CN118191294B - A non-invasive uric acid colorimetric test paper and its manufacturing method and detection method - Google Patents
A non-invasive uric acid colorimetric test paper and its manufacturing method and detection method Download PDFInfo
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Abstract
The invention relates to a non-invasive uric acid colorimetric test paper, a manufacturing method and a detection method thereof, which comprises a hydrophilic liner and a hydrophobic liner, wherein the hydrophilic liner is fixedly arranged at a position close to one end of the hydrophobic liner; the hydrophilic pad is saturated with a mixed reagent comprising a color developing agent, a multifunctional modifier, uricase and horseradish peroxidase. Uric acid levels are detected using specific chromogenic agents and multifunctional modifiers using methods that combine enzymatic, oxidative and catalytic reactions. In solutions containing uric acid, three main consecutive reactions lead to a change in the color of the hydrophilic pad from colorless to blue. Higher concentrations of uric acid produce higher concentrations of H 2O2, forming higher concentrations of diamine-diimine Charge Transfer (CT) complexes, resulting in a more intense blue color. From the difference in blue intensity, uric acid levels in biological fluids, particularly in urine samples, can be determined. The uric acid can be subjected to noninvasive rapid field test, and the rapid real-time acquisition of uric acid level detection results is realized.
Description
Technical Field
The invention belongs to the technical field of medical detection equipment, and particularly relates to non-invasive uric acid colorimetric test paper, a manufacturing method thereof and a detection method thereof.
Background
Uric acid is the end product of purine metabolism in humans, with normally about one third of uric acid excreted through the gastrointestinal tract and the remaining two thirds excreted through urine; a condition known as hyperuricemia occurs when uric acid in urine is excessive. Hyperuricemia is not only associated with excessive uric acid levels in the blood (i.e., hyperuricemia), but also may be associated with gout disease, and may further have other independent important effects on human health; elevated uric acid levels in urine can lead to the formation of uric acid and calcium oxalate stones in the kidneys and bladder. Furthermore, sustained high levels of uric acid may also contribute to the development of Chronic Kidney Disease (CKD). Therefore, in addition to the ongoing effort to detect hyperuricemia, attention must be paid to the identification of hypouricemia and hyperuricemia (lower and higher uric acid levels in urine). By monitoring and treating uric acid levels in urine, healthcare professionals can better assess risk of kidney stone formation and kidney disease, thereby improving prognosis and overall health of patients.
The traditional method for measuring uric acid level in urine by clinical examination usually adopts a special urine biochemical analyzer, a urine sample collected on site or collected for 24 hours is introduced into equipment, a reagent mixture in a liquid form is added, and the change of light absorbance is measured by a spectrometer in the device; the patient to be detected needs to be kept for 24 hours to obtain a urine sample, refrigerated and sent to a pathology clinic within one hour, and a complex examination processing procedure is carried out in a laboratory to obtain an optimal detection result so as to ensure that the detection result is accurate and reliable; the urine biochemical analyzer is only configured in related professional places such as pathology clinics, is inconvenient to operate and long in time consumption, cannot realize on-site rapid inspection, and cannot easily repeat test operation; the on-site rapid test can rapidly obtain the detection result in real time, so that the treatment measures are taken in real time according to the detection result, for example, if the detection result shows that the uric acid level is high, patients are required to be recommended to pay attention to diet, take low-purine diet and exercise, keep body moisture, and repeat the detection after a few months besides the related drug treatment; for many common people, strict management of lifestyles is not easy to realize; rapid and immediate detection by home helps individuals track their daily diet and lifestyle impact on their health.
In the prior art, POCT detection devices and detection methods capable of rapidly detecting on site and rapidly obtaining detection results can only be used for POCT detection of blood uric acid level, but not for POCT detection of uric acid; while the prior art urine reagent strips are capable of qualitatively and semi-quantitatively detecting various analytes in urine, including white blood cells, urobilinogen, microalbumin, proteins, bilirubin, glucose, ascorbic acid, specific gravity, ketone bodies, nitrite, creatinine, pH, blood and calcium, which operate on a colorimetric (typically visual) basis, urine acid detection has also been missed; the prior art home POCT test strips have been commercialized for detecting different analytes in urine, but they have also missed the urine acid detection. For example, patent publication number CN116482090a, a new uric acid test paper, discloses a technical scheme that OPD is used as a color developing agent for uric acid detection, paper sheets containing OPD substrates are used as substrate sheets, the color change of DAP visible in the detection process is from colorless to yellow, the yellow color change condition of uric acid with different concentrations is not easy to clearly observe by naked eyes, and the change of light absorption can be detected by means of a spectrometer.
The clinical and hospital examinations of the prior art are expensive, time consuming, inconvenient to operate and cannot be repeated daily or infrequently. Therefore, there is a lack of a POCT test device in the prior art that can achieve rapid in-situ testing of uric acid.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention is directed to a non-invasive uric acid colorimetric test strip, a manufacturing method and a detection method thereof, which are used for detecting biological liquid samples, particularly urine samples; the method has the advantages of no need of going to clinical and hospital professional detection sites, low cost, less time consumption and convenient operation, and can realize non-invasive on-site rapid detection of uric acid, thereby rapidly obtaining urine acid level detection results in real time.
