Dissolvable fecal leukocyte esterase test paper

Abstract

A dissolvable fecal leukocyte esterase test paper is provided, comprising a substrate layer, and a front detection layer and a back detection layer disposed on opposite sides of the substrate layer, respectively. The substrate layer has hydrophilic and hydrophobic properties, allowing the test paper to float on a liquid surface during testing, and includes a colorimetric reference area. The front detection layer and the back detection layer are each coated with a chemical reagent configured to react with fecal leukocyte esterase and produce a color change. During testing, a user can place the test paper on a fecal sample, capture the color development results with a handheld device, and use the colorimetric reference area as a colorimetric reference for conversion. This test paper effectively blocks interfering substances in the fecal sample and provides a non-invasive, easy-to-operate, and quantifiable home testing solution.

Description

DISSOLVABLE FECAL LEUKOCYTE ESTERASE TEST PAPERBACKGROUND OF THE INVENTION FIELD OF THE PRESENT DISCLOSURE

[0001] The present disclosure relates to a test paper, and more particularly to a dissolvable test paper for detecting leukocyte esterase in a fecal sample.BACKGROUND

[0002] Inflammatory Bowel Disease (IBD) is a chronic and recurrent inflammatory bowel disease, primarily comprising Crohn's disease and ulcerative colitis. For IBD patients, effective and close evaluation of treatment efficacy to achieve aggressive disease control is crucial. Clinically, while achieving clinical remission is the primary goal to improve patients' quality of life, clinical remission does not fully equate to the disappearance of intestinal inflammation. Therefore, developing objective and quantifiable detection methods to accurately assess the degree of intestinal inflammation has become a major clinical challenge.

[0003] Current clinical methods for assessing the degree of intestinal inflammation primarily rely on invasive endoscopy and fecal calprotectin testing. Although endoscopy is considered the gold standard, it is invasive, costly, requires prior bowel preparation, and can cause significant discomfort to patients, leading to low patient compliance and making frequent follow-up monitoring difficult. While fecal calprotectin testing is non-invasive, its detection process is relatively cumbersome, requiring the collection of fecal samples for laboratory analysis, which typically involves several days of waiting time and cannot provide immediate test results, thus preventing patients from immediately understanding their disease status.

[0004] In recent years, fecal leukocyte esterase (FLE) has been proven to be a potential biomarker for assessing intestinal inflammatory activity. Leukocyte esterase is primarily released by neutrophils. When inflammation is present in the intestine, a large number of neutrophils accumulate at the inflammatory site and release leukocyte esterase. Therefore, the content of leukocyte esterase in feces can indirectly reflect the degree ofintestinal inflammation. Existing leukocyte esterase tests are mainly based on urine test strips, the principle of which is to utilize chemical reagents on the test strip to react with leukocyte esterase, producing a color change.

[0005] However, directly applying urine test strips to fecal testing faces numerous difficulties. Fecal samples have complex compositions, including large amounts of dietary fiber, undigested residues, red blood cells, and other proteins. These substances directly interfere with the color development reaction of the test strip, leading to difficulties in color interpretation or producing false positive / false negative results. Furthermore, the viscosity and heterogeneity of feces also make it difficult for the test strip to uniformly contact the analyte, affecting the accuracy and reproducibility of the test.

[0006] To address the aforementioned problems, some research has proposed a structure where the outer layer of the test paper is covered with a filter membrane, as disclosed in a US provisional application. This technique utilizes a composite transparent biodegradable biofilter membrane to filter interfering substances from feces. Although this structure partially solves the interference problem, its three-layer structure (filter membrane, detection layer, filter membrane) has a relatively complex manufacturing process. Moreover, controlling the pore size of the filter membrane and its adhesion to the detection layer may affect the final detection performance and stability. Therefore, how to develop a home-use FLE detection tool with a simpler structure, more intuitive operation, and effective avoidance of fecal sample interference remains a technical problem that urgently needs to be solved.SUMMARY OF THE INVENTION

[0007] In view of the problems existing in the aforementioned prior art, the primary objective of the present disclosure is to provide a dissolvable fecal leukocyte esterase test paper. Its structural design effectively avoids direct interference from solid fecal matter and simplifies the operating procedure, providing a non-invasive, low-cost, easy-to-operate, highly effective, easy-to-record, and real-time home testing solution.This facilitates self-health management for patients and assists medical personnel in evaluating the treatment efficacy of Inflammatory Bowel Disease (IBD).

