correspond to the relevant target analyte. They can be converted into concentrations and quantitative or semi-quantitative evaluations. This can be achieved. The relationship shown in Figure 2 is representative and refers to a specific 5 It does not represent the experimental data set. In another application, microbiome-derived metabolites and kidney function Numerical values ​​of related biomarkers are normalized and a weighted computational analysis is performed. combined through a model called the Microbiome–Renal Index (MRI). A combined evaluation index is being created. This index includes microbiome 10 in evaluating the relationship between metabolic activity and renal function It is available for use. In composite assessment approaches such as the Microbiome–Renal Index (MRI), microbiome-derived metabolites and biomarkers associated with kidney function The numerical values ​​are normalized and a weighted computational model is used to calculate 15 can be combined. The index in question is the obtained biomarker and / or metabolite. as an optional computational output for the combined evaluation of data It is available for use. Biomarker values ​​in alternative applications: machine learning-based models analyzed by 20 to detect renal dysfunction or chronic kidney disease. An output regarding the risk can be generated, and the results obtained can be used in a mobile diagnostic application. can be presented to the user via this platform. Figure 3 shows the digital imaging of the colorimetric signal generated on the biosensor platform. Numerical analysis is performed by evaluating the image through image preprocessing, color analysis, and calibration. The process flow for converting it to the final result is shown schematically. Digital 25 Image and data processing operations performed by the analysis module (5) The resulting signal values ​​are appropriate reference and / or calibration data. They can be converted to target analyte concentrations using this method. In the modular analysis structure of the invention, the biological sample is placed in the sample application module. (1) is implemented, in the enzymatic conversion module (2) conversion of the target analyte 30 is performed and the color signal is measurable in the colorimetric reaction modulus (3). is created. The color signal can be compared with the reference / calibration module (4), 31 The digital analysis module (5) processes the data and generates the analysis result (6). In the test strip format, the sample pad (7), enzymatic reaction area (8), colorimetric reaction region (9), control region (10), reference / calibration region (11), absorbent pad (12) and carrier layer (13) as separate structural and functional components It can be edited. 5 In one application of the production structure, the reagent preparation module and buffer preparation module are used. module, enzyme preparation module, colorimetric reagent preparation module, reagent application module, drying and stabilization module, platform assembly module, quality The control module, packaging module, and final product module are separate modules. It is located in the reagent preparation module. The reagent system to be used in the analysis is 10. is being prepared. In the buffer preparation module, the appropriate pH and temperature for the enzymatic reaction are determined. An ionic environment is created, and one of the preferred applications is a phosphate-citrate buffer. The system can be used. In the enzyme preparation module, the conversion of the target analyte takes place. One or more enzymes are prepared and supplied with a suitable buffer system. They are mixed. In this context, in addition to β-glucuronidase, arylsulfatase, esterase, 15 Glycosidase or similar hydrolytic enzymes, alone or in combination. It is available for use. In the colorimetric reagent preparation module, the result of enzymatic conversion is an oxidation system that converts the reaction product into a visible color change is being prepared. The reagents prepared in the reagent application module are used in carrier material 20 dripping, spraying, impregnation, printing, slot coating, inkjet application It can be applied via a microdispenser system. Drying and stabilization. in the module at room temperature, controlled temperature, low humidity, under vacuum Drying and stabilization can be achieved by lyophilization and / or other methods. In the platform mounting module, sample pad (7), reaction zones, control zone (10), 25 The reference area and absorbent pad (12) are combined on the carrier layer (13). In the quality control module, reagent loading quantity, homogeneity, reaction performance, Color development, mechanical strength, and other quality parameters are verified. In the packaging module, the product has protective barrier properties against moisture and oxygen. The product is enclosed in packaging material and a ready-to-use product is obtained in the final product module. 30 In another production structure, the reagent preparation unit and the carrier material are used. preparation unit, reagent application unit, drying unit, stabilization unit, assembly 32 unit, quality control unit, packaging unit, storage unit and final biosensor The platform can be used as separate production units and outputs. Enzymatic reaction solution in a suitable buffer system in the reagent preparation unit. A colorimetric reaction solution can be prepared with it. In the enzyme preparation unit, β- Glucuronidase solution can be prepared, and the prepared reagents are placed in the reagent application unit. 5 It can be applied to the carrier material. Drying and stabilization in the drying unit. enzyme and reagent stabilization in the unit, platform layers in the assembly unit assembly, quality verification in the quality control unit, packaging unit in protective packaging and storage unit under suitable temperature and humidity conditions Preservation can be achieved. Enzymatic conversion and colorimetric reaction, single 10 reaction zone architecture, dual reaction zone architecture, sequential reaction architecture, simultaneous reaction architecture, microfluidic reaction setup, multichamber reaction the system, modular reaction platform or alternative platform architecture It can be accomplished. In evaluating reaction kinetics, the reaction initiation is taken as the basis and time is divided by 15. The associated color change is monitored, a reaction curve is created, and the reaction rate is determined. maximum color intensity, plateau region, reference signal, calibration curve, and The calculated analysis result (6) can be