The technical scheme adopted by the invention is as follows:
the first technical scheme is as follows: the noninvasive uric acid colorimetric test paper comprises a hydrophilic liner and a hydrophobic liner, wherein the hydrophilic liner is of a rectangular structure, the hydrophobic liner is of a strip-shaped structure, and the hydrophilic liner is fixedly arranged on the upper surface of the hydrophobic liner at a position close to one end;
the hydrophilic pad is provided with a mixed reagent; the mixed reagent is used for continuous selective enzyme reaction and catalytic oxidation reaction when exposed to uric acid samples;
the mixed reagent at least comprises uricase, horseradish peroxidase, a color developing agent and a multifunctional modifier;
the uricase is used for selectively capturing uric acid molecules and then converting the uric acid molecules into 5-ureido hydantoin, carbon dioxide and hydrogen peroxide serving as an oxidant;
The color developing agent is used for carrying out single-electron oxidation reaction with hydrogen peroxide, firstly generating a free radical cation, and then forming a blue diamine-diimine charge transfer compound;
the horseradish peroxidase is used for catalyzing and promoting a single-electron oxidation reaction of the color developing agent in the hydrophilic liner;
The multifunctional modifier is used for fixing uricase and horseradish peroxidase in the hydrophilic pad through crosslinking and grafting, so as to increase the stability of the color developing agent and the distribution uniformity of pigment in the hydrophilic pad, and the wettability of the hydrophilic pad.
Further, the multifunctional modifier is one or a mixture of more of D (+) -trehalose dihydrate, gelatin, chitosan, glutaraldehyde, methoxypolyethylene glycol propionaldehyde, ethylenediamine tetraacetic acid, polyethylene glycol octylphenyl ether, polypropylene glycol and glycerol.
Further, the material of the hydrophilic liner is absorbent paper, filter paper or chromatographic paper.
Further, the material of the hydrophobic liner is a hydrophobic material.
Further, the color-developing agent is 3,3', 5' -tetramethyl biphenyl diamine.
Further, the area of the hydrophilic liner is 16-49mm 2, and the color developing agent is saturated and impregnated in the hydrophilic liner.
Further, the hydrophilic pad is saturated with 6-30ul of the mixing agent.
The second technical scheme is as follows: provided is a method for manufacturing noninvasive uric acid colorimetric test paper, which is used for manufacturing noninvasive uric acid colorimetric test paper and comprises the following manufacturing steps:
s01, preparing rectangular hydrophilic gaskets and strip-shaped hydrophobic gaskets;
s02, preparing a mixed reagent, impregnating a hydrophilic liner by the mixed reagent, and drying;
Preparing a mixed reagent by a buffer solution with the pH of 8-9, 20-140u/mL uricase, 0.1-0.3mg/mL horseradish peroxidase, 3', 5' -tetramethyl biphenyl diamine with the concentration of 5-25mM and a multifunctional modifier with the concentration of 10-30%; saturated impregnation of the hydrophilic pad with the mixed reagent, drying and sealing storage;
Or preparing a solution I, a solution II, a solution III and a solution IV; dissolving chitosan powder in diluted acetic acid to obtain chitosan solution I with the concentration of 0.5-3 mg/ml; dissolving 3,3', 5' -tetramethyl biphenyl diamine solution in alcohol solution to obtain solution II with concentration of 5-25 mM; dissolving horseradish peroxidase in a buffer solution with the pH value of 6-7 to obtain a solution III; dissolving uricase in a buffer solution with the pH value of 8-9 to obtain a solution IV; sequentially soaking the hydrophilic pad in the solution I, the solution II, the solution III and the solution IV, respectively airing, and hermetically storing;
s03, fixedly bonding a hydrophilic pad to one end of a hydrophobic pad;
s04, preparing the noninvasive uric acid colorimetric test paper.
The third technical scheme is as follows: another method for manufacturing the noninvasive uric acid colorimetric test paper is provided, and the method is used for manufacturing the noninvasive uric acid colorimetric test paper and comprises the following manufacturing steps:
s01, preparing hydrophilic paper raw materials and hydrophobic sheet raw materials;
s02, manufacturing hydrophilic paper raw materials into a plurality of rectangular hydrophilic liners with the side length of 4-7 mm through a punching and shearing cutting process;
S03, manufacturing a plurality of strip-shaped hydrophobic gaskets with the thickness of 4-7 mm multiplied by 50-100 mm from the hydrophobic sheet material through a punching and shearing cutting process;
s04, dissolving uricase in a buffer solution to prepare uricase solution;
S05, dissolving a proper amount of horseradish peroxidase in a buffer solution to prepare a horseradish peroxidase solution;
S06, dissolving 3,3', 5' -tetramethyl benzidine in an alcohol solution of methanol or ethanol to prepare a color reagent solution;
S07, dissolving chitosan powder in diluted acetic acid to prepare chitosan solution;
s08, taking uricase solution, horseradish peroxidase solution, color developing agent solution and chitosan solution, and mixing to prepare a mixed solution;
S09, depositing 6-30ul of mixed solution on each hydrophilic pad;
S10, incubating the hydrophilic pad for 15-90 minutes at the temperature of 25-40 ℃;
S11, drying to immobilize the reagent;
S12, fixedly bonding the hydrophilic pad to one end of the hydrophobic pad;
s13, preparing the noninvasive uric acid colorimetric test paper.
The fourth technical scheme is as follows: the detection method of the noninvasive uric acid colorimetric test paper comprises the following manufacturing steps of:
P01, presetting a plurality of different blue depth levels according to the color change condition of the color developing agent in the process of changing from colorless to blue;
p02, preparing uric acid samples by using buffer solution or artificial urine;
p03, dropping uric acid sample onto hydrophilic pad of non-invasive uric acid colorimetric test paper, or immersing hydrophilic pad into uric acid sample for 1 second;
p04, uric acid concentration is different, and blue intensity is also different; uric acid concentration of uric acid samples is determined by visual comparison of color with a standard color chart or by computer vision digital analysis.