[0008] To achieve the above objective, one aspect of the present disclosure provides a dissolvable fecal leukocyte esterase test paper, comprising: a substrate layer; a front detection layer disposed on one side of the substrate layer; and a back detection layer disposed on the opposite side of the substrate layer. The substrate layer is made of a material degradable in an aqueous environment and possesses hydrophilic and hydrophobic properties, such that the dissolvable fecal leukocyte esterase test paper floats on the surface of a liquid sample to be tested during detection. The front detection layer and the back detection layer are each independently coated with a chemical reagent, wherein the chemical reagent is configured to react with fecal leukocyte esterase and produce a color change. Furthermore, a colorimetric reference area is disposed on the substrate layer to serve as a reference for color interpretation.

[0009] By virtue of the technical solution described above, when performing a test, the test paper of the present disclosure can be placed on top of the fecal sample. Supported by the substrate layer, only the detection layers will contact and absorb the liquid sample from the toilet water via capillary action, while solid interfering substances from the fecal sample itself are effectively blocked. Concurrently, the design of dualsided detection layers allows users to use the test paper without needing to distinguish between the front and back sides, significantly enhancing operational convenience. The colorimetric reference area disposed on the substrate layer allows users or a handheld device to perform color comparison to calculate the corresponding fecal leukocyte esterase concentration, providing a more objective quantitative result.

[0010] Hereinafter, the technical features of the present disclosure will be described in detail with reference to specific embodiments and the accompanying drawings, so that those skilled in the art can readily understand the objectives, technical features, and advantages of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic perspective view of an embodiment of the dissolvable fecal leukocyte esterase test paper of the present disclosure.FIG. 2 is a schematic exploded view of an embodiment of the dissolvable fecal leukocyte esterase test paper of the present disclosure.FIG. 3 is a schematic exploded bottom view of an embodiment of the dissolvable fecal leukocyte esterase test paper of the present disclosure.FIG. 4 is a schematic view of another embodiment of the dissolvable fecal leukocyte esterase test paper of the present disclosure, further illustrating filter layers disposed respectively above the front detection layer and below the back detection layer.FIG. 5 is a schematic view of the dissolvable fecal leukocyte esterase test paper of the present disclosure in use.FIG. 6 is a schematic diagram of the results of a feasibility test performed with the test paper of the present disclosure, wherein FIG. 6(A) and FIG. 6(B) show tests performed using commercially available urine test strips of two different brands.FIG. 7 is a schematic diagram of the results of a sensitivity test performed with the test paper of the present disclosure.FIG. 8 is a schematic diagram of the results of a permeability and adsorption test performed with the test paper of the present disclosure.FIG. 9 is a schematic diagram of a standard curve for different leukocyte concentrations established using the test paper of the present disclosure in conjunction with image analysis software.DESCRIPTION OF THE EMBODIMENTS

[0012] To facilitate the understanding of the technical features, content, advantages, and achievable effects of the present disclosure by the esteemed examiner, the present disclosure will be described in detail below with reference to the accompanying drawings and in the form of embodiments. The purpose of the drawingsused herein is merely for illustration and to assist the description, and does not necessarily represent the true proportions and precise arrangements of the present disclosure in actual implementation. Therefore, the scope of rights of the present disclosure in actual implementation should not be interpreted or limited by the proportions and arrangement relationships of the accompanying drawings. This is stated first.

[0013] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense, unless expressly so defined herein.

