Whole blood visual colorimetric test strip for detecting multiple biomarkers

Abstract

The invention provides a whole blood visual colorimetric test strip for detecting multiple biomarkers, and relates to the technical field of medical detection, the whole blood visual colorimetric test strip comprises a test paper structure composed of a top protection layer, a bottom protection layer and a middle functional layer, and the middle functional layer comprises a bifunctional blood distribution layer, a plasma extraction layer and a detection layer; the detection layer is used for generating color change through interaction of a biomarker in plasma and a reagent mixture, and the detection layer comprises an immobilized enzyme, a color developing agent, an enzyme protective agent, a coherence agent, a plasma stabilizer, a secondary anti-interference material and a surfactant; the reagent mixture is optimized aiming at each biomarker and comprises glucose oxidase, uricase, cholesterol esterase, cholesterol oxidase, horse radish peroxidase, a color developing agent, an enzyme protective agent, a coherence agent, a plasma stabilizer, a secondary anti-interference material and a surfactant. The whole blood visual colorimetric test paper provided by the invention is used for household blood detection, especially for measuring blood sugar, uric acid and total cholesterol, and improves the selectivity and accuracy of the test.

Description

Technical Field

[0001] The invention relates to the technical field of medical detection, and in particular to a whole blood visual colorimetric test strip for detecting multiple biomarkers. Background Art

[0002] Measuring blood glucose levels, blood uric acid levels, and total cholesterol levels is essential for comprehensive health monitoring and early detection of potential health problems. The levels of these biomarkers play a key role in the assessment and management of multiple health conditions, such as diabetes, gout, and cardiovascular disease.

[0003] Blood sugar levels are a key indicator of metabolic health. High blood sugar levels may indicate diabetes or its pre-symptoms. If not treated promptly, it may lead to serious complications such as cardiovascular disease, kidney damage and nerve damage. Therefore, regular monitoring of blood sugar levels is essential for effective management of diabetes.

[0004] Monitoring of blood uric acid levels is essential for detecting and managing gout, which also affects cardiovascular and renal health. Elevated uric acid levels can lead to the formation of urate crystals, which can cause gout attacks. In addition, high uric acid levels are associated with an increased risk of hypertension, cardiovascular disease, and chronic kidney disease. Monitoring uric acid levels can help manage these risks, allowing early intervention through dietary modifications and medications to control uric acid levels.

[0005] Total cholesterol levels are an important marker of cardiovascular health. Elevated levels of LDL cholesterol may lead to plaque buildup in the arteries, increasing the risk of atherosclerosis, heart attack, and stroke. Regular monitoring of total cholesterol levels helps assess cardiovascular risk and take preventive measures, such as lifestyle changes and taking statins, to maintain healthy cholesterol levels.

[0006] Clinical methods for measuring blood glucose, uric acid, and total cholesterol typically use a biochemistry analyzer to measure these biomarkers. Blood glucose levels are typically measured using a glucose oxidase assay, where glucose reacts with glucose oxidase to produce hydrogen peroxide, which then reacts with a chromogenic agent to produce a measurable color change. This can be detected using a rapid home test using a glucometer or using a laboratory spectrophotometer for precise readings. Blood uric acid levels are measured using a uricase method, where uric acid reacts with uricase to produce allantoin and hydrogen peroxide, which reacts with a chromogenic agent in the presence of peroxidase to produce a color change that can be measured using a spectrophotometer or point-of-care device. Total cholesterol is measured enzymatically, where cholesterol esterase breaks down cholesterol esters into free cholesterol and fatty acids, and cholesterol oxidase oxidizes free cholesterol into cholesterol-4-en-3-one and hydrogen peroxide, which then reacts with a chromogenic agent in the presence of peroxidase to produce a color change. The intensity of this color change correlates with cholesterol concentration and can be measured using a laboratory spectrophotometer or point-of-care device.

[0007] Technical issues associated with clinical and home testing methods for biomarkers such as glucose, uric acid, and total cholesterol include several significant challenges. Clinical methods often require expensive biochemistry analyzers, which limits their access to well-equipped laboratories and trained personnel. These methods also typically require larger sample volumes and longer processing times, making them less convenient for frequent monitoring.

[0008] While more convenient and cost-effective, at-home testing devices also face technical issues. They require power and batteries, which can be a limitation in terms of portability and long-term use. Additionally, these devices are not as compact as people would like, making them less practical for people who need to carry them around for frequent testing. While more compact than clinical devices, at-home devices are still not small enough to fit easily in a pocket, which limits their portability and convenience during travel or daily activities.

[0009] An emerging solution to these technological issues is the development of rapid blood test strips that do not require additional equipment for analysis. This approach represents a major advancement in everyday health monitoring as it eliminates the need for instrumentation, making it possible to perform testing anytime, anywhere. Designed to be as portable as possible, these strips are small enough to fit in a pocket and require no batteries or power sources, further reducing costs and increasing accessibility. By reducing the cost of home testing and making it feasible in more locations, these test strips offer a practical and user-friendly alternative for everyday health monitoring, ensuring individuals can stay healthy more effectively and conveniently.

[0010] Visual colorimetric test strips have been commercially developed for the detection of several biomarkers in urine; however, visual colorimetric test strips for blood face challenges because the red color of blood can interfere with the results and disrupt visual colorimetry. Therefore, whole blood separation is necessary for blood colorimetric analysis. For example, in clinical methods that use the Trinder reaction to detect glucose, uric acid, and total cholesterol, blood samples - usually large volumes (several milliliters) - are collected in special test tubes filled with anticoagulant materials. Plasma or serum is then separated by clinical methods and tested using a liquid reagent mixture and a biochemical analyzer. Proper sample dilution and professional operators adhering to clinical protocols are essential for running these tests. Colorimetric analysis is performed using optical equipment implemented in the biochemical unit.

[0011] Home uric acid and total cholesterol testing devices that are not based on electrochemical methods typically use colorimetric analysis to determine the concentration of these biomarkers. For uric acid, a small blood sample is applied to a test strip or test pad containing specific reagents that react with the uric acid to produce a color change. The intensity of this color change is proportional to the uric acid concentration, and the device uses photometric detection, where a light source illuminates the test area and a sensor measures the amount of light absorbed or reflected by the color change. This information is then converted into a uric acid concentration value that is displayed to the user. Similarly, for total cholesterol, the test strip or test pad contains enzymes such as cholesterol esterase and cholesterol oxidase, which react with the cholesterol in the blood sample to produce hydrogen peroxide. This hydrogen peroxide reacts with a chromogenic agent in the presence of peroxidase, resulting in a color change. The device uses photometric detection to measure this color change, evaluating light absorption or reflection. The intensity of the color is proportional to the cholesterol concentration, and the device displays this value to the user. These colorimetric methods use the principles of light absorption or reflection to quantify the target analyte concentration, providing a convenient and reliable method for monitoring health at home.

[0012] If colorimetry is used for home blood testing, whole blood separation is also necessary and more challenging. Given the very small amount of blood obtained from a finger prick (typically 5-30 microliters), special attention must be paid to the separation process. If the device reads and analyzes the results colorimetrically, there is the more flexible option of using chromogens that change color in the UV-visible range, meaning that the color change does not need to be detectable and obvious to the naked eye. However, as mentioned above, the use of equipment, especially optical equipment, makes the method more expensive, less portable, and more difficult to use by everyone.