obtained. Images are taken at fixed intervals, continuously It can be created using video recording or time-lapse video. Digital Image acquisition module, image preprocessing module, 20 in the evaluation system Reaction region determination module, reference region determination module, color analysis module, numerical evaluation module, results generation module, and reporting. The modules exist as separate modules. Image acquisition module for portable optical reader, desktop reader, smartphone camera, tablet computer, industrial camera or similar imaging system 25 It can record images through it. The image preprocessing module includes white balance, Background correction, contrast adjustment, perspective correction, brightness normalization, one of the following operations: geometric correction, noise reduction, and region of interest selection. He is able to accomplish several of them. The reaction site determination module identifies the reaction site as 30 degrees below the reference site. The module determines the reference region automatically or semi-automatically. It can determine the following. The color analysis module supports RGB, HSV, HSL, CIELAB, XYZ, YUV, CMYK, YCbCr can utilize grayscale and / or hyperspectral imaging data, optically. 33 density, average channel values, color intensity, and channel ratios. It is able to calculate. The quantitative evaluation module uses mathematical models, statistical methods, and digital methods. image processing algorithms, artificial intelligence algorithms, machine learning and / or deep It can evaluate data using learning methods. Conclusion generation 5 The module generates the analyte concentration and / or evaluation output. The reporting module presents the generated results to the user in numerical, graphical and / or formats. It presents the output in a classified format. In the digital analysis example, the image acquisition unit records the color change, the image The preprocessing unit processes the image, and the color analysis unit adjusts the color parameters to 10. The calculation and numerical evaluation unit analyzes the obtained data. The results generation unit determines the analyte concentration, and the reporting unit reports the result. It presents the user with a calibration curve for the analytical concentration calculation unit. It can also calculate the quantitative result from that. In the internal reference and calibration system, the reference zone, calibration zone, color 15 standard, calibration curve, analyte concentration calculation module, composite evaluation algorithm, decision support module, results reporting module, external data The transfer module and the user interface are separate components. The reference region is a fixed color area, a standard color scale, a control area, or an optical system. It can be configured as a calibration structure. Calibration zone color 20 It can be used in normalization. Color standard display conditions. This contributes to reducing the resulting variability in analyte concentration. The calculation module obtains the quantitative result using the calibration curve. The composite evaluation algorithm combines multiple numerical values ​​to create a composite evaluation. It can generate an evaluation. The decision support module processes the obtained data in 25 steps. It can interpret the results. The results reporting module can generate the results report. The external data transfer module transmits results to laboratory information management systems and mobile devices. It can send the results to devices or other digital platforms. The user interface displays the results. for the purpose of being viewed by the user and interacting with the system. It is available. 30 34 In a multi-analyte platform, there is a primary reaction site, a secondary reaction site, and a third reaction site. reaction region, common reference region, control region (10), common digital analysis module (5) includes multiple outcome evaluation modules and final analysis output. Different Signals from reaction regions are analyzed separately in the common digital analysis module (5) It is being processed and in the multiple results evaluation module, a single report or multiple analysis output is generated. 5 They can be combined as follows. In other words, the same reaction signals can be combined into a common digital format. It can be evaluated in the analysis unit and a single report in the multiple results evaluation unit. It can be transformed into this form. Analysis platform test strip format, lateral flow platform, microfluidic cartridge, microplate format, paper-based analysis system, platform operating with optical reader, 10 in the form of a portable digital analysis platform or integrated analysis system This can be achieved. In microplate applications, optical reading is done using a 450-620 nm wavelength. This can be done within a certain height range; in an example application, incubation takes 30-60 minutes. And the color reaction can be applied within 10-30 minutes. Portable reader Device display, status / charging indicator, read button, power on / off and operation menu, data 15 It may include a data transfer and charging port and a rechargeable battery compartment. Data transfer and The charging port can be implemented using a USB-C interface. The modular reader configuration includes the top cover, test module, reader module, processor, and The control module, battery module, and housing / bottom case are separate modules. The reader module uses an LED and camera and / or detector 20 for optical image acquisition. It may include a processor and control module for signal processing and communication operations. It is capable of operating and the battery module can provide a rechargeable power source. The test module is compatible with test strips, hydraulic platforms, flow channels, cartridges, or microplates. It can provide a changeable layout. The modular structure allows for different analyzers and... platform compatibility, ease of maintenance and service, reduced production and logistics costs. 25 This can be achieved through system integration using portable reader devices and mobile applications. Wireless communication, cloud server, and data management are separate system components. It is available. The mobile application includes results display and user interaction functions. It is able to do this, the user interface is separate from the mobile application, and it interacts with the user. It enables visual and functional interaction between the systems. 