The beneficial effects of the invention are as follows:
A noninvasive uric acid colorimetric test paper, a manufacturing method thereof and a detection method thereof comprise a hydrophilic pad and a hydrophobic pad, wherein the hydrophilic pad is fixedly arranged on the upper surface of the hydrophobic pad at a position close to one end; saturated dipping a mixed reagent comprising a color reagent, a multifunctional modifier, uricase and horseradish peroxidase on a hydrophilic liner, adopting a method of combining enzymatic reaction, oxidation reaction and catalytic reaction, and detecting uric acid level by using a specific color reagent and the multifunctional modifier; in the presence of a solution containing uric acid, three main consecutive reactions lead to a change in the color of the hydrophilic pad from colorless to blue; high concentrations of uric acid produce high concentrations of H 2O2, which in turn form high concentrations of diamine-diimine Charge Transfer (CT) complexes, referred to herein as TMB (CT-OX), resulting in a deeper blue color. Uric acid at different concentrations, which has different blue intensities, is visually detectable. Obvious color differences can be observed by naked eyes, particularly no, lower limit and upper limit of uric acid level in urine samples; can realize noninvasive on-site rapid detection of uric acid, thereby rapidly obtaining urine acid level detection results in real time.
The noninvasive uric acid colorimetric test paper provided by the invention has the advantages of low cost, simplicity in operation and easiness in use, and the POCT detection method is free from going to clinical and hospital professional detection places, is low in cost, less in time consumption and convenient to operate, can be repeated for many times at any place every day, and can track and monitor the influence of the life style of a patient on the uric acid level.
Drawings
FIG. 1 is a schematic diagram of the structure of a non-invasive uric acid colorimetric test strip of the present invention;
FIG. 2 is a schematic diagram of a uric acid testing solution of the non-invasive uric acid colorimetric test strip of the present invention showing two testing methods: dropping uric acid onto the hydrophilic pad or immersing the hydrophilic pad in the uric acid sample for 1 second;
FIG. 3 is a schematic diagram of the present invention showing the chemical steps and mechanisms for detecting uric acid using a non-invasive uric acid colorimetric test strip;
FIG. 4 is a linear schematic of the correlation between uric acid concentration and delta (gray scale) of the non-invasive uric acid colorimetric test paper of the present invention upon addition of uric acid solutions of different concentrations;
FIG. 5 is a schematic of the blue depth scale of six different uric acid levels of the non-invasive uric acid colorimetric test strips of the present invention upon addition of different concentrations of uric acid solutions.
Detailed Description
The following description of the technical solution in one embodiment of the present invention is clear and complete, and it is obvious that the described embodiment is only a part of embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art without the inventive effort, are intended to be within the scope of the present invention.
The invention aims to provide a noninvasive test paper sensor which is low in cost and friendly to users and is used for detecting uric acid in urine and contributing to human health, and particularly provides a noninvasive test paper sensor which enables patients to test conveniently and repeatedly, so that life style and medicines of the patients can be effectively managed to improve health. The non-invasive bar sensor of the present invention allows a patient individual to perform and repeat tests at any time and place, helping the patient to reasonably control and manage his lifestyle and medication use to better maintain health. It is an object of the present invention to provide a home point of care (POCT) device enabling the individual of the patient to monitor his daily habits at any time for health effects.
The invention aims to solve the problems in the prior art, and the specific planning scheme is as follows:
The first technical scheme provides noninvasive uric acid colorimetric test paper which is used as a dry chemical test paper sensor for detecting uric acid in biological fluid, in particular to a dry chemical test paper sensor for detecting uric acid in urine; or as a dry-chemical strip test sensor for detecting low uric acid urine and high uric acid urine.
The non-invasive uric acid colorimetric test strip of the present invention comprises a hydrophilic pad 1 for fixing a reagent mixture and a hydrophobic pad 2 for manipulating a test strip sensor, as shown in fig. 1; the hydrophilic liner 1 is fixedly arranged on the upper surface of the hydrophobic liner 2 at a position close to one end; the hydrophilic liner 1 has a rectangular structure, and the material of the hydrophilic liner 1 is absorbent paper, chromatographic paper, filter paper or chromatographic paper. The hydrophobic liner 2 has a long strip structure, the material of the hydrophobic liner 2 is a hydrophobic material composed of a hydrophobic polymer, such as plastic, and PVC (polyvinyl chloride) may be particularly preferred as the material of the hydrophobic liner 2.
Disposing a mixed reagent (i.e., reagent mixture) on a hydrophilic pad (hereinafter hydrophilic pad, hydrophilic pad); the mixed reagent at least comprises a color development agent, a multifunctional modifier (multifunctional modifier, nonreactive stabilizer, multifunctional stabilizer), uricase and horseradish peroxidase;
Hydrogen peroxide can be produced by enzymatic reaction with uric acid by uricase; that is, uric acid molecules can be selectively captured by uricase, which converts them to 5-allophycocyanin, carbon dioxide (CO 2) and hydrogen peroxide (H 2O2) as an oxidizing agent.
The developer can undergo an oxidation reaction with hydrogen peroxide (H 2O2) to first generate a radical cation and then form a blue diamine-diimine Charge Transfer (CT) complex. And blue colors of different depths are displayed according to the concentration of the hydrogen peroxide; specific color developing agent is adopted as the color developing agent, and 3,3', 5' -tetramethyl benzidine (TMB) can be adopted.