[0013] The use of a single drop of blood from a finger prick (usually about 101 l) for visual colorimetric detection of multiple biomarkers at home without the need for batteries or equipment presents several technical challenges: 1) Effective separation: Plasma or serum must be effectively separated from the whole blood sample, especially when only a single drop of blood from a finger prick is used. Without this separation, red blood cells can interfere with the colorimetric results. The process should be performed without any sample pretreatment, centrifugation, or use of washing or dilution buffers. 2) Rapid processing: The separation of plasma or serum must be completed quickly to prevent the plasma / serum from drying on the extraction layer. 3) Sufficient yield: The yield of separated plasma / serum must be sufficient to uniformly and completely saturate the detection area in the detection layer, and the reaction should be completed before the plasma evaporates. 4) Reagent optimization: The reagent mixture must be optimized for a small amount of extracted plasma / serum (as low as 11 l) to form a clear, uniform color visible to the naked eye in the detection area. 5) Fast reaction time: The reaction in the detection zone should occur rapidly (from a few seconds to 1-2 minutes) to prevent the plasma / serum from evaporating before the reaction is complete. 6) Visible color differences: Color differences between biomarkers at different concentrations must be easily visible, even when the plasma / serum volumes are small. 7) Minimize the coffee ring effect: The reagent mixture should be optimized to eliminate the coffee ring effect, which is particularly difficult to prevent when the test sample (plasma / serum) volume is very low (as low as 11L). The coffee ring effect occurs when a drop of liquid dries on a solid surface, causing the particles suspended in it to settle in a circular pattern along the edges. This phenomenon is commonly observed when a drop of coffee dries and is caused by capillary flow moving the particles from the center to the edges as the coffee evaporates. Although it has wide-ranging implications in various fields such as printing, biology, and material assembly, this effect complicates the creation of uniform coatings. In colorimetric dry chemistry methods, this results in uneven color distribution in the detection area, with the colors at the edges being darker than those in the center.

[0014] Methods developed by Dr. P. Trinder in the 1960s provided the basis for these measurements by measuring glucose, uric acid, and cholesterol in biological fluids through enzymatic reactions and auxiliary reactions (colorimetric reactions catalyzed by peroxidase). Although these methods are widely adopted in clinical practice, they require sample pretreatment, dilution, centrifugation, and specific incubation times or temperatures.

[0015] The development of dry chemistry paper-based test strips aims to overcome these challenges by optimizing the structure and reagent mixture to detect different biomarkers in biological fluids. Nevertheless, the home blood testing market still mainly relies on electrochemical or optical devices, which can be more expensive and less portable than electrochemical devices.

[0016] Therefore, the development of a battery-free, low-cost visual colorimetric test strip for home blood testing, especially for measuring glucose, uric acid, and total cholesterol, has become an important technical need. Such a test strip needs to address key issues including effective plasma separation, rapid processing, sufficient yield, reagent optimization, fast reaction time, visible color difference, and reduced coffee ring effect to achieve rapid blood testing without the need for additional equipment for analysis. Summary of the invention

[0017] In view of this, in order to solve the above problems, the present invention proposes a battery-free, low-cost whole blood visual colorimetric test strip for detecting multiple biomarkers for home blood testing, especially for measuring blood glucose, uric acid and total cholesterol. Enzyme reactions are selected to measure these biomarkers to improve the selectivity and accuracy of the test. The blood test strip does not rely on electrochemical measurements, so there are no electrodes in the test strip. Instead, the test strip utilizes dry chemistry, colorimetry and microfluidics. The test strip uses enzyme reactions to improve the selectivity and accuracy of the test, does not rely on electrochemical measurements, and does not require batteries or external equipment for analysis.

[0018] The present invention is implemented by the following technical solutions:

[0019] In a first aspect, the present invention provides a whole blood visual colorimetric test strip for detecting multiple biomarkers, the whole blood visual colorimetric test strip comprising:

[0020] Test strip structure: The test strip structure consists of a top protective layer, a bottom protective layer and an intermediate functional layer. The intermediate functional layer includes a dual-functional blood distribution layer, a plasma extraction layer and a detection layer;

[0021] Dual-function blood distribution layer: impregnated with anti-interference material and hydrophilic material, used to eliminate interfering compounds and transfer blood vertically and evenly to the plasma extraction layer;

[0022] Plasma Extraction Layer: Using vertical microfluidics and size-based capture capabilities, blood cells are captured in the plasma extraction layer, allowing for rapid extraction and transfer of plasma to the detection layer;

[0023] Detection layer: used to produce color change through the interaction between biomarkers in plasma and the reagent mixture, the detection layer contains immobilized enzymes, color developers, enzyme protectors, coherent agents, plasma stabilizers and surfactants;

[0024] Reagent mixture: A reagent mixture optimized for each biomarker, including glucose oxidase, uricase, cholesterol esterase, cholesterol oxidase, horseradish peroxidase, chromogen, enzyme protectant, coherent agent, plasma stabilizer, secondary anti-interference material and surfactant.

[0025] As a further solution of the present invention, the test paper structure is also provided with a blood inlet and an observation window, and there are at least three key layers between the blood inlet and the observation window: a blood distribution layer, a plasma extraction layer and a detection layer: a dual-function blood distribution layer, a plasma extraction layer and a detection layer; the blood inlet is a hole arranged on the top protective layer, which is used for capillary blood sampling and transmission through a disposable capillary dropper, or includes a siphon blood unit integrated into the test strip, which is used to attract blood drops from the blood inlet to the blood distribution layer.

[0026] As a further embodiment of the present invention, the preferred substrate of the bifunctional blood distribution layer is a natural protein fiber, in particular a silk screen, which is sequentially impregnated with a main anti-interference material consisting of ascorbic acid oxidase, bilirubin oxidase, potassium hexacyanoferrate (II), dodecasodium phytate, and nitrilotriacetic acid; and an aqueous solution, which is an aqueous mixture of 0.1% to 5% by weight, selected from the following materials: collagen powder, agarose, Triton X-114, polyvinyl pyrrolidone, polyacrylamide, polyvinyl alcohol, and Tween 80.

[0027] As a further embodiment of the present invention, the plasma extraction layer is located below the dual-function blood distribution layer, and is used to directly transfer blood drops to the plasma extraction layer below. The plasma extraction layer is made of an asymmetric glass fiber membrane or an asymmetric polyethersulfone membrane, with a thickness ranging from 100 to 800 microns and a water absorption capacity of 25-180 mg / cm1. The membrane structure can effectively capture blood cells and particles larger than 3 microns.

[0028] As a further solution of the present invention, the dual-functional blood distribution layer is provided with a mesh impregnated with a hydrophilic material or a combination of materials for quickly and evenly transferring blood to the plasma extraction layer. The dual-functional blood distribution layer is preferably a natural protein fiber, especially a silk mesh.

[0029] As a further embodiment of the present invention, the detection layer is a hydrophilic paper for absorbing and retaining the reagent. The reagent mixture contains an enzyme protectant, a coherent agent, a plasma stabilizer, a secondary anti-interference material and a surfactant to achieve uniform fixation of polymeric enzymes and monomeric enzymes and color developers, maintain enzyme activity and stability of color developers in a dry state, eliminate interfaces, ensure complete reaction, uniform color formation, and obtain accurate results at very low plasma volumes.

[0030] As a further embodiment of the present invention, the developer is at least one of N-ethyl-N-(3-propylsulfonyl)aniline sodium salt (ALPS), N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt (TODB), 3,5-dichloro-2-hydroxybenzenesulfonate sodium, 2,4,6-tribromo-3-hydroxybenzoic acid (TBHB), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt (EHSPT / TOOS), 4-chlorophenol, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (F-DAOS), and N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt (ESPAS).

[0031] As a further embodiment of the present invention, the enzyme protector used in the uric acid and glucose reagent mixture is selected from the following materials: sodium salt of bis(sulfosuccinimidyl) suberate (BS3), polyacrylic acid (PLA), polyglycerol, poly(2-hydroxyethyl methacrylate) (PHEMA), N,N'-methylenebisacrylamide (Bis), poly(acrylic acid-co-maleic acid), polyethylene glycol-co-polyglycolic acid (PEG-co-PGA), polyethylene imine, tartrate, ethylene glycol bis(succinimidyl succinate) (EGS), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Su l fo-SMCC); and the enzyme protector used in the cholesterol reagent mixture is selected from the following materials: polyaniline, tartrate, methyl vinyl ether, polyethylene imine, poly(methyl vinyl ethyl ether), polylactic acid-glycolic acid copolymer (PLGA), polylactic acid, polyglycerol, Eudragit RS100.

[0032] As a further embodiment of the present invention, the coherent agent in the mixture of uric acid, glucose and cholesterol reagents is selected from the following compounds: poly (2-ethyl-2-oxazoline), 2-hydroxyethyl cellulose, pullulan, polyvinyl pyrrolidone, PEG6000, carrageenan, polyvinyl alcohol-vinyl acetate copolymer and polyethylene oxide (PEO).