30 Wireless communication can be achieved via Bluetooth and / or Wi-Fi. Wireless data Cloud communication enables the transfer of analytical data between devices. The server center can provide data services, and cloud storage provides analytics data. 35 It can store and synchronize data after offline use. When provided, it enables the synchronization of local records with the cloud environment. Optical Imaging via LED illumination and image via camera and / or detector. This may include the acquisition of optical signals. Data analysis software can analyze images, colors, and optics. It can process density, kinetic and / or concentration data. Reporting software 5 the creation of the analysis result as a numerical, graphical or classified report (6) and can ensure its preservation. Electronic identifiers include barcodes, QR codes, or test type, lot, calibration data and analysis protocol in electronic identification format can be associated. Platform standard lighting during digital imaging. It can be viewed under these conditions and at a fixed distance and angle. The image is high 10 It can be obtained in high-resolution RAW and / or JPEG format and with a timestamp. It can be saved. During the preprocessing stage, white balance, noise reduction, and background are performed. Plan correction, geometric correction, and selection of the area of ​​interest can be applied. Sample pad (7), enzymatic region, colorimetric test region, reference / calibration region (11) and The absorbent region can be defined as separate analysis areas. Color analysis 15 During this process, the average RGB value for each region, the individual R, G, and B channel values, and the color Density and channel ratios such as R / G, R / B, or G / B can be calculated. Quantitative In the evaluation, the optical density of the test region is compared with the optical density of the reference region. ratio to intensity, internal normalization approach and / or color intensity based Transformation relationships can be used. Concentration, a calibration function, 20 linear or nonlinear regression, multiple color channel relation, or machine learning This can be calculated using a learning-based model. In calibration and verification studies, linearity or proper model fit is determined. analytical parameters such as limit, limit of detection, repeatability, observation interval, recovery, and bias. Performance parameters can be evaluated. The calibration curve is for different target analytes 25 using the colorimetric signal values ​​obtained at different concentrations can be generated and with appropriate positive, negative and / or reaction controls. This can be verified. In this context, the calibration representation given in Figure 2 is only valid. It is illustrative / schematic in nature. Accuracy in image analysis quality control, precision based on repeated measurements, reference 30 correction based on regions, signal-to-noise ratio, and valid / invalid result decision. The algorithm can be used. The results report includes the analysis number, user ID, date / time, test description, area information, optical density, concentration, reference value, and Normalization values ​​can be stored; results archive, cloud data and user record. 36 Traceability can be ensured. Different approaches in the integrated assessment method. normalized values ​​obtained from biomarkers or different analytical regions It can be largely combined and a composite index like MRI It can be calculated. The steps involved in the process described in the invention are as follows: 5  Fixed display of the analysis platform under standard lighting conditions Viewing under distance and angle (1001),  The image should be obtained in high-resolution RAW and / or JPEG format, and recorded with a timestamp (1002),  White balance, noise reduction, background correction, contrast adjustment to the image. related to adjustment, brightness normalization, geometric and / or perspective correction. implementation of regional selection (1003),  Test area, control area (10), reference area and calibration area automatic or semi-automatic determination and segmentation (1004),  RGB, HSV, HSL, CIELAB, XYZ, YUV, CMYK, YCbCr 15 from the specified regions and / or grayscale parameters, optical density and color channel ratios removal (1005),  Reference region of the test signal, calibration region, internal reference dye and / or normalization using the internal normalization region (1006),  Ratio method of normalized data, calibration curve, linear regression, 20 nonlinear model, mathematical model, statistical model and / or conversion to analyte concentration using machine learning model (1007),  Obtained at fixed time intervals from the start of the reaction images, time-lapse viewing, continuous viewing and / or video 25 Monitoring time-dependent color change and reaction kinetics via recording. determination (1008),  Image processing of color, optical density, feature and / or reaction kinetics data, statistical classification, artificial intelligence, machine learning and / or deep learning evaluation using algorithms (1009), 30  The accuracy, repeatability, signal-to-noise ratio, detection limit of the analysis result (6), determination limit, recovery, sensitivity, and valid / invalid result decision criteria. undergoing quality control (1010), 37  Analysis regarding analyte concentration and / or evaluation result the creation of the result (6) (1011),  The analysis result in (6) numerical, graphical and / or risk classification form reporting (1012),  The analysis result (6) with date, time, analysis number and user information 5 recording and archiving (1013),  Analysis results (6) mobile application, laboratory information management system, wireless data communication and / or transfer via cloud server (1014),  Analysis data recorded in offline conditions is transferred to the cloud when a connection is established. Synchronization with the server and / or other data management systems (1015), 10  Signals obtained from multiple reaction regions of the same sample multiple analysis outputs are evaluated within a common digital analysis framework. creation (1016). The application methods described above are only illustrative examples of the invention. It constitutes. The invention, while preserving the basic working principle, has different structural characteristics. 15 This can also be achieved through regulations and implementation methods. Testing in an application The strip contains a single reaction line. In alternative applications, the same carrier structure Different analytical regions are obtained by creating multiple reaction lines on it. It is possible. 25