The oxidation reaction of the color developing agent can be catalyzed and accelerated by horseradish peroxidase; i.e., single electron oxidation of TMB in an aqueous pad can be catalyzed and promoted by horseradish peroxidase (HRP).
The area of the hydrophilic pad is 16-49mm 2, and the color developing agent is saturated and immersed in the hydrophilic pad. In particular, 6 to 30ul of the color-developing agent can be saturated and immersed in a hydrophilic liner of 16 to 49mm 2.
The multifunctional modifier can fix uricase and horseradish peroxidase in the hydrophilic pad through crosslinking and grafting, so that the stability of the color developing agent and the distribution uniformity of pigment in the hydrophilic pad as well as the wettability of the hydrophilic pad are improved.
The multifunctional modifier is one or more of D (+) -trehalose dihydrate, gelatin, chitosan, glutaraldehyde, methoxy polyethylene glycol propionaldehyde, ethylenediamine tetraacetic acid, polyethylene glycol octyl phenyl ether, polypropylene glycol and glycerol.
The dry chemical and colorimetry combined principle is adopted to manufacture dry chemical strip test paper of POCT uric acid, and a series of enzyme reactions, catalysis, single electron oxidation reactions and balanced charge transfer complex formation reactions are utilized to respond to a detection target; uric acid levels are detected by a combination of enzymatic, oxidative and catalytic reactions using specific chromogenic agents and multifunctional stabilizers. The noninvasive uric acid colorimetric test strip of the invention has a strip-shaped test strip structure, and is fixedly attached to a hydrophobic pad through a hydrophilic pad impregnated with a reagent. The preferred material for the hydrophobic liner is a hydrophobic material, preferably PVC; the preferred major component of the hydrophilic backing is absorbent paper, preferably chromatographic paper or filter paper. The reagent mixture is saturated in the hydrophilic pad and then dried, that is, the reagent mixture is saturated in the pad and dried to stabilize fixation. A mixed reagent is formulated and optimized to obtain optimal results, the mixed reagent containing two enzymes: uricase and horseradish peroxidase; (one of the two enzymes is uricase, which is used for capturing specific uric acid, namely, for selectively interacting with uric acid, and the other is horseradish peroxidase, which is used as a dye reagent oxidation catalyst, namely, for catalytic oxidation of a color developing agent), a color developing agent (namely, a dye reagent, 3', 5' -tetramethyl benzidine or TMB) and a multifunctional modifier. The multifunctional modifier may be one or a combination of two or more of the following materials: d (+) -trehalose dihydrate, gelatin, chitosan, glutaraldehyde, methoxypolyethylene glycol propionaldehyde, ethylenediamine tetraacetic acid (acetic acid), triton X-100, polypropylene alcohols (polypropylene glycols) and glycerol; the multifunctional modifier has various functions, such as enhancing enzyme immobilization by crosslinking, improving the stability of the coloring agent, ensuring uniform color distribution and enhancing wettability. The specific volume of the impregnating agent mixture in the hydrophilic pad is within the range of from 6 to 30ul of the mixed agent in the hydrophilic pad having an area of from 16 to 49mm 2 to achieve saturated impregnation. The mixed reagent is stably fixed in the hydrophilic pad after being dried, thereby forming the dry test strip paper of POCT uric acid which is easy to operate.
The mixed reagent is integrated and stably fixed in the hydrophilic liner, so that the (POCT) colorimetric uric acid dry chemical strip test paper convenient for on-site rapid detection application is formed; the colorimetric uric acid dry chemical strip test strip, when contacted with a uric acid-containing sample, causes the color of the hydrophilic pad to change from colorless to blue by three different sequential reactions, which are shown in the following three equations:
Reaction formula one:
Reaction formula II:
Reaction III:
First, uric acid molecules are captured by uricase catalyzed selective enzyme reactions immobilized within a hydrophilic pad. This interaction converts uric acid molecules into 5-ureidohydantoin, carbon dioxide (CO 2), and hydrogen peroxide (H 2O2) as an oxidizing agent. Subsequently, a one-electron oxidation reaction occurs between the oxidizing agent in the previous step and the chromogenic reagent 3,3', 5' -Tetramethylbenzidine (TMB) in the hydrophilic pad. This second oxidation reaction is catalyzed and promoted by HRP catalyst also present in the hydrophilic liner. The result is the formation of the colorless radical cationic form of pigment TMB ·+. Finally, the rapid equilibration reaction produces a blue diamine-diimine Charge Transfer (CT) complex known as TMB (CT-OX). This complex is responsible for the formation of blue color within the hydrophilic backing. Higher uric acid concentrations give more TMB (CT-OX), resulting in a more pronounced and darker blue hue. I.e. the intensity of the blue color varies with different uric acid concentrations so that visual detection with the naked eye is possible.
The selective interaction between uric acid and uricase immobilized within the hydrophilic pad of the sensor results in the production of 5-ureidohydantoin, carbon dioxide (CO 2), and hydrogen peroxide (H 2O2). Among them, hydrogen peroxide is an oxidizing agent that oxidizes 3,3', 5' -Tetramethylbenzidine (TMB), a developer immobilized in a hydrophilic pad. The latter reaction is slow at ambient conditions; thus, the reaction was further accelerated by horseradish peroxidase (HRP) catalyst immobilized in a hydrophilic pad. After oxidation, a Charge Transfer (CT) complex of TMB forms, which is blue in color, known as TMB (CT-ox). High concentrations of uric acid produce high concentrations of H 2O2, which in turn form high concentrations of TMB (CT-OX), resulting in a deeper and deeper blue color. Uric acid at different concentrations, which has different blue intensities, is visually detectable. Obvious color differences, particularly the absence, lower and upper limits of uric acid levels in urine samples, can be observed with the naked eye.