[0033] As a further embodiment of the present invention, the plasma stabilizer is selected from materials including dextran, protein powder, starch, guar gum, polyvinyl pyrrolidone, alginate, polyvinyl alcohol, hydroxyethyl starch and hyaluronic acid.

[0034] As a further embodiment of the present invention, the secondary anti-interference material is iodic acid, potassium bromate, iodate, sodium citrate, triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid. The secondary anti-interference material eliminates the residual interference of reducing agents and metal ions in the plasma.

[0035] As a further embodiment of the present invention, the glucose reagent is a buffer solution, and its components include glucose oxidase>10 ku / l, horseradish peroxidase>500 u / l, 0.1%-10% 4-aminoantipyrine, 0.1%-10% color developer, 0.01%-8% enzyme protectant, 0.06%-12% coherent agent, 0.01%-6% plasma stabilizer, and 0.01%-6% secondary anti-interference material; the uric acid reagent mixture is a buffer solution, and its components include uricase>200 u / l, horseradish peroxidase>800 u / l, 0.1%-10% 4-aminoantipyrine, 0.1%-10% color developer, and 0.01%- 8% enzyme protectant, 0.06%-12% coherent agent, 0.01%-6% plasma stabilizer, 0.01%-8% secondary anti-interference material; the cholesterol reagent mixture is a buffered ethanol solution, and its components include cholesterol esterase>200u / l, cholesterol oxidase>90u / l, horseradish peroxidase>120u / l, 0.1%-10% 4-aminoantipyrine, 0.1%-10% color developer, 0.01%-8% enzyme protectant, 0.03%-12% coherent agent, 0.01%-6% plasma stabilizer, 0.02%-6.2% surfactant, and 0.01%-4% secondary anti-interference material. As a further embodiment of the present invention, the developer is at least one of N-ethyl-N-(3-propylsulfonyl)aniline sodium salt (ALPS), N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt (TODB), 3,5-dichloro-2-hydroxybenzenesulfonate sodium, 2,4,6-tribromo-3-hydroxybenzoic acid (TBHB), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt (EHSPT / TOOS), 4-chlorophenol, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (F-DAOS), and N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt (ESPAS).

[0036] As a further embodiment of the present invention, the enzyme protector for the mixture of uric acid and glucose reagents is selected from bis(sulfosuccinimidyl) suberate sodium salt (BS3), polyacrylic acid (PLA), polyglycerol, polymethacrylate-2-hydroxyethyl ester (PHEMA), N,N'-methylenebisacrylamide (Bis), poly(acrylic acid-co-maleic acid), polyethylene glycol-co-polyglycolic acid (PEG-co-PGA), polyethyleneimine, tartrate, ethylene glycol bis(succinimidyl succinate) (EGS), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Su l fo-SMCC).

[0037] As a further embodiment of the present invention, the enzyme protectant of the cholesterol reagent mixture is selected from the following compounds: polyaniline, tartrazine, methyl vinyl ether, polyethyleneimine, poly(methyl vinyl ethyl ether), polylactic acid-glycolic acid copolymer (PLGA), polylactic acid, polyglycerol, Eudragit RS100.

[0038] As a further embodiment of the present invention, the surfactant in the reagent mixture is selected from: docusate sodium salt (aerosol-ot), polyoxyethylene lauryl ether, sodium lauryl sulfate, sodium cholate, Tween 20, Tween 80, CHAPS, hexadecyltrimethylammonium bromide and triton x-114.

[0039] The detection layer showed response to as little as 0.1 μl of plasma, and the amount of plasma required to cover the detection layer depended on the detection layer area. A drop (5-30 μl) of whole blood was ideal for adequate plasma extraction and colorimetric detection.

[0040] As a further embodiment of the present invention, the plasma stabilizer in the reagent mixture is selected from dextran, protein powder, starch, guar gum, polyvinyl pyrrolidone, alginate, polyvinyl alcohol, hydroxyethyl starch and hyaluronic acid.

[0041] Compared with the prior art, the whole blood visual colorimetric test strip for detecting multiple biomarkers provided by the present invention has the following beneficial effects:

[0042] The present invention is a whole blood visual colorimetric test strip for detecting multiple biomarkers, which provides an accurate, low-cost, battery-free solution for home blood testing, especially for measuring blood glucose, uric acid and total cholesterol using a single drop of blood sample. The enzyme reaction improves the selectivity and accuracy of the test. This blood test strip does not rely on electrochemical measurements, eliminating the need for electrodes. It can operate without batteries and does not require external equipment (electrochemical or optical) for analysis. The design extracts plasma from a drop of blood pricked by the finger. This ensures that red blood cells do not interfere with the colorimetric process, thereby achieving accurate visual colorimetry for blood testing.

[0043] Users can interpret the results based on the color change, providing a simple method for home health monitoring without the need for complex instrumentation or power supplies. This innovation is designed to make health monitoring more accessible and user-friendly, especially in environments where traditional testing equipment may be impractical or inaccessible.

[0044] Simultaneous measurement of blood glucose, blood uric acid, and total cholesterol levels during at-home point-of-care testing (POCT) is essential for a comprehensive health assessment. Combining these measurements provides a comprehensive understanding of an individual's metabolic, cardiovascular, and renal health. This integrated approach enables individuals to conveniently monitor their health at home, promoting early detection of potential health issues and timely intervention. It enables individuals to take proactive steps to manage their health, leading to better health outcomes and improved quality of life. Home POCT devices that measure these three parameters provide a valuable tool for ongoing health monitoring and personalized healthcare management. This innovative invention introduces an accurate, ultra-compact solution that fits in your pocket, providing low-cost, battery-free home whole blood testing.

[0045] These and other aspects of the present invention will be more clearly understood in the following description of the embodiments. It should be understood that the above general description and the following detailed description are only exemplary and explanatory and cannot limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or related technologies, the following briefly introduces the drawings required for use in the exemplary embodiments or related technical descriptions. The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0047] Figure 1 The figure is a schematic diagram of the structure of a whole blood visual colorimetric test strip for detecting multiple biomarkers according to an embodiment of the present invention.

[0048] Figure 2 This is a schematic structural diagram of a whole blood visual colorimetric test strip for detecting multiple biomarkers according to another embodiment of the present invention.

[0049] Reference numerals in the figures:

[0050] 1-blood inlet; 2-top protective layer; 3-blood distribution layer; 4-plasma extraction layer; 5-detection layer; 6-bottom protective layer; 7-observation window; 8-hydrophilic membrane siphon upper layer; 9-siphon groove layer; 10-siphon support lower layer. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0052] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The sequence numbers of the operations, such as 1, 2, etc., are only used to distinguish between different operations, and the sequence numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0053] In order to solve the problems of existing test strips, the present invention provides a whole blood visual colorimetric test strip for detecting multiple biomarkers, and develops battery-free, low-cost visual colorimetric test strips for home blood testing, especially for measuring blood sugar, uric acid and total cholesterol. Enzyme reactions are selected to measure these biomarkers to improve the selectivity and accuracy of the test. Blood test strips do not rely on electrochemical measurements, so there are no electrodes in the test strips. Instead, the test strips utilize dry chemistry, colorimetry and microfluidics. One problem with enzyme reactions in dry chemistry is the proper fixation of the enzyme and the retention of enzyme activity in a dry and fixed form. In addition, the reaction rate is very important for providing rapid results and achieving obvious color differences for biomarkers of different concentrations in a short time. Plasma retention and uniform plasma distribution in the detection area are other challenges in designing extremely low blood volume blood test strips based on visual colorimetry. In order to solve problems related to enzyme fixation, enzyme activity and enzyme stability, previous studies tend to use nanozymes instead of enzymes, such as Prussian blue or other nanozymes instead of peroxidase. Although nanozymes (mimicking enzyme performance) are more stable, their use reduces the selectivity of the test strips and affects the accuracy of the results. Nanozymes can interact with many analytes and biomarkers in biological fluids such as plasma, leading to selectivity issues for blood testing. For example, it has been repeatedly reported that dyes such as Prussian blue can bind to albumin. This may interfere with the results of colorimetric test strips used to detect other biomarkers such as glucose, uric acid, and cholesterol. Fortunately, these problems have been addressed in the work of the present invention by using enzymes and color developers and optimized amounts of modifiers, which perform various functions in the test strips. These modifiers include enzyme protectors, coherent materials, two sets of anti-interference materials, and plasma stabilizers. pH adjustment has been taken into account to ensure effective intermolecular interactions in the detection layer, especially between enzymes and biomarkers. The present invention introduces an optimized test strip structure with an optimized reagent mixture, in which the enzyme is immobilized, stabilized, and its activity is maintained by using appropriate modifiers. The modification of the dual-functional blood distribution layer and the addition of plasma retention materials (plasma stabilizers) in the reagent mixture further ensure effective and uniform color development of blood visual colorimetry, even without using a smartphone for analysis. This design ensures a rapid response to specific biomarkers in the blood.