Claims

38 REQUESTS 1. Colorimetric measurement of Indoxyl-β-D-Glucuronide (IDG) levels in urine samples. It is a test strip for determining its characteristics, and its feature is that it has at least one supporting structure and the load-bearing structure in question must contain at least one reactive zone and The aforementioned reactive region contains at least one β-glucuronidase enzyme and at least one oxidizing agent. It contains agents.

2. Test strip conforming to Claim 1, characterized by a reactive zone of 0.2–2 U per test strip. β-glucuronidase enzyme and 0.1–2 mg of oxidizing agent at a pH range of 5.5–6.

5. It contains 1–3 µg of buffer, 5–20 µg of stabilizer, and 2–10 µg of moisture regulator.

3. A test strip conforming to Claim 2, characterized by its stabilizer being trehalose, albumin, or 10. combinations of these; the oxidizing agent being a ferric / ferrocyanide system, peroxidase, manganese dioxide (MnO₂), oxygen or combinations thereof It is the selection process.

4. A test strip that conforms to any of the previous requirements, and whose characteristic is; the carrier structure Selection of nitrocellulose, polyvinylidene fluoride (PVDF) or cellulose-based materials, 15 Formation of the reactive region on a porous cellulose-based reactive pad and β- This means that the glucuronidase enzyme is immobilized onto the reactive pad in question.

5. A test strip suitable for any of the previous requirements, and featuring a reagent pad. polyvinyl alcohol (PVA), chitosan or combinations thereof between the carrier structure at least one polyester-based backing film with at least one polymer binder layer 20 It must include at least one protective and waterproof backing on the upper surface of the test strip. the presence of a layer, and at least one spreading layer and / or at the bottom of the test strip The presence of a hydrophobic barrier and a test strip width of 3–5 mm are important factors.

6. A test strip suitable for any of the previous requirements, characterized by its suitability for sample application. The test strip 25 includes the reactive zone and the indicator zone. Arranged on it in such a way that they follow each other in the direction of the flow of the sample or The reactive region and the indicator region coexist within the same reaction matrix. is the creation of.

7. A test strip suitable for any of the previous requirements, characterized by its ability to show the test result. It is presented along with a reference color scale for evaluation. 30 39 8. A diagnostic kit suitable for any of the previous requirements, featuring a test strip. It includes a reference color scale, instructions for use, and a sample collection container.

9. Colorimetric measurement of Indoxyl-β-D-Glucuronide (IDG) levels in urine samples. It is a method for producing a test strip for determining β- 5 A reactive system containing the glucuronidase enzyme and at least one oxidizing agent preparation and placement of the prepared reactive system on at least one carrier structure It involves steps to apply it to at least one reactive site.