In-home point of care (POCT) urine dipstick tests, although used to detect various analytes in urine, do not involve the detection of uric acid. Methods for clinical and hospital use of urine levels typically employ specialized urine biochemical analyzers that are costly, time consuming, uncomfortable to test, and do not facilitate daily or intermittent repetition. According to the technical scheme, a point-of-care test (POCT) method combining dry chemistry and colorimetry is applied to the development of uric acid detection, and an economical and practical POCT method easy to use is provided and is used for detecting uric acid in urine. The method is simple to operate and can be repeated a plurality of times in a day, so that individuals of patients can monitor the influence of life style on uric acid level in urine, and no matter where the individuals are.
The dry chemical and colorimetry combined principle is adopted to manufacture the dry test strip paper for POCT uric acid, and enzymes used in the mixed reagent are uricase (also called uricase) and peroxidase. Uricase is used to specifically and selectively react with uric acid. And (3) oxidizing the color developing agent by taking horseradish peroxide as a catalyst. Wherein 3,3', 5' -Tetramethylbenzidine (TMB) is used as a color developer. Uric acid interacts with uricase immobilized within the hydrophilic pad, resulting in the production of 5-ureidohydantoin, CO 2, and H 2O2. The H 2O2 produced is an oxidizing agent that oxidizes TMB to free radical cations (TMB ·+) and then rapidly forms a diamine-diimine Charge Transfer (CT) complex, referred to herein as TMB (CT-OX). TMB is colorless and TMB (CT-OX) is blue. High concentrations of uric acid produce more H 2O2 and thus more TMB (CT-OX), resulting in a darker blue color. The multifunctional modifier used in this experiment has a number of advantages, including better immobilization of the reagent, especially the enzyme on the hydrophilic pad, higher developer stability, uniform color distribution within the hydrophilic pad, and pH stability. Better immobilization of the reagent, in particular increasing the stability of the enzyme and the color-developing agent, and maintaining its activity on the hydrophilic pad.
The second technical scheme provides a manufacturing method of the noninvasive uric acid colorimetric test paper, which is used for manufacturing the noninvasive uric acid colorimetric test paper and comprises the following manufacturing steps of:
s01, preparing hydrophilic paper raw materials and hydrophobic sheet raw materials;
s02, manufacturing hydrophilic paper raw materials into a plurality of rectangular hydrophilic liners with the side length of 4-7 mm through a punching and shearing cutting process;
s03, manufacturing a plurality of strip-shaped hydrophobic gaskets with the thickness of (4-7) mm and the thickness of (50-100) mm from the hydrophobic sheet material through a punching and shearing cutting process;
s04, dissolving uricase in a buffer solution to prepare uricase solution;
S05, dissolving a proper amount of horseradish peroxidase in a buffer solution to prepare a horseradish peroxidase solution;
S06, dissolving 3,3', 5' -tetramethyl benzidine in an alcohol solution of methanol or ethanol to prepare a color reagent solution;
S07, dissolving chitosan powder in diluted acetic acid to prepare chitosan solution;
s08, taking uricase solution, horseradish peroxidase solution, color developing agent solution and chitosan solution, and mixing to prepare a mixed solution;
S09, depositing 6-30ul of mixed solution on each hydrophilic pad;
S10, incubating the hydrophilic pad for 15-90 minutes at the temperature of 25-40 ℃;
S11, drying to immobilize the reagent;
S12, fixedly bonding the hydrophilic pad to one end of the hydrophobic pad;
s13, preparing the noninvasive uric acid colorimetric test paper.
Further, in step S08, a multifunctional modifier is also added to prepare a mixed solution;
The multifunctional modifier is one or more of D (+) -trehalose dihydrate, gelatin, chitosan, glutaraldehyde, methoxypolyethylene glycol propionaldehyde, edetic acid and glycerol.
The third technical scheme is as follows:
s01, preparing rectangular hydrophilic gaskets and strip-shaped hydrophobic gaskets;
s02, preparing a mixed reagent, impregnating a hydrophilic liner by the mixed reagent, and drying;
Preparing a mixed reagent by a buffer solution with the pH of 8-9, 20-140u/mL uricase, 0.1-0.3mg/mL HRP, TMB with the concentration of 5-25mM and a multifunctional modifier with the concentration of 10-30%; saturated impregnation of the hydrophilic pad with the mixed reagent, drying and sealing storage;
Or preparing a solution I, a solution II, a solution III and a solution IV; dissolving chitosan powder in diluted acetic acid to obtain chitosan solution I with the concentration of 0.5-3 mg/ml; dissolving TMB solution in alcohol solution to obtain solution II with the concentration of 5-25 mM; dissolving HRP in a buffer solution with pH of 6-7 to obtain a solution III; dissolving uricase in a buffer solution with the pH value of 8-9 to obtain a solution IV; sequentially soaking the hydrophilic pad in the solution I, the solution II, the solution III and the solution IV, respectively airing, and hermetically storing;
s03, fixedly bonding a hydrophilic pad to one end of a hydrophobic pad;
s04, preparing the noninvasive uric acid colorimetric test paper.