[0014] Please refer to FIG. 1 to FIG. 3, which are a perspective schematic view, an exploded structural schematic view, and an exploded bottom view, respectively, of a dissolvable fecal leukocyte esterase test paper according to a preferred embodiment of the present disclosure. As shown in the figures, the dissolvable fecal leukocyte esterase test paper 100 of the present disclosure may have an overall structure that is sheet-like, circular, square, or any other suitable shape. It is primarily composed of a front detection layer 110, a substrate layer 120, and a back detection layer 130.

[0015] The front detection layer 110 is disposed on one side (e.g., the upper surface) of the substrate layer 120, and the back detection layer 130 is disposed on the opposite side (e.g., the lower surface) of the substrate layer 120, such that the substrate layer 120 is sandwiched between the front detection layer 110 and the back detection layer 130, forming a three-layer symmetric or asymmetric structure. The advantage of this double-sided design is that users do not need to deliberately distinguish between the front and back sides of the test paper 100 during testing; contact with the sample on either side can achieve the detection effect, which significantly enhances operational convenience and fault tolerance.

[0016] In this embodiment, the materials selected for the front detection layer 110 and the back detection layer 130 must possess good water absorption and liquid conductivity to facilitate rapid and uniform absorption of the liquid sample within a short period. In a preferred embodiment, the front detection layer 110 and the back detection layer 130 are paper materials with filtering properties, such as cellulose paper material or glass fiber paper material. Cellulose is the primary choice because it combines key characteristics such as filtration, pore control, resistance to organic solvents during processing, and low protein adsorption. The grade level of the cellulose paper material determines its pore size, paper thickness, and adsorption properties. Therefore, a cellulose paper material sufficient to filter fecal water, allow reasonable passage of fecal leukocyte esterase (FLE), and meet detection requirements can be selected according to actual needs, such as Whatman filter paper or other equivalent products.

[0017] The front detection layer 110 and the back detection layer 130 are the core regions where chemical reactions occur and color changes are produced. A chemical reagent is coated on the surface of this region using a suitable coating method, such as spraying, spotting, dipping, or printing. The selection of this chemical reagent is one of the keys to the present disclosure; it must be capable of specifically or semi-specifically reacting with fecal leukocyte esterase (FLE) and producing a color change discernible by the naked eye. In a primary embodiment, the chemical reagent comprises an indoxyl ester derivative as a substrate and a diazonium salt compound as a chromogenic agent.

[0018] The reaction principle is as follows: when fecal leukocyte esterase (FLE) in the sample contacts the detection layer (110 or 130), it hydrolyzes the indoxyl ester substrate, releasing indoxyl or its derivatives. Subsequently, the generated indoxyl reacts with the diazonium salt compound in an alkaline environment through a coupling reaction, forming a deeply colored azo dye, typically appearing purple or blue-purple. The intensity of the color is positively correlated with the concentration of FLE in the sample; that is, a higher FLE concentration results in more azo dye formation and a deeper color. Thereby, the FLE content in the sample can be semi-quantitatively or quantitatively evaluated by observing the color change.

[0019] To broaden the scope of protection of the present disclosure and considering factors such as sample solubility, process stability, color development capability, and reaction specificity, the present disclosure covers various combinations of chemical reagents. The chemical reagents of the present disclosure may include the following variations:

[0020] Variation 1: Modified Indoxyl Ester Substrate. The original indoxyl ester substrate is 3-(N-Tosyl-L-alanyloxy)indole (TAI). However, to improve its water solubility or adjust reaction characteristics, its structure can be modified. A preferred modification involves replacing Alanine with another amino acid (Xaa) to form a general formula Tos-Xaa-Y, wherein Tos represents a Tosyl group, Xaa is any single amino acid or a short peptide composed of multiple amino acids, and Y is an Indolyl group or its derivative. For example, Alanine can be replaced with Proline to form Tos-Pro-Y, or with Valine to form Tos-Val-Y. Such modifications can not only adjust the affinity between the substrate and the enzyme, thereby affecting reaction speed and specificity, but may also improve the water solubility of the reagent itself by introducing different amino acid side chains. This is a significant advantage for aqueous-based paper processes, as it avoids the use of highly polar organic solvents.