10. The production method conforms to claim 9, and its characteristic is that the reactive system also has a pH value of 5.5– 6.5 range contains at least one buffer, at least one stabilizer and at least one moisture regulator 10 It is prepared to include.

11. The production method conforming to claim 9 or 10, characterized by the presence of the β-glucuronidase enzyme. Dissolving in phosphate buffer, leaving the oxidizing agent in a separate solution phase. Preparation, mixing the prepared enzyme solution with the oxidizing agent solution 15 bringing in the solution, adding the stabilizer to the resulting mixture, and preparing the reagent. This includes the stages of stabilizing the system.

12. The manufacturing method conforming to either of claims 9-11, characterized by its reactive system. Application to the reactive region by immersion and / or impregnation method and β-20 This involves immobilizing the glucuronidase enzyme on a reactive site.

13. The production method is in accordance with claim 12, and its characteristic is that it involves the application of the reactive system. then the test strip is dried and the dried test strip is placed in moisture-proof packaging 25 It includes the stages of placing it inside.

14. Using a test strip conforming to any of the requirements 1-7, in the urine sample found Its use in determining indoxyl-β-D-glucuronide levels.

15. Use in accordance with Claim 14, characterized by; obtained from the target analyte level. the result is an assessment or screening of the relevant biological or clinical condition, It is used in classification and / or monitoring.

16. Use in accordance with claim 14 or 15, characterized by the fact that the use has an effect on humans and / or 35 It is performed on animal species used in veterinary medicine. 40 17. The level of Indoxyl-β-D-Glucuronide (IDG) found in a urine sample, as measured on a test strip. converting the resulting colorimetric signal into digital image data and the aforementioned by processing image data together with reference and / or calibration data electronically through software running on an electronic device It is an electronic analysis system for determining, and its feature is; 5  An imaging system displays the colorimetric signal generated on the test strip. an image acquisition module that records the image data as digital image data by capturing it from the medium,  digital image data recorded by the image acquisition module measures to reduce variability caused by imaging conditions Image preprocessing module, 10, creates processed image data by applying processing.  on the processed image data generated by the image preprocessing module By determining the reaction region, the area of ​​analysis where the colorimetric signal occurs. Module for identifying the reaction region that generates data,  on the processed image data generated by the image preprocessing module 15 in normalizing and / or calibrating reaction zone data Generating reference region data by defining the reference area to be used. Reference region determination module,  Color parameters from reaction region data and / or reference region data A color analysis module that generates digital color data by extracting optical density data.  The numerical color data generated by the color analysis module is used as a reference area 20 normalize with data and / or calibration data and perform mathematical, statistical, by applying image processing and / or machine learning-based assessment Numerical data that generates a quantitative assessment of the IDG level. evaluation module,  Numerical assessment generated by the numerical assessment module 25 generating results that transform data into analytical output regarding IDG level module,  The analysis output generated by the results generation module is displayed to the user. converting into a transmissible digital, graphical and / or classified output format Reporting module 30 It includes.

18. An electronic analysis system in accordance with Claim 17, whose feature is image acquisition. module, portable optical reader, desktop reader, smartphone camera, 41 at least a tablet computer, industrial camera and / or laboratory-type optical reader one of them captures and receives high-resolution RAW and / or JPEG images. It is a module that records the image with a timestamp.

19. An electronic analysis system conforming to Claim 17, featuring image preprocessing 5. the module's digital image data recorded by the image acquisition module white balance, background correction, contrast adjustment, brightness normalization, noise reduction, geometric correction, perspective correction and / or region of interest It is a module that creates processed image data by applying the selection.

20. An electronic analysis system conforming to Claim 17, characterized by its ability to analyze the reaction zone. the determination module, processed by the image preprocessing module test corresponding to the colorimetric reaction of the test strip on the image data automatically or semi-automatically identifies the region and compares that region with other It is a module that creates reaction region data by separating it from image areas. 15 21. An electronic analysis system in accordance with Claim 17, characterized by its function of the reference region. the determination module, processed by the image preprocessing module the region on the image data used for reference and / or calibration purposes automatic or semi-automatic detection and reaction 20 of the region in question It is a module that defines the data as a separate reference region from the region.