[0054] The present invention introduces a single-drop blood colorimetric test strip that operates without batteries and does not require external equipment for analysis. The present invention simplifies the blood testing process by allowing the user to drop a drop of blood directly on the test strip. Capillary action (siphon action) transfers the blood drop from the fingertip to the distribution layer. The dual-functional blood distribution layer is impregnated with hydrophilic materials and anti-interference materials, ensuring that the blood is vertically and evenly transferred to the plasma extraction layer, and it can also eliminate some interfering substances in the blood before the blood reaches the bottom layer. Blood cells, including red blood cells, are trapped in the fiber structure of the plasma extraction layer by binding and encapsulation, while the plasma gradually passes vertically and reaches the detection layer impregnated with a reagent mixture. The biomarkers (glucose, uric acid or cholesterol) in the extracted plasma interact with the reagent mixture fixed in the detection layer. This interaction causes color development, which can be observed with the naked eye through the observation window below the detection layer. Users can interpret the results based on the color change, providing a simple method for home health monitoring without the need for complex instruments or power supplies. This innovation aims to make health monitoring more accessible and user-friendly, especially in environments where traditional testing equipment may be impractical or inaccessible.

[0055] The importance of simultaneously measuring blood glucose, blood uric acid, and total cholesterol levels for home point-of-care testing (POCT) lies in the comprehensive health assessment it provides. Combining these measurements provides a comprehensive understanding of an individual's metabolic, cardiovascular, and renal health. This comprehensive approach enables individuals to monitor their health from the convenience of their own home, facilitating early detection of potential health issues and timely intervention. It enables individuals to take proactive steps to manage their health, leading to better health outcomes and improved quality of life. Home POCT devices that measure these three biomarkers provide a valuable tool for ongoing health monitoring and personalized healthcare management.

[0056] The following will be combined with the drawings in the exemplary embodiments of the present invention to clearly and completely describe the technical solutions in the exemplary embodiments of the present invention. Obviously, the exemplary embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0057] See also Figure 1As shown, a whole blood visual colorimetric test strip for detecting multiple biomarkers provided in an embodiment of the present invention comprises a test strip structure, which is composed of a top protective layer 2, a bottom protective layer 6 and an intermediate functional layer, wherein the intermediate functional layer comprises a dual-functional blood distribution layer 3, a plasma extraction layer 4 and a detection layer 5; the dual-functional blood distribution layer 3 is impregnated with an anti-interference material and a hydrophilic material for eliminating interfering compounds and vertically and uniformly transferring blood to the plasma extraction layer 4, wherein the plasma extraction layer 4 uses vertical microfluidics and size-based capture functions to obtain blood from the plasma extraction layer 4. Blood cells are captured in the extraction layer 4, allowing rapid extraction and transfer of plasma to the detection layer 5; the detection layer 5 is used to produce color changes through the interaction of biomarkers in the plasma with the reagent mixture, and the detection layer 5 contains immobilized enzymes, color developers, enzyme protectants, coherent agents, plasma stabilizers and surfactants; the reagent mixture is a reagent mixture optimized for each biomarker, including glucose oxidase, uricase, cholesterol esterase, cholesterol oxidase, horseradish peroxidase, color developers, enzyme protectants, coherent agents, plasma stabilizers, secondary anti-interference materials and surfactants.

[0058] In one embodiment, see Figure 2 As shown, the test paper structure is also provided with a blood inlet 1 and an observation window 7, and there are at least three key layers between the blood inlet 1 and the observation window 7: a blood distribution layer 3, a plasma extraction layer 4 and a detection layer 5: a dual-function blood distribution layer 3, a plasma extraction layer 4 and a detection layer 5; the blood inlet 1 is a hole arranged on the top protective layer 2, which is used for capillary blood sampling and transmission through a disposable capillary dropper, or includes a siphon blood unit integrated into the test strip, which is used to attract blood drops from the blood inlet 1 to the blood distribution layer 3, wherein the siphon blood unit includes a hydrophilic membrane siphon upper layer 8, a siphon groove layer 9 and a siphon support lower layer 10.

[0059] The preferred substrate of the dual-functional blood distribution layer 3 is a natural protein fiber, in particular a silk screen, which is sequentially impregnated with a main anti-interference material consisting of ascorbic acid oxidase, bilirubin oxidase, potassium hexacyanoferrate (II), phytic acid sodium salt, and nitrilotriacetic acid; and a hydrophilic solution, which is an aqueous mixture of 0.1%-5% by weight, selected from the following materials: collagen powder, agarose, Triton X-114, polyvinyl pyrrolidone, polyacrylamide, polyvinyl alcohol, and Tween 80. The plasma extraction layer 4 is located below the dual-functional blood distribution layer 3, and is used to directly transfer blood drops to the plasma extraction layer 4 below. The plasma extraction layer 4 is made of an asymmetric glass fiber membrane or an asymmetric polyethersulfone membrane, with a thickness ranging from 100 to 800 microns and a water absorption capacity of 25-180 mg / cm3. The membrane structure can effectively capture blood cells and particles larger than 3 microns.

[0060] The blood inlet 1 is a hole designed for capillary blood sampling and transmission through a disposable capillary dropper, or comprises a siphon blood unit integrated into the test strip, for attracting blood drops from the blood inlet 1 to the blood distribution layer 3. In another embodiment, in order to further simplify the process and eliminate the need for a separate disposable capillary dropper, an independent capillary unit is directly integrated into the test strip.

[0061] By means of capillary action (siphoning blood), a drop of blood is attracted to the dual-functional blood distribution layer 3. This layer is a mesh impregnated with a hydrophilic material or a combination of materials, which is saturated with a hydrophilic solution, then dried at 37-70°C for 10-50 minutes, and stored in a container with a desiccant for later use, ensuring that the blood is quickly and evenly transferred to the plasma extraction layer 4. Various mesh types can be used for the dual-functional blood distribution layer 3, and the preferred substrate of the dual-functional blood distribution layer 3 is a natural protein fiber, in particular a silk screen, which is sequentially impregnated with a main anti-interference material consisting of ascorbic acid oxidase, bilirubin oxidase, potassium hexacyanoferrate (II), phytic acid dodecasodium salt, and nitrilotriacetic acid; and a hydrophilic solution, which is an aqueous mixture of 0.1% to 5% by weight, selected from the following materials: collagen powder, agarose, Triton X-114, polyvinyl pyrrolidone, polyacrylamide, polyvinyl alcohol, and Tween 80.

[0062] The grid layer ensures that the blood droplets are evenly transferred to the plasma extraction layer 4. The plasma extraction layer 4 is located below the dual-functional blood distribution layer 3 and its working principle is to filter blood cells vertically, which requires two key conditions to be met: first, the blood must evenly cover the entire surface of the plasma extraction layer 4, and second, this must happen simultaneously across the entire layer. This means that lateral blood flow across the surface of the plasma extraction layer 4 should not be promoted, and the vertical plasma passage rate should remain consistent throughout the depth of the plasma extraction layer 4. If these conditions are not met, it will result in uneven plasma distribution within the detection layer 5. In addition, if the plasma reaches one side of the detection layer 5 faster than the other side, lateral flow will occur, resulting in uneven color distribution.