[0021] Variation 2: Non-Indoxyl Ester Substrates. In addition to indoxyl ester substrates, other compounds that can be hydrolyzed by esterase and produce a detectable signal are also within the scope of protection of the present disclosure. For example, alpha-naphthyl acetate can also serve as an esterase substrate. Upon hydrolysis, it produces alpha-naphthol, which then reacts with a diazonium salt (e.g., Fast Blue B Salt or the aforementioned 2-methoxy-4-(N-morpholino)benzene diazonium salt (MMB)) to form a colored azo dye. The structure of such substrates differs significantly from indoxyl esters, but they can achieve similar detection purposes.

[0022] Variation 3: Self-Chromogenic Substrates. Another simpler design employs "self-chromogenic" substrates, where the product itself, after enzymatic hydrolysis, possesses color, eliminating the need for a reaction with a second reagent (such as a diazonium salt). A typical example is p-nitrophenyl acetate. This compound is colorless itself, but when hydrolyzed by esterase, it produces p-nitrophenol. In an alkalineenvironment, p-nitrophenol dissociates into p-nitrophenolate, which exhibits a distinct yellow color. The advantage of this design is the simplicity of the reagent formulation, requiring only one compound to complete the detection, which can reduce costs and improve process stability.

[0023] As for the selection of diazonium salts, in addition to the aforementioned 2-methoxy-4-(N-morpholino)benzene diazonium salt (MMB), any diazonium salt compound capable of forming stable colored products with reactants such as indoxyl or naphthol can be applied in the present disclosure, such as Fast Blue B Salt, Fast Red TR Salt, etc. In a preferred embodiment, the chemical reagent coated on the detection layer (110, 130), in addition to the aforementioned substrate and chromogenic agent, may also include a buffer to maintain the appropriate pH value required for the reaction (e.g., pH 7.5 to 8.5), as well as a surfactant to promote the dissolution and uniform distribution of the reagent.

[0024] Next, the substrate layer 120 of the present disclosure will be described. The substrate layer 120 is one of the cores of the structural design of the present disclosure and plays multiple key roles. First, the substrate layer 120 is made of a material that is degradable in an aqueous environment. The term "degradable" herein may refer to physical disintegration, such as the structure falling apart upon contact with water, or decomposing into small fragments under the external force of flushing the toilet; it may also refer to chemical or biological degradation (biodegradation). This characteristic allows the test paper 100, after use, to be directly flushed down the toilet like ordinary toilet paper, without causing environmental burden or clogging pipes, meeting the requirements of environmental protection and convenience. Suitable materials may include, but are not limited to, water-soluble paper, flushable non-woven fabric, or composite materials containing water-soluble polymers.

[0025] Secondly, the substrate layer 120 possesses special functions through the control of hydrophilic / hydrophobic coatings. In a preferred embodiment, the main body of the substrate layer 120 may be hydrophilic, but its surface facing the test environmental liquid may be coated with a hydrophobic coating. This design can effectively separate the main body of the test paper 100 from the large amount of toiletwater below within a set time (e.g., the few minutes required for testing), allowing it to float stably on the liquid surface and only allowing the detection layer (110 or 130) to contact the liquid surface. This allows for precise control of the path and amount of liquid entering the test paper, preventing the entire test paper from being soaked, which could lead to structural damage or uncontrolled reactions.

[0026] Furthermore, the substrate layer 120 has sufficient background color blocking capability. The color of feces varies in depth; if its color directly penetrates the test paper, it will severely interfere with the color interpretation of the detection layer (110, 130). Therefore, the substrate layer 120 needs to have sufficient thickness, density, or be added with an opacifier, such as titanium dioxide (TiCh) or calcium carbonate (CaCCh), to provide sufficient reflection or absorption capability, so that the color of the liquid below the water surface does not directly penetrate to the detection layer above, ensuring that the interpretation of the color development result is based on the chemical reaction itself, rather than background interference.