22. It is an electronic analysis system in accordance with Claim 17, and its feature is; the color analysis module, Reaction region determination module and reference region determination module RGB, HSV, HSL, CIELAB, XYZ, YUV, 25 are regional data generated by [company name]. The most suitable of the CMYK, YCbCr, grayscale and / or hyperspectral imaging parameters. extracting at least one of these parameters, namely optical density, color intensity and / or chromaticity. It is a module that calculates channel ratio.

23. An electronic analysis system in accordance with Claim 17, characterized by its numerical evaluation. 30 The module references the numerical color data generated by the color analysis module. area, calibration area, internal reference dye and / or internal normalization normalizes the data obtained from the region and the normalized data calibration curve, linear regression, nonlinear model, mathematical 42 analytics through model, statistical model and / or machine learning model It is a module that converts concentration.

24. It is an electronic analysis system in accordance with Claim 17, and its characteristic is numerical evaluation. 5 modules taken at specific time intervals from the start of the reaction timestamped images, time-lapse images, continuous images and / or by determining the time-dependent color change from video recordings, the reaction the reaction curve that is formed and from that reaction curve the reaction rate, maximum color density, plateau region and / or area under the reaction curve It is a module that extracts at least one of its parameters. 10 25. An electronic analysis system compliant with Claim 17, characterized by its ability to generate results. the module, the numerical evaluation module, generated by the numerical evaluation module. Evaluation data includes analyte concentration, biomarker level, and disease risk. and / or by associating the analysis output regarding IDG level with clinical decision support 15 It is a module that creates 26. It is an electronic analysis system compliant with Claim 17, and its feature is that the reporting module, The analysis output generated by the results generation module is numerical and graphical. and / or converting it into a classified report format and including the date, time, 20 Analysis number and user information, test description, zone information, optical density, analyte. concentration, reference value and / or normalization value with at least one of them It is a module that records data in a form that can be presented to the user by associating it with other data.

27. An electronic analysis system in accordance with Claim 17, characterized by its imaging conditions being 25. a reference providing a fixed color area or standard color scale for correction the region, calibration data that provides the basis for color normalization region, color standard that provides a predefined color value for optical calibration, Calibration curve that relates normalized measurement data to concentration, Analyte 30 calculates analyte concentration using the calibration curve. The concentration calculation module weights multiple numerical values. composite evaluation that creates composite evaluation data by combining them The algorithm combines composite evaluation data with clinical evaluation criteria. A decision support module that generates decision support output by associating decisions with other tools. The results reporting module, which converts the output into a report, sends the generated report to laboratory 35. 43 transferring to information management systems, mobile devices and / or other digital platforms external data transfer module and visual and functional interface between the user and the system. It includes a user interface that enables interaction.

28. An electronic analysis system conforming to Claim 17, characterized by its ability to analyze 5 different samples from the same sample. The first reaction site generates distinct colorimetric signals for the analytes, the second reaction site and third reaction site, the reaction sites in question a common reference region that provides data for normalizing the reaction a common digital system that processes signals from different regions into separate data channels analysis module (5), processed data channels in single report and / or multiple analysis output 10 combining multiple outcome assessment module and multiple outcome assessment module It includes the final analysis output generated by.

29. It is an electronic analysis system in accordance with claim 17, and its feature is that it displays the analysis result (6) The screen displayed to the user shows the device status and battery charge level. Status / charge indicator, read button that initiates the optical reading process, device power-on, The power on / off and operation controls allow for the management of the operation menu and its shutdown. The menu provides data transfer and a USB-C connection for battery charging. and the rechargeable port that allows for the placement of the rechargeable battery. It includes a portable reader device with a battery compartment. 20 30. An electronic analysis system in accordance with Claim 29, characterized by access to the test module. providing top cover, test strip, hydraulic platform, flow channel, cartridge and / or Test module with interchangeable connection via microplate, test module with LED illuminating and receiving an image or optical signal via a camera and / or detector 25 The reader module processes the signal obtained by the reader module and the system The processor and control module that manages communication operations, rechargeable power. battery module and the housing / base that mechanically supports those modules It includes a safe.