[0063] In one embodiment, the plasma extraction layer 4 is located below the blood distribution layer 3, and the blood distribution layer 3 is used to transfer blood droplets to the plasma extraction layer 4 directly below. The plasma extraction layer 4 is made of an asymmetric glass fiber membrane or an asymmetric polyethersulfone membrane. The asymmetric glass fiber membrane is an asymmetric glass fiber membrane with a thickness ranging from 100 to 8001m, a water absorption capacity of 25-180mg / cm3, and a fiber structure that effectively captures blood cells and particles larger than 31m.

[0064] In one embodiment, the enzyme protector used in the uric acid and glucose reagent mixture is selected from the following materials: bis(sulfosuccinimidyl) suberate sodium salt (BS3), polyacrylic acid (PLA), polyglycerol, poly(2-hydroxyethyl methacrylate) (PHEMA), N,N'-methylenebisacrylamide (Bis), poly(acrylic acid-co-maleic acid), polyethylene glycol-co-polyglycolic acid (PEG-co-PGA), polyethylene imine, tartrate, ethylene glycol bis(succinimidyl succinate) (EGS), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Su l fo-SMCC); and the enzyme protector used in the cholesterol reagent mixture is selected from the following materials: polyaniline, tartrate, methyl vinyl ether, polyethylene imine, poly(methyl vinyl ethyl ether), polylactic acid-glycolic acid copolymer (PLGA), polylactic acid, polyglycerol, Eudragit RS100.

[0065] Specifically, in the embodiment with independent capillary units, the blood droplets sucked into the strip should first spread on the mesh surface (distribution layer) and then move downward in the vertical direction. The modified polar attraction between the mesh surface and the blood helps the blood droplets spread completely and evenly on the mesh surface.

[0066] The present invention can evenly distribute the blood droplets on the surface of the plasma extraction layer by selecting a grid layer and impregnating it with a hydrophilic solution, thereby ensuring the uniform distribution of plasma in the detection layer 5. In the experiment, the performance of three types of test strips (without a grid layer, with an original grid layer, and with a modified grid layer) was evaluated, and each test was repeated three times. The results show that plasma extracted from a single drop of blood can only be evenly distributed in the detection layer 5 after the grid layer is modified with a hydrophilic solution. The design of this distribution layer helps to improve the overall performance and accuracy of the system.

[0067] As described above, the bifunctional blood distribution layer 3 transfers the blood droplet to the plasma extraction layer 4 located directly below it. Blood cells, including red blood cells, are captured within this layer using vertical microfluidic principles, size-based capture and in combination with layer texture. The plasma extraction layer can be made of an asymmetric glass fiber membrane or an asymmetric polyethersulfone membrane. The preferred thickness range is 100 to 8001 μm. Within this thickness range, the water absorption capacity is 25-180 mg / cm3, and the fiber structure effectively captures blood cells and particles larger than 31 μm. Blood cells bind to the fiber matrix both on the surface and inside the fiber structure, where they are securely captured. The plasma is then extracted and vertically transferred to the detection layer 5 at a rapid speed of 30-120 seconds depending on the membrane size.

[0068] This vertical flow plasma extraction layer 4 has advantages over other widely used methods such as lateral flow plasma separation, especially for visual colorimetry using a single drop of blood volume. First, it enables the plasma to pass quickly through the detection layer 5, minimizing plasma losses. Second, it ensures that the plasma / serum is evenly absorbed over the entire surface of the detection layer 5, preventing uneven color distribution and lateral washout of reagent mixtures that can occur in lateral flow designs. In addition, it allows for a larger detection area, which is ideal for visual colorimetry.

[0069] In this embodiment, in the detection layer 5, the biomarkers in the plasma (including glucose, uric acid and cholesterol) interact with the reagent mixture, resulting in a color change that can be observed from the bottom observation window 7. It is worth noting that by using an appropriate reagent mixture in the detection layer 5, this test strip design can be applied to detect any biomarker in the blood, which can be identified by the color change of the detection layer 5. This detection method is based on an enzyme oxidation reaction and has high selectivity. However, the visual whole blood colorimetric design of a single drop of blood sample volume needs to consider more than just enzymes and color developers.

[0070] The detection layer 5 is a hydrophilic paper used to absorb and retain reagents. A modifier is used in the detection layer 5 to fix the enzyme unit in the thin paper of the hydrophilic paper to maintain enzyme activity. The modifier is not only conducive to the cross-linking of the enzyme unit, but also forms an enzyme protector combined with the detection paper.

[0071] Among them, the pretreatment and surface modification of paper increase the manufacturing cost and time for preparing the test strips. Secondly, materials such as Triton X-100, gelatin and trehalose do not solve the problem of color uniformity. For example, although Triton X-100 increases color intensity and improves the lower limit of detection, it reduces the color difference between different concentrations and limits the detection range of the test strip. The present inventors also observed that gelatin and trehalose slowed down the reaction rate, resulting in a slower detection speed, while the color intensity of a single drop of blood test was not significantly improved. Some researchers have suggested an additional second impregnation with a hydrophobic film layer of methylcellulose or ethylcellulose. However, this method not only increases manufacturing costs, but also introduces a hydrophobic barrier, which negatively affects the interaction of low-volume plasma with the reagent mixture, which requires an aqueous environment.

[0072] Given the thinner test paper and low blood sample volume, the goal of the present invention was to achieve uniform color distribution and clear color difference at low and high levels of blood biomarkers, and several technical solutions were considered. The first solution was to use a modifier to immobilize more enzyme units within the thin paper while maintaining enzyme activity. Uricase usually functions as a tetramer, composed of four identical subunits, making it a polymeric enzyme. Glucose oxidase is a dimer with two identical subunits, while cholesterol esterase and cholesterol oxidase usually function as monomers.

[0073] The modifier should be able to form significant intermolecular attractions not only between the enzyme units, but also between the enzyme units and the molecular structure of the paper. This means that the modifier should not only act as an effective cross-linking agent to collect the enzyme units, but also uniformly fix them within the detection layer 5. Although weak physical fixation of the enzyme will lead to enzyme loss from the paper surface, uncontrolled strong covalent bonds between the enzyme and the paper may inactivate the enzyme or reduce the enzyme activity. For example, for uricase, an incorrect connection point will lead to conformational changes, destroy the interaction between the subunits, destabilize the tetramer structure, and lead to dissociation into inactive monomers or dimers.

[0074] The first solution of the present invention to fix more enzyme units into the thin paper involves the use of a modifier (referred to as "enzyme guard" in the present invention) that can cross-link the enzyme units and form a significant bond with the detection paper. The strength of the enzyme-enzyme interaction is comparable to the enzyme-paper interaction. This ensures that more enzymes can be concentrated in the detection layer 5 without distorting the natural structure of the enzyme or reducing its activity. In addition, due to the increase in the amount of active enzyme fixed in the detection layer 5, the enzyme guard accelerates the color development in the detection layer 5.

[0075] The present invention observed that the use of enzyme guards results in a rapid reaction within the detection layer 5: as soon as the sample contacts the detection layer 5, the color begins to appear, and a clear color difference between the concentrations is visible within 10 seconds. The color stabilizes within 1 to 2 minutes, which is suitable for comparison.

[0076] In this embodiment, the enzyme protector for the uric acid and glucose reagent mixture in the reagent mixture is selected from: bis(sulfosuccinimidyl) suberate sodium salt (BS3), polyacrylic acid (PLA), polyglycerol, polymethacrylate-2-hydroxyethyl ester (PHEMA), N,N'-methylenebisacrylamide (Bis), poly(acrylic acid-co-maleic acid), polyethylene glycol-co-polyglycolic acid (PEG-co-PGA), polyethyleneimine, tartrate, ethylene glycol bis(succinimidyl succinate) (EGS), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Su l fo-SMCC).

[0077] The enzyme protector of the cholesterol reagent mixture in the reagent mixture is selected from: polyaniline, lemon yellow, methyl vinyl ether, polyethyleneimine, poly (methyl vinyl ethyl ether), polylactic acid-glycolic acid copolymer (PLGA), polylactic acid, polyglycerol, Eudragit RS100. The above-mentioned persistent problem is the existence of the coffee ring effect. Although this effect is improved after using the enzyme guard, the color depth in the center of the detection layer 5 is still low. In order to solve this problem, the present invention introduces a second type of modifier in the system, which the present invention refers to as a "coherent agent". These reagents contain chemical functional groups that can form significant intermolecular interactions between the enzyme protector and other components in the reagent mixture (mainly the developer).