[0027] As shown in FIG. 1 to FIG. 3, in a preferred embodiment of the present disclosure, a colorimetric reference area 140 is disposed on both the front and back sides of the substrate layer 120. The colorimetric reference area 140 may be an area coated or printed with standard colors, which may correspond to specific fecal leukocyte esterase concentrations. When interpreting the results, users can directly compare the color of the detection layer (110 or 130) with the adjacent colorimetric reference area 140 to obtain a semi-quantitative result. This design not only facilitates visual interpretation but also serves as a built-in calibration reference for subsequent image analysis using a smartphone APP, to correct color cast caused by different smartphone camera models and different ambient light sources, greatly improving the accuracy and reproducibility of digital image analysis. As shown in FIG. 3, this design ensures that a colorimetric reference area 140 is available for reference regardless of whether the front or back side is used for testing.

[0028] Please refer to FIG. 4, which is a schematic view of the dissolvable fecal leukocyte esterase test paper according to another embodiment of the present disclosure. This embodiment is substantially the same as the previous embodiment, with thedifference being that a filter layer 150 is further disposed above the front detection layer 110 and below the back detection layer 130, respectively. The filter layer 150 may be made of a material with a larger pore size than the front detection layer 110 and the back detection layer 130, to preliminarily filter out larger solid particles or impurities in the fecal sample, while allowing the liquid containing fecal leukocyte esterase to penetrate to the detection layers for reaction. This design can further reduce contamination and interference of the sample on the detection layer, and is suitable for samples with particularly high impurity content.

[0029] Please refer to FIG. 5, which is a schematic view of the dissolvable fecal leukocyte esterase test paper of the present disclosure in use. As shown in the figure, after using the toilet, the user directly places the test paper 100 of the present disclosure on the surface of the toilet water above the fecal sample 160. Due to the hydrophobicity and buoyancy design of the substrate layer 120, the test paper 100 floats stably on the water surface. At this time, the detection layer in contact with the fecal sample 160 (e.g., the front detection layer 110 or the back detection layer 130) will contact and absorb the toilet water dissolved with fecal excretions through capillary action. The liquid to be tested then diffuses in the detection layer and reacts with the chemical reagent to develop color. Subsequently, the user can use a handheld device 170 (e.g., a smartphone) to photograph the color-developed dissolvable fecal leukocyte esterase test paper 100, and use the color of the colorimetric reference area 140 on the substrate layer 120 as a reference to analyze and calculate the corresponding fecal leukocyte esterase concentration through an application (APP).

[0030] The benefit of this process is that solid particles, fibers, red blood cells, and other macromolecular interfering substances in the fecal sample 160 itself, due to their physical size or diffusion speed limitations, cannot effectively enter and pass through the pore structure of the detection layer (110 or 130), and are physically blocked out. At the same time, the back of the color development reaction is protected by the waterproof substrate layer 120, completely isolated from the toilet water below. In this way, what actually reaches the detection layer is mainly the small-molecule analyte dissolved in water, such as the target analyte fecal leukocyte esterase (FLE), while mostof the color and solid interference is excluded, thereby ensuring the specificity and accuracy of the test.

[0031] The following will further illustrate the technical efficacy of the present disclosure through specific experimental data; however, these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure.

[0032] Example 1: Feasibility Test of Commercially Available Urine Test Strips.

[0033] To verify the feasibility of detecting leukocyte esterase (LE) using commercially available test strips, we selected two common brands of urine test strips (labeled Arkray and Siemens, respectively) for preliminary testing. Since it is difficult to directly obtain fecal samples containing different concentrations of FLE, we used peritoneal dialysis (PD) fluid as a source of LE. Peritoneal dialysis fluid is rich in white blood cells (WBC), and the liquid itself is transparent in color, which does not interfere with color development, making it an ideal simulated sample.