The fourth technical scheme is as follows: the detection method of the noninvasive uric acid colorimetric test paper adopts the noninvasive uric acid colorimetric test paper, and comprises the following manufacturing steps:
P01, presetting a plurality of different blue depth levels according to the color change condition of the color developing agent in the process of changing from colorless to blue;
p02, preparing uric acid samples by using buffer solution or artificial urine;
p03, dropping uric acid sample onto hydrophilic pad of non-invasive uric acid colorimetric test paper, or immersing hydrophilic pad into uric acid sample for 1 second;
p04, uric acid concentration is different, and blue intensity is also different; uric acid concentration of uric acid samples is determined by visual comparison of color with a standard color chart or by computer vision digital analysis.
In the actual operation process, in the step P01, six different blue depth levels may be preset according to the color change condition in the process of changing the color of the color developer from colorless to blue, or more or less different blue depth levels may be preset;
In step P04, after the color change of the hydrophilic pad is stable, comparing with preset six different blue depth levels, or comparing with more different blue depth levels, or comparing with fewer different blue depth levels, and judging and confirming the uric acid concentration of the uric acid sample.
The invention relates to a non-invasive uric acid colorimetric test paper, a manufacturing method and a detection method thereof, which comprises a hydrophilic liner and a hydrophobic liner, wherein the hydrophilic liner is fixedly arranged on the upper surface of the hydrophobic liner at a position close to one end; saturated dipping a mixed reagent comprising a color reagent, a multifunctional stabilizer, uricase and horseradish peroxidase on a hydrophilic liner, adopting a method of combining enzymatic reaction, oxidation reaction and catalytic reaction, and detecting uric acid level by using a specific color reagent and the multifunctional stabilizer; the color of the hydrophilic pad is changed from colorless to blue by three consecutive reactions in the presence of a solution containing uric acid; high concentrations of uric acid produce high concentrations of H 2O2, which in turn form high concentrations of TMB (CT-OX), resulting in a deeper and deeper blue color; the three consecutive reactions are shown in the following three equations:
Reaction formula one:
Reaction formula II:
Reaction III:
Uric acid at different concentrations, which has different blue intensities, is visually detectable. Obvious color differences can be observed by naked eyes, particularly no, lower limit and upper limit of uric acid level in urine samples; can realize noninvasive on-site rapid detection of uric acid, thereby rapidly obtaining urine acid level detection results in real time.
The noninvasive uric acid colorimetric test paper provided by the invention has the advantages of low cost, simplicity in operation and easiness in use, and the POCT detection method is free from going to clinical and hospital professional detection places, is low in cost, less in time consumption and convenient to operate, can be repeated for many times at any place every day, and can track and monitor the influence of the life style of a patient on the uric acid level.
Embodiment one:
Example one embodiment of the invention according to the proposed solution, a mixed reagent, i.e. a reagent mixture, for preparing a non-invasive uric acid colorimetric test strip was tested, and the specific procedure was as follows:
The raw materials for preparing the reagent mixture are as follows: a buffer solution having a pH in the range of 8 to 9; 20-140u/mL uricase, 0.1-0.3mg/mL HRP, and TMB at a concentration of 5-25mM, and 10-30% (v/v) of a multifunctional modifier (multifunctional modifier). Multifunctional modifiers are materials that do not interfere with the primary reaction and provide several benefits, including uniform distribution of color within the hydrophilic pad, improved enzyme immobilization and stability, and increased wettability. They act as stabilizers, surfactants and complexing agents simultaneously. The multifunctional modifier adopts one or a combination of two or more of the following materials: d (+) -trehalose dihydrate, gelatin, chitosan, glutaraldehyde, methoxypolyethylene glycol propionaldehyde, ethylenediamine tetraacetic acid, triton X-100, polyglycerol ether, and glycerol. The buffer solution may be selected from phosphate buffer, british-robinson buffer, and citric acid buffer, among which phosphate buffer is most preferred. The hydrophilic pad is impregnated and saturated with the above mixture, dried, and then stored in a sealed container with a desiccant.
Embodiment two:
Example two according to the inventive concept of the proposed solution, a hydrophilic pad for preparing a non-invasive uric acid colorimetric test paper was tested, and the specific operation procedure is as follows:
Two different sets of reagent mixtures were prepared, labeled "Mixture I" and "Mixture II", respectively. "Mixture I" consists of buffer solution, uricase, HRP and multifunctional modifier, "Mixture II" consists of chromogenic reagent (specifically 3,3', 5' -tetramethylbenzidine) and alcoholic solution (alcoholic solution preferably methanol or ethanol solution). The hydrophilic pad is impregnated with "Mixture I" and "Mixture II" in this order. First, the hydrophilic pad is impregnated with a hydrophilic pad "Mixture II" and dried. Then, the hydrophilic pad is impregnated with "Mixture I" and dried. The drying process is carried out at a temperature of 25-75 ℃. According to the planning scheme, the ideal operation time of each step is 1-10 minutes.
Embodiment III:
Example three the inventive concept according to the proposed solution, another hydrophilic pad for preparing a non-invasive uric acid colorimetric test paper was tested as follows: the mixed reagent is added layer by layer onto the hydrophilic pad.
Four different impregnating solutions were prepared:
and I) dissolving chitosan powder in diluted acetic acid to obtain chitosan solution I with the concentration of 0.5-3 mg/ml.
II) dissolving TMB solution in alcohol solution to obtain solution II with concentration of 5-25 mM; the alcohol solution may be a methanol or ethanol solution.
III) HRP was dissolved in a buffer pH 6-7 to prepare a HRP solution at a concentration of 0.1-0.6 mg/ml. Solution III was obtained.
IV) dissolving uricase in a buffer solution with pH of 8-9 to generate uricase solution with activity of 20-140U/ml per volume. Obtaining solution IV.