[0078] The enzyme guard helps to evenly distribute high concentration enzymes in the paper and prevents the enzymes from accumulating at the edge of the detection layer 5, but the color developer tends to migrate to the edge, resulting in uneven distribution and a persistent coffee ring effect. The coherent agent acts as an intermolecular bridge between the enzyme guard and the color developer, ensuring that the enzyme units and the color developer are evenly distributed within the detection layer 5. This effectively solves the coffee ring effect.

[0079] Furthermore, the combination of the enzyme protectant and the coherent agent allows higher concentrations of reagents (enzyme and chromogenic agent) to be adsorbed from the reagent mixture into the thin detection layer 5. This capability enables the measurement of higher concentrations of blood biomarkers, thereby expanding the detection range.

[0080] The coherent agent for the uric acid, glucose and cholesterol reagent mixture is selected from the following compounds: poly(2-ethyl-2-oxazoline), 2-hydroxyethylcellulose, pullulan, polyvinyl pyrrolidone, PEG6000, carrageenan, polyvinyl alcohol-vinyl acetate copolymer and polyethylene oxide (PEO).

[0081] Since cholesterol reagents must interact with lipids, which are hydrophobic biomarkers encapsulated in plasma lipoproteins, detergents (surfactants) are required in addition to enzyme protectants and coherent agents for the detection of total cholesterol. Detergents ensure uniform color development in the presence of hydrophobic molecules, resulting in accurate and consistent results.

[0082] In the present invention, the surfactant in the reagent mixture is selected from: docusate sodium salt (aerosol-ot), polyoxyethylene lauryl ether, sodium lauryl sulfate, sodium cholate, Tween 20, Tween 80, CHAPS, hexadecyltrimethylammonium bromide and triton x-114.

[0083] When very small amounts of blood (5-101 l) are used, the reaction in the detection layer 5 is not completed for higher biomarker concentrations, while this problem does not occur for blood samples greater than 101 l. This problem is caused by the evaporation of plasma, and the interaction between the plasma biomarkers and the reagent mixture in the detection layer 5 stops due to lack of water. In order to solve this problem, the present invention introduces plasma stabilizers, which help to maintain moisture in the detection layer 5 when the plasma reaches the detection layer 5. These stabilizers expand the plasma volume in the detection layer 5, ensuring accuracy over a wider concentration range even at very low blood volumes (5-101 l). This is particularly beneficial when multiple biomarkers need to be detected.

[0084] In the present invention, the plasma stabilizer in the reagent mixture is selected from dextran, protein powder, starch, guar gum, polyvinyl pyrrolidone, alginate, polyvinyl alcohol, hydroxyethyl starch and hyaluronic acid.

[0085] In the present invention, the glucose reagent is a buffer solution, and its components include glucose oxidase>10 ku / l, horseradish peroxidase>500 u / l, 0.1%-10% 4-aminoantipyrine, 0.1%-10% color developer, 0.01%-8% enzyme protectant, 0.06%-12% coherent agent, 0.01%-6% plasma stabilizer, and 0.01%-6% secondary anti-interference material; in order to prepare the glucose detection layer 5, the hydrophilic paper is saturated with the glucose reagent mixture, dried at 25-60°C for 10-55 minutes, and stored in an opaque container with a desiccant.

[0086] In the present invention, the uric acid reagent mixture is a buffer solution, and its components include uricase>200u / l, horseradish peroxidase>800u / l, 0.1%-10% 4-aminoantipyrine, 0.1%-10% color developer, 0.01%-8% enzyme protectant, 0.06%-12% coherent agent, 0.01%-6% plasma stabilizer, and 0.01%-8% secondary anti-interference material; in order to prepare the uric acid detection layer 5, the hydrophilic paper is saturated with the uric acid reagent mixture, dried at 25-60°C for 10-55 minutes, and stored in an opaque container with a desiccant.

[0087] In the present invention, the cholesterol reagent mixture is a buffered ethanol solution, and the components include cholesterol esterase>200u / l, cholesterol oxidase>90u / l, horseradish peroxidase>120u / l, 0.1%-10% 4-aminoantipyrine, 0.1%-10% color developer, 0.01%-8% enzyme protectant, 0.03%-12% coherent agent, 0.01%-6% plasma stabilizer, 0.02%-6.2% surfactant, and 0.01%-4% secondary anti-interference material. In order to make the cholesterol detection layer 5, the hydrophilic paper is saturated with the cholesterol reagent mixture, dried at 30-65°C for 10-55 minutes, and stored in an opaque container with a desiccant.

[0088] In the present invention, the developer is at least one of N-ethyl-N-(3-propylsulfonyl)aniline sodium salt (ALPS), N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt (TODB), 3,5-dichloro-2-hydroxybenzenesulfonate sodium, 2,4,6-tribromo-3-hydroxybenzoic acid (TBHB), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt (EHSPT / TOOS), 4-chlorophenol, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (F-DAOS), and N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt (ESPAS).

[0089] The preferred color developing agent for the small volume blood sample used in the present invention is ALPS.

[0090] In the present invention, the enzyme protectant used for the mixture of uric acid and glucose reagents is selected from bis(sulfosuccinimidyl) suberate sodium salt (BS3), polyacrylic acid (PLA), polyglycerol, poly(2-hydroxyethyl methacrylate) (PHEMA), N,N'-methylenebisacrylamide (Bis), poly(acrylic acid-co-maleic acid), polyethylene glycol-co-polyglycolic acid (PEG-co-PGA), polyethyleneimine, tartrate, ethylene glycol bis(succinimidyl succinate) (EGS), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Su l fo-SMCC).

[0091] In the present invention, the enzyme protectant of the cholesterol reagent mixture is selected from the following compounds: polyaniline, tartrazine, methyl vinyl ether, polyethyleneimine, poly(methyl vinyl ethyl ether), polylactic acid-co-glycolic acid (PLGA), polylactic acid, polyglycerol, Eudragit RS100. In the present invention, the coherent agent used for the uric acid, glucose and cholesterol reagent mixture in the reagent mixture is selected from poly(2-ethyl-2-oxazoline), 2-hydroxyethylcellulose, pullulan, polyvinyl pyrrolidone, PEG6000, carrageenan, polyvinyl alcohol-co-vinyl acetate and polyethylene oxide (PEO).

[0092] For the glucose detection layer 5, the buffer solution may be any buffer solution buffered to pH = 6-7.5, most preferably pH = 6-7.2. Preferred buffer solutions are acetate buffer solution, phosphate buffer solution and succinate buffer solution.

[0093] For the uric acid detection layer 5, the buffer solution can be any buffer solution buffered to pH = 7-9, most preferably pH = 7.5-8.5. Preferred buffer solutions are acetate buffer solution, phosphate buffer solution and succinic acid buffer solution.

[0094] For the cholesterol detection layer 5, the buffer solution may be any buffer buffered to pH = 6-8, most preferably pH = 6.5-7.5. Preferred buffer solutions are MES buffer solution, Good buffer solution and phosphate buffer solution.

[0095] The reagent mixture for modifying the dual-functional blood distribution layer 3 consists of 0.1-5 wt% of a hydrophilic material dissolved in a buffer solution. The hydrophilic material is selected from agar collagen powder, agarose, Triton X-114, polyvinyl pyrrolidone, polyacrylamide, polyvinyl alcohol and Tween 80.

[0096] In the present invention, the dual-function blood distribution layer 3 can be selected from various mesh types, and the preferred material is natural protein fiber. The most preferred mesh layer is a silk screen. The mesh layer is saturated with a hydrophilic solution and impregnated with a main anti-interference material consisting of ascorbic acid oxidase, bilirubin oxidase, potassium hexacyanoferrate (II), phytic acid dodecasodium salt, and nitrilotriacetic acid, then dried at 37-70°C for 10-50 minutes and stored in a container with a desiccant for later use. The dual-function blood distribution layer 3, the plasma extraction layer 4, and the detection layer 5 are cut into appropriate sizes with an area range of 7-29 mm2.