31. It is an electronic analysis system in accordance with Claim 17, and its feature is that the analysis result is (6) allows the user to view and input user feedback. mobile application, device and / or analytics system separate from the mobile application a user interface that provides visual and functional interaction between the user and the system. interface, wireless communication that transmits analysis data via Bluetooth and / or Wi-Fi, 35 44 A cloud server that centrally processes and / or makes accessible the transferred analytical data. Data that enables the association of analysis data with users, devices, and analysis records. management, cloud storage that remotely stores analytical data, and offline recording. Local analytics data is transferred to the cloud server and / or other data management system when a connection is established. It includes data synchronization that matches with the systems. 5 32. An electronic analysis system conforming to Claim 31, whose features include image, color, and optical capabilities. Quantitative evaluation by processing density, kinetic and / or concentration data. Data analysis software that generates data, the analysis result (6) numerical, graphical and / or Reporting software and test type that creates and stores classified reports, lot 10 by electronically defining the information, calibration data and analysis protocol. Enabling the system to select calibration and analysis information. electronic identifier in the form of a barcode, QR code and / or electronic ID It includes.

33. The colorimetric signal generated on a biosensor platform can be used for electronic imaging and... It is a method for quantitatively analyzing data using data processing. feature;  Fixed display of the analysis platform under standard lighting conditions Viewing under distance and angle (1001), 20  The image should be obtained in high-resolution RAW and / or JPEG format, and recorded with a timestamp (1002), • White balance, noise reduction, background correction, contrast adjustments are applied to the image. related to adjustment, brightness normalization, geometric and / or perspective correction. regional selection implementation (1003), 25  Test area, control area (10), reference area and calibration area automatic or semi-automatic determination and segmentation (1004),  RGB, HSV, HSL, CIELAB, XYZ, YUV, CMYK, YCbCr from the specified regions and / or grayscale parameters, optical density and color channel ratios removal (1005), 30  Reference region of the test signal, calibration region, internal reference dye and / or normalization using the internal normalization region (1006),  Ratio method for normalized data, calibration curve, linear regression, nonlinear model, mathematical model, statistical model and / or 45 conversion to analyte concentration using machine learning model (1007),  Obtained at fixed time intervals from the start of the reaction images, time-lapse viewing, continuous viewing and / or video Monitoring time-dependent color change and reaction kinetics via recording 5 determination (1008),  Image processing of color, optical density, feature and / or reaction kinetics data, statistical classification, artificial intelligence, machine learning and / or deep learning evaluation using algorithms (1009),  The accuracy, repeatability, signal-to-noise ratio, detection limit of the analysis result (6) 10 determination limit, recovery, sensitivity, and valid / invalid result decision criteria. undergoing quality control (1010),  Analysis regarding analyte concentration and / or evaluation result the creation of the result (6) (1011),  The analysis result (6) in the form of numerical, graphical and / or risk classification 15 reporting (1012),  The analysis result with (6) date, time, analysis number and user information recording and archiving (1013),  Analysis results (6) mobile application, laboratory information management system, wireless data communication and / or transfer via cloud server (1014), 20  Analysis data recorded in offline conditions is transferred to the cloud when a connection is established. Synchronization with the server and / or other data management systems (1015),  Signals obtained from multiple reaction regions of the same sample multiple analysis outputs are evaluated within a common digital analysis framework. creation (1016) 25 It includes the steps of the process.

34. This method complies with Claim 33 and its characteristic feature is the LED illumination of the analysis platform. and images or optical signals are received from that platform via camera and / or detector. The signal reception process includes the following steps. 30 35. The method is in accordance with claim 33 and is characterized by the calibration curve being drawn for at least five different analytes. the formation of the concentration and the calibration in question is positive control, negative 46 Verification process using at least one of control and flow / reaction control. It includes the steps.

36. The method is in accordance with claim 33 and its characteristic is that it obtains more than one sample from the same sample. normalization of biomarker and / or reaction site values ​​and normalization 5 by combining the obtained values ​​using predetermined weights Creating a composite evaluation index involves the process steps.

37. A method that complies with claim 33, and whose characteristic is related to the test strip or analysis platform. Reading the electronic identifier and the type of test derived from the electronic identifier, 10 Relevant calibration according to lot information, calibration data and / or analysis protocol. The selection of analysis parameters involves the process steps.

38. The method is in accordance with claim 33 and is characterized by the fact that the analysis platform is a microplate and / or optical If implemented in reader format, the colorimetric signal will be displayed using the color 15 in a predetermined optical wavelength range suitable for the reaction system It includes the reading process step.

Images