The hydrophilic pad is sequentially saturated with the four solutions in four steps: solution I, solution II, solution III, solution IV. Firstly, soaking a hydrophilic pad into the solution I and then airing; soaking the solution II and airing; then soaking the solution III for airing; then soaking the solution IV and airing, and finally storing the solution IV in a closed container with a drying agent.
And the noninvasive uric acid colorimetric test paper is prepared according to the invention conception of the planned technical scheme.
Embodiment four:
example four according to the inventive concept of the planned technical scheme, preparing uric acid samples; the specific operation process is as follows:
uric acid standard solutions were prepared in buffer solutions with varying concentrations, covering uric acid concentrations of 0.0mM (control sample), 0.02mM, 0.1mM, 0.4mM, 1.3mM and 4.5mM, representing low to high levels of uric acid. Sampling 3-20 μl uric acid sample to meet detection requirements and obtain detection results.
Fifth embodiment:
example five according to the inventive concept of the planned technical scheme, preparing uric acid samples; the specific operation process is as follows:
Using another experimental approach, a reference similar experiment was performed in which uric acid standard samples including uric acid concentrations of 0.0mM (control sample), 0.02mM, 0.1mM, 0.4mM, 1.3mM, and 4.5mM were prepared using artificial urine solutions.
Example six:
Example six according to the inventive concept of the planned technical scheme, test verification of uric acid level was performed by the hydrophilic pad for preparing the non-invasive uric acid colorimetric test paper and the non-invasive uric acid colorimetric test paper (test strip) prepared in example three; the hydrophilic pad of the non-invasive uric acid colorimetric test paper prepared in example three was dropped with each uric acid sample prepared in examples four and five, as shown in fig. 2.
In the experimental operation process, a uric acid sample (3-20 mu l of uric acid sample with smaller amount as described in experiment 4 and experiment 5) is dripped into a hydrophilic liner through a micropipette, so that a detection result is obtained; therefore, the hydrophilic pad of the noninvasive uric acid colorimetric test paper is immersed into a urine sample in the daily use process, so that the response of the test paper sensor is sufficiently triggered, then the test paper is taken out and put on white napkin paper or white paper to obtain a result more reliably, and the subsequent expansion of the application range of detection is facilitated.
In the experimental process, a gray value linear graph is drawn according to the test result, and as shown in fig. 3, the test strips treated by uric acid with different concentrations generate obvious color change. These color differences can be observed by the naked eye. The response speed is high, and the color changes once uric acid samples reach the hydrophilic pad. In all cases, the color change stabilized in less than 2 minutes, sufficient for visual inspection or digital analysis. This color stability provides a significant advantage because it reduces the time difference that an individual needs to complete testing and comparing colors. It is noted that the examples containing the multifunctional modifier have longer color stability. The noninvasive uric acid colorimetric test strips of the present invention are capable of detecting uric acid levels as low as 0.02mM and as high as 4.5mM uric acid concentrations. Because uric acid concentrations exceeding 4.2mM over 24 hours can lead to the risk of kidney stones, gout and other kidney dysfunction, the noninvasive uric acid colorimetric test strips of the present invention provide for routine monitoring of uric acid levels and maintenance of physical health. In addition, it has been found experimentally that the preparation of the non-invasive uric acid colorimetric test strips of the present invention can be accomplished in one step using a single impregnating solution, thereby effectively reducing the time and cost of the manufacturing process.
FIG. 4 shows the detection response effect of the non-invasive uric acid colorimetric test strips of the invention and illustrates the visual colorimetric results obtained in our experiments. Photographs were taken using an iPhone 13 camera placed in parallel over a strip of colorimetric paper on a white paper background. Although solutions without uric acid do not change the color on the pad, solutions of different concentrations of uric acid exhibit blue intensities at different depths. The color difference due to the difference in uric acid concentration can be observed by naked eyes. In addition, color/density calibration can be extended later to make reference color charts. In addition, the digital analysis technology can be expanded later, and the method can be used for quantitative measurement of uric acid in more biological fluids, particularly urine. The analysis can be performed by computer software or a smart phone after expansion. Color analysis is performed, for example, using ImageJ software to evaluate gray values, particularly average gray values. Then, the average gradation value of 0.0mM (no uric acid) was subtracted from the average gradation value of each color to obtain Δ (gradation value). The correlation between uric acid concentration and delta (gray value) was evaluated, and as shown in fig. 5, the linear correlation between the logarithm of uric acid concentration and delta (gray value) gave a corresponding color in addition to gray color for comparison.
A linear correlation was also observed between the logarithm of uric acid concentration and delta (gray scale value), with an R2 value of 0.9379. These results indicate that the logarithm of uric acid concentration is linearly related to Δ (gray scale value) calculated by color analysis. These correlations are useful in developing more quantitative analyses of uric acid levels in biological samples such as urine.
In example six, six blue intensity levels were finally determined, as shown in FIG. 5, with the dark blue indicating higher uric acid concentrations, representing different uric acid concentration levels from 0.0mM to 4.5mM, respectively.
In the actual operation process, a plurality of different blue depth levels can be preset according to the color change condition of the color developing agent in the process of changing from colorless to blue;
The uric acid concentration is different, and the intensity of blue is also different. The uric acid concentration of the uric acid sample can be determined by comparing colors with a color chart through naked eyes, and the uric acid concentration can be determined through digital analysis and a computer vision system along with development and perfection of an intelligent control program and an operation system.
The invention is not limited to the above-described alternative embodiments, and any person who may derive other various forms of products in the light of the present invention, however, any changes in shape or structure thereof, all falling within the technical solutions defined in the scope of the claims of the present invention, fall within the scope of protection of the present invention.