[0097] In one embodiment, the dual-functional blood distribution layer 3, plasma extraction layer 4, and detection layer 5 are placed and fixed between the blood inlet 1 portion of the top protective layer 2 and the observation window 7 of the bottom protective layer 6. In this configuration, a single drop of blood sample from a finger prick is applied to the blood inlet 1 portion, and the color appears in the detection area and is visible to the naked eye through the observation window 7. Disposable capillary droppers can be used for sample transfer. For multiplex detection of biomarkers, the top protective layer 2 includes multiple blood inlet 1 portions, and the bottom protective layer 6 includes multiple observation windows 7. The blood distribution layer, plasma extraction layer 4, and detection layer 5 are placed and fixed between each blood inlet 1 portion and the observation window 7. The detection layer 5 for each biomarker is different; for example, the separate detection layers 5 for glucose, uric acid, and cholesterol each contain a specific reagent mixture.

[0098] In another embodiment, the test strip has a self-contained capillary unit on the top layer. The dual-function blood distribution layer 3, plasma extraction layer 4 and detection layer 5 are placed and fixed between the self-contained capillary unit and the viewing window 7 of the high-end bottom layer. This embodiment further simplifies the test for the user and eliminates the need for a separate disposable capillary dropper. When the self-contained capillary unit contacts a drop of blood from a finger prick, the sample is automatically transferred directly to the dual-function blood distribution layer 3. The color then develops in the detection area and is visible to the naked eye through the viewing window 7.

[0099] The developed color is compared with a standard color card to determine the concentration of the biomarker in the blood. It is worth noting that by using the whole blood test strip of the present invention, a visual color detection algorithm and a smartphone application platform can also be developed for digital detection and analysis of the results.

[0100] The technical solution of the present invention is further described below in conjunction with specific embodiments:

[0101] Example 1, when making the detection layer 5, glucose oxidase solution>8ku / l, horseradish peroxidase>800u / l, 0.3% 4-aminoantipyrine, 2% N-acetyl-N-(3-propylsulfonyl)aniline sodium salt (ALPS), 1.2% polyvinylamine (PVAm), 2% poly(2-ethyl-2-oxazoline), 0.5% pullulan and 0.1% potassium bromate were prepared in a phosphate buffer at pH=7.2. First, glucose oxidase and horseradish peroxidase were dissolved in a buffer solution. Then polyvinylamine (PVAm) was added to the solution, stirred and incubated for at least 10 minutes to form crosslinks. Poly(2-ethyl-2-oxazoline), 4-aminoantipyrine and N-ethyl-N-(3-propylsulfonyl)aniline sodium salt (ALPS) were then added to the mixture and stirred at 37°C. Finally, pullulan and potassium bromate were added to the solution and mixed thoroughly. The filter paper was saturated with the above solution, dried and stored in an opaque container with a desiccant.

[0102] Uricase solution >320u / l, horseradish peroxidase >700u / l, 0.5% 4-aminoantipyrine, 1.5% ALPS and EHSPT mixture (according to a weight ratio of 1:1), 3% poly(2-aminoethyl methacrylate) (PAEMA), 1.4% poly(vinyl alcohol-vinyl acetate copolymer), 1.4% poly(vinyl pyrrolidone), 0.8% sodium alginate and 0.2% ammonium iodate were prepared in phosphate buffer at pH=8. First, uricase and horseradish peroxidase were dissolved in phosphate buffer solution at pH=8. Poly(2-aminoethyl methacrylate) (PAEMA) was added, the solution was stirred and incubated for at least 10 minutes to achieve crosslinking. Poly(vinyl alcohol-vinyl acetate copolymer), 1.4% poly(vinyl pyrrolidone), 4-aminoantipyrine, ALPS and EHSPT) were then stirred at 37°C. Finally, sodium alginate and ammonium iodate were added to the solution and mixed thoroughly. The filter paper was saturated with the above solution, dried and stored in an opaque container with a desiccant.

[0103] Cholesterol esterase solution>200u / l, cholesterol oxidase>100u / l, horseradish peroxidase>300u / l, 0.7% 4-aminoantipyrine, 0.7% N-bis(4-methyl)-3-methylaniline disodium salt (TODB), 1.5% polyethyleneimine, 6% polyvinylpyrrolidone, 4% PEG6000, 0.5% sodium cholate, 0.2% dextran, 0.1% iodic acid and 0.1% triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid were prepared in MES buffer at pH=6.2. First, cholesterol esterase, cholesterol oxidase and horseradish peroxidase were added to the mixture. Peroxidase was dissolved in MES buffer solution at pH 6.2. Polyethyleneimine was added to this solution and stirred for at least 10 minutes for cross-linking. This solution was labeled Solution I. Sodium cholate solution was prepared separately in a 8:1 buffered ethanol mixture and then gradually added to Solution I. Once a homogeneous mixture was obtained, polyvinylpyrrolidone, PEG6000, 4-aminoantipyrine and N-bis(4-methyl)-3-methylaniline disodium salt were added (TODB), followed by dextran, iodic acid and triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid and stirred again at 37°C. The filter paper was saturated with the above solution, dried and stored in an opaque container with a desiccant.

[0104] Embodiment 2, preparation concentration is the glucose standard solution of 50mg / dl, 100mg / dl, 140mg / dl, 180mg / d, 250mg / dl, 500mg / dl and 1000mg / dl.Preparation concentration is the uric acid standard solution of 119umol / l, 297umol / l, 420umol / l, 595umol / l and 1784umol / l.Preparation concentration is respectively the cholesterol standard solution of 70mg / dl, 100mg / dl, 150mg / dl, 200mg / dl and 250mg / dl.By dripping every kind of standard solution of 1-10 microliter to glucose detection layer 5, uric acid detection layer 5 and cholesterol detection layer 5 respectively, color map is prepared for glucose detection strip, uric acid detection strip and cholesterol detection strip.The color of each detection zone changes from colorless or light yellow to blue or purple / violet. The intensity of the color is proportional to the concentration of the standard solution.

[0105] Example 3, the distribution layer can also be modified as follows: 3% of the anti-interference material (ascorbic acid oxidase, bilirubin oxidase and phytic acid sodium salt) is mixed in a ratio of 1:1:0.1 and dissolved in a buffer solution of pH = 7.5. 0.8% collagen powder, 2% polyvinyl pyrrolidone and 0.1% Triton X-114 are dissolved in a phosphate buffer solution of pH = 7.5. The mesh layer is saturated with the solution, then dried in a vacuum oven at 37-70°C for 10-50 minutes, and stored in a container with a desiccant for later use.

[0106] Example 4, the dual-function blood distribution layer 3, the plasma extraction layer 4 and the detection layer 5 are cut into appropriate sizes with an area of ​​7-29 mm2. These three layers are placed and fixed in order from top to bottom below the blood inlet 1 part: blood distribution layer, plasma extraction layer 4 and detection layer 5. 33 separate test strips were made: 11 with glucose detection layer 5, 11 with uric acid detection layer 5, and 11 with cholesterol detection layer 5. A sample volume of 5-30 microliters is sufficient to complete the test of each test strip with the size.

[0107] Example 5, in order to verify effective blood separation, two preliminary experiments were conducted. In one experiment, the detection layer 5 in the test strip was replaced by blank white paper. In another experiment, it was replaced by a universal pH test paper. Since the pH value of blood is usually between 7.35 and 7.45, and there is no significant difference between people, the color change on the pH test paper can well indicate the effect of blood separation. The test strips are divided into three groups, test strips without a distribution layer (mesh layer), test strips with a distribution layer (mesh layer), and test strips with a distribution layer modified with a hydrophilic material. In the test, a drop of blood (10 microliters) was dropped into the blood inlet 1 part of each test strip. After one minute, the test strip was turned over and the observation window 7 was checked. No red was observed on the blank paper, indicating that the plasma extraction was effective, and the test strip with a distribution layer (mesh layer) modified with a hydrophilic material provided the best uniform green on the pH test paper detection layer 5, indicating that the extracted plasma and color distribution were uniform.

[0108] Example 6, three different rat blood samples were collected, wherein the glucose, uric acid and cholesterol levels were predetermined, and the concentrations of glucose, uric acid and cholesterol levels are shown in Table 1. Then one drop (10 microliters) of each sample was added to 9 test strips (3 glucose test strips, 3 uric acid test strips and 3 cholesterol test strips). The test strips were kept for 1 minute, and after one minute, they were turned over to observe the color change in the observation window 7. The developed color was compared with the color chart provided in Experiment 2.

[0109] The results of the present invention indicate that the color block values ​​in the colorimetric results match the predetermined values. If the color block values ​​obtained from the colorimetric results match the predetermined values, the results are considered to be 100% matched. Based on the results of the present invention, the colorimetric test can successfully classify these predetermined biomarker values.

[0110] Table 1 Concentration of glucose, uric acid and cholesterol levels

[0111]

[0112] Example 7, in order to test the wider, higher range of biomarker concentrations that can be obtained in human blood, 2 ml of each sample 1 and sample 2 are transferred to two separate vials. By dissolving an appropriate amount of these biomarkers in the blood sample, the concentrations of glucose, uric acid and cholesterol are increased. The final concentrations are shown in Table 2. A drop (10 microliters) of each blood sample is added to 4 test strips. After 1 minute, the color development at the observation window 7 is checked and compared with the color card to evaluate the colorimetric results. The results match the known concentrations, and the colors fall into the correct color blocks, indicating that the colorimetric results are effective and accurate.

[0113] Table 2 Final concentration table

[0114]

[0115] Example 8, human blood samples from three volunteers were tested at two different times of the day: once before breakfast (fasting) and the other two hours after breakfast. One volunteer was a healthy individual with no record of hyperglycemia, hyperuricemia or high total cholesterol levels. Another volunteer had hyperuricemia for nearly two years, and the third volunteer had a history of hyperglycemia for nearly 20 years. For each test, a commercial analyzer (glucose meter, uric acid meter and total cholesterol meter) was used to analyze the blood samples from the finger pricks. At the same time, the samples were tested using the test strips of the present invention, and the results were compared, as shown in Table 3.

[0116] Table 3. Test concentrations of biomarkers correctly predicted by comparison with the standard color map of Experiment 2.

[0117]

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A whole blood visual colorimetric test strip for detecting multiple biomarkers, the whole blood visual colorimetric test strip is used to separate plasma from a drop of blood and detect target biomarkers, characterized in that: The whole blood visual colorimetric test strips include: Test strip structure: The test strip structure consists of a top protective layer, a bottom protective layer and an intermediate functional layer. The intermediate functional layer includes a dual-functional blood distribution layer, a plasma extraction layer and a detection layer; Dual-function blood distribution layer: impregnated with anti-interference material and hydrophilic material, used to eliminate interfering compounds and transfer blood vertically and evenly to the plasma extraction layer; Plasma Extraction Layer: Using vertical microfluidics and size-based capture capabilities, blood cells are captured in the plasma extraction layer, allowing for rapid extraction and transfer of plasma to the detection layer; Detection layer: used to produce color changes through the interaction of biomarkers in plasma with the reagent mixture; Reagent Cocktail: A reagent cocktail optimized for each biomarker, including glucose oxidase, uricase, cholesterol esterase, cholesterol oxidase, horseradish peroxidase, chromogen, enzyme protectant, coherent agent, plasma stabilizer, secondary anti-interference material, and surfactant.

2. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The test paper structure is also provided with a blood inlet and an observation window, and there are at least three key layers between the blood inlet and the observation window: a blood distribution layer, a plasma extraction layer and a detection layer: a dual-function blood distribution layer, a plasma extraction layer and a detection layer; the blood inlet is a hole arranged on the top protective layer, which is used for capillary blood sampling and transmission through a disposable capillary dropper, or includes a siphon blood unit integrated into the test strip, which is used to attract blood drops from the blood inlet to the blood distribution layer.

3. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The preferred substrate of the dual-functional blood distribution layer is a natural protein fiber, in particular a silk screen, which is sequentially impregnated with a main anti-interference material consisting of ascorbic acid oxidase, bilirubin oxidase, potassium hexacyanoferrate (II), phytic acid dodecasodium salt, and nitrilotriacetic acid; and an aqueous solution, which is a 0.1%-5% by weight aqueous mixture selected from the following materials: collagen powder, agarose, Triton X-114, polyvinyl pyrrolidone, polyacrylamide, polyvinyl alcohol, and Tween 80.

4. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The plasma extraction layer is located below the dual-functional blood distribution layer and is used to transfer blood droplets directly to the plasma extraction layer below. The plasma extraction layer is made of an asymmetric glass fiber membrane or an asymmetric polyethersulfone membrane with a thickness ranging from 100 to 800 microns and a water absorption capacity of 25-180 mg / cm3. The membrane structure can effectively capture blood cells and particles larger than 3 microns.

5. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The color developer is at least one of N-ethyl-N-(3-propylsulfonyl)aniline sodium salt (ALPS), N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt (TODB), 3,5-dichloro-2-hydroxybenzenesulfonate sodium, 2,4,6-tribromo-3-hydroxybenzoic acid (TBHB), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt (EHSPT / TOOS), 4-chlorophenol, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (F-DAOS), and N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt (ESPAS).

6. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The enzyme protectants used in the uric acid and glucose reagent mixtures are selected from the following materials: bis(sulfosuccinimidyl) suberate sodium salt (BS3), polyacrylic acid (PLA), polyglycerol, poly(2-hydroxyethyl methacrylate) (PHEMA), N,N'-methylenebisacrylamide (Bis), poly(acrylic acid-co-maleic acid), polyethylene glycol-co-polyglycolic acid (PEG-co-PGA), polyethylene imine, tartrazine, ethylene glycol bis(succinimidyl succinate) (EGS), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC); and the enzyme protectants used in the cholesterol reagent mixture are selected from the following materials: polyaniline, tartrazine, methyl vinyl ether, polyethylene imine, poly(methyl vinyl ethyl ether), polylactic acid-glycolic acid copolymer (PLGA), polylactic acid, polyglycerol, Eudragit RS100.

7. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The coherent agent in the uric acid, glucose and cholesterol reagent mixture is selected from the following compounds: poly (2-ethyl-2-oxazoline), 2-hydroxyethyl cellulose, pullulan, polyvinyl pyrrolidone, PEG6000, carrageenan, polyvinyl alcohol-vinyl acetate copolymer and polyethylene oxide (PEO).

8. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The plasma stabilizer is selected from materials including dextran, protein powder, starch, guar gum, polyvinyl pyrrolidone, alginate, polyvinyl alcohol, hydroxyethyl starch and hyaluronic acid.

9. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The secondary anti-interference materials are iodic acid, potassium bromate, iodate, sodium citrate, and triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid.

10. The whole blood visual colorimetric test strip for detecting multiple biomarkers according to claim 1, characterized in that: The reagent mixture is composed of: a glucose reagent is a buffer, and its components include glucose oxidase>10 ku / l, horseradish peroxidase>500 u / l, 0.1%-10% of 4-aminoantipyrine, 0.1%-10% of a color developer, 0.01%-8% of an enzyme protectant, 0.06%-12% of a coherent agent, 0.01%-6% of a plasma stabilizer, and 0.01%-6% of a secondary anti-interference material; a uric acid reagent mixture is a buffer, and its components include uricase>200 u / l, horseradish peroxidase>800 u / l, 0.1%-10% of 4-aminoantipyrine, 0.1%-10% of a color developer, 0.01%-8% of an enzyme protectant, 0.06%-12% of a coherent agent, 0.01%-6% of a plasma stabilizer, and 0.01%-8% of a secondary anti-interference material; The cholesterol reagent mixture is a buffered ethanol solution, and its ingredients include cholesterol esterase>200u / l, cholesterol oxidase>90u / l, horseradish peroxidase>120u / l, 0.1%-10% 4-aminoantipyrine, 0.1%-10% color developer, 0.01%-8% enzyme protectant, 0.03%-12% coherent agent, 0.01%-6% plasma stabilizer, 0.02%-6.2% surfactant, and 0.01%-4% secondary anti-interference material.

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