Claims (6)
1. A non-invasive uric acid colorimetric test strip, characterized in that: the hydrophilic pad is of a rectangular structure, the hydrophobic pad is of a strip-shaped structure, and the hydrophilic pad is fixedly arranged on the upper surface of the hydrophobic pad at a position close to one end;
The hydrophilic liner is prepared by the following steps: four different impregnating solutions were prepared; dissolving chitosan powder in diluted acetic acid to obtain chitosan solution I with the concentration of 0.5-3 mg/ml; dissolving TMB solution in alcohol solution to obtain solution II with the concentration of 5-25 mM; the alcohol solution is methanol or ethanol solution; dissolving HRP in buffer solution with pH of 6-7 to prepare HRP solution with concentration of 0.1-0.6 mg/ml; obtaining a solution III; dissolving uricase in a buffer solution with the pH value of 8-9 to generate uricase solution with the activity of 20-140U/ml per volume; obtaining a solution IV; soaking the hydrophilic pad in the solution I and then airing; soaking the solution II and airing; then soaking the solution III for airing; soaking the solution IV, airing, and finally storing in a sealed container with a drying agent;
the hydrophilic pad is provided with a mixed reagent; the mixed reagent is used for continuous selective enzyme reaction and catalytic oxidation reaction when exposed to uric acid samples;
The mixed reagent at least comprises uricase, horseradish peroxidase, a color developing agent and a multifunctional modifier, and the multifunctional modifier is chitosan;
the uricase is used for selectively capturing uric acid molecules and then converting the uric acid molecules into 5-ureido hydantoin, carbon dioxide and hydrogen peroxide serving as an oxidant;
The color developing agent is used for carrying out single-electron oxidation reaction with hydrogen peroxide, firstly generating a free radical cation, and then forming a blue diamine-diimine charge transfer compound;
the horseradish peroxidase is used for catalyzing and promoting a single-electron oxidation reaction of the color developing agent in the hydrophilic liner;
The multifunctional modifier is used for fixing uricase and horseradish peroxidase in the hydrophilic pad through crosslinking and grafting, so as to increase the stability of the color developing agent and the distribution uniformity of pigment in the hydrophilic pad, and the wettability of the hydrophilic pad.
2. The non-invasive uric acid colorimetric test strip of claim 1, wherein: the hydrophilic pad is made of absorption paper, filter paper or chromatographic paper.
3. The non-invasive uric acid colorimetric test strip of claim 1, wherein: the material of the hydrophobic liner is a hydrophobic material.
4. The non-invasive uric acid colorimetric test strip of claim 1, wherein: the area of the hydrophilic pad is 16-49mm 2.
5. A method for manufacturing a non-invasive uric acid colorimetric test strip according to any one of claims 1 to 4, characterized in that: the method comprises the following manufacturing steps:
Preparing a solution I, a solution II, a solution III and a solution IV; dissolving chitosan powder in diluted acetic acid to obtain chitosan solution I with the concentration of 0.5-3 mg/ml; dissolving 3,3', 5' -tetramethyl benzidine solution in alcohol solution to obtain solution II with the concentration of 5-25 mM; dissolving horseradish peroxidase in a buffer solution with the pH value of 6-7 to obtain a solution III; dissolving uricase in a buffer solution with the pH value of 8-9 to obtain a solution IV; sequentially soaking the hydrophilic pad in the solution I, the solution II, the solution III and the solution IV, respectively airing, and hermetically storing;
fixedly bonding a hydrophilic pad to one end of a hydrophobic pad;
and (5) preparing the noninvasive uric acid colorimetric test paper.
6. A method for detecting a non-invasive uric acid colorimetric test strip using the non-invasive uric acid colorimetric test strip according to any one of claims 1 to 4, characterized in that: the method comprises the following manufacturing steps:
P01, presetting a plurality of different blue depth levels according to the color change condition of the color developing agent in the process of changing from colorless to blue;
p02, preparing uric acid samples by using buffer solution or artificial urine;
p03, dropping uric acid sample onto hydrophilic pad of non-invasive uric acid colorimetric test paper, or immersing hydrophilic pad into uric acid sample for 1 second;
p04, uric acid concentration is different, and blue intensity is also different; uric acid concentration of uric acid samples is determined by visual comparison of color with a standard color chart or by computer vision digital analysis.
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| CN106404758A (en) * | 2015-07-28 | 2017-02-15 | 珠海和凡医药股份有限公司 | Test paper for detecting uric acid content range in urine |
| CN108568278A (en) * | 2017-03-13 | 2018-09-25 | 广州市芯检康生物科技有限公司 | A kind of novel instant aerogel microball and preparation method thereof |
| CN108593633A (en) * | 2018-04-19 | 2018-09-28 | 中山大学 | A kind of Test paper for quickly detecting saliva uric acid |
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| CN117607418A (en) * | 2023-11-27 | 2024-02-27 | 中科康磁医疗科技（苏州）有限公司 | Reagent and test strip for detecting uric acid, and preparation method and application thereof |
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| CN106404758A (en) * | 2015-07-28 | 2017-02-15 | 珠海和凡医药股份有限公司 | Test paper for detecting uric acid content range in urine |
| CN108568278A (en) * | 2017-03-13 | 2018-09-25 | 广州市芯检康生物科技有限公司 | A kind of novel instant aerogel microball and preparation method thereof |
| CN108593633A (en) * | 2018-04-19 | 2018-09-28 | 中山大学 | A kind of Test paper for quickly detecting saliva uric acid |
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