[0034] The experimental procedure is as follows:

[0035] Step 1: Carefully cut out the reaction block for detecting white blood cells from the commercially available urine test strip and paste it onto a piece of standard toilet paper to simulate the simple structure of the test paper of the present disclosure.

[0036] Step 2: Serially dilute the original peritoneal dialysis fluid to obtain white blood cell suspensions of different concentrations, specifically 500, 250, 75, and 25 cells / pL.

[0037] Step 3: Drop 15 pL of PD solution of different concentrations onto the reaction block of the test strip, and observe and record the color change within 90 seconds after dropping.

[0038] Please refer to FIG. 6, which shows the test results of two urine test strips. As shown in FIG. 6(A), the Arkray brand urine test strip exhibited excellent reactivity and discrimination. As the white blood cell concentration increased, its color gradually changed from light yellow to different shades of purple, and its color change was in good agreement with the reference colors provided by the manufacturer. This result indicatesthat the chemical formulation of the Arkray urine test strip can accurately detect different concentrations of white blood cells in PD fluid, providing an important technical basis for the subsequent development of home-use fecal leukocyte esterase test paper.

[0039] In contrast, as shown in FIG. 6(B), when the Siemens brand urine test strip was tested in the same manner, its color development reaction was very inconspicuous, and it was almost impossible to distinguish samples of different concentrations. This result indicates that the formulation or structure of the Siemens urine test strip may not be suitable for this type of detection. Therefore, subsequent experiments will mainly refer to the formulation of the Arkray test strip for development and verification.

[0040] Example 2: Sensitivity Test.

[0041] Based on the results of Example 1, we further conducted a sensitivity test on the better-performing Arkray urine test strip. We used PD solution with a concentration of 500 cells / pL for repeated experiments (n=4), and recorded the time required to reach the deepest color level (3+).

[0042] Please refer to FIG. 7, the experimental results show that the average time for the 4 experiments to produce a 3+ reaction was only 62.5 seconds, significantly faster than the manufacturer's recommended reading time of 90 seconds. This result strongly indicates that the chemical reaction system used by the Arkray urine test strip has very high sensitivity and can produce a saturated color reaction to high concentrations of LE in a very short time, which is a great advantage for home testing products that require rapid results.

[0043] Example 3: Permeability-Adsorption Test of Degradable Filter Membrane (Toilet Paper).

[0044] One of the design concepts of the present disclosure is to use the material of the detection layer itself (such as cellulose paper material) to achieve filtering and liquid guiding effects. To simulate this scenario, we designed an experiment to evaluate the effect of ordinary toilet paper (as a sample of a degradable absorbent layer) on detection. This experiment used a two-layer structure, i.e., covering a layer of toilet paperon the reaction block of the Arkray test strip, to evaluate whether the adsorption of the toilet paper would affect the color development reaction of the test strip below.

[0045] The experimental method is as follows: The experiment also used WBC solutions with concentrations of 500, 250, 75, and 25 cells / pL. The experimental group covered a layer of toilet paper on the test strip, while the control group did not; then, WBC solutions of different concentrations were dropped onto the test strip from above the toilet paper, and color changes were photographed and recorded at different time points (60 seconds, 90 seconds, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes). The results are shown in Table 1 and Table 2, where Table 1 is the control group and Table 2 is the experimental group.

[0046] Table 1

[0047] Table 2

[0048] Please refer to FIG. 8, which shows the detailed experimental results. In the figure, CO represents the control group with a WBC solution concentration of 0 / pL; and TO represents the experimental group with a WBC solution concentration of 0 / pL, and so on. The experimental data from Table 1, Table 2, and FIG. 8 show that at lowconcentrations (<75 cells / pl.), the experimental group with toilet paper covering did show a slight delay and slightly lighter color in the color development reaction compared to the control group without covering. This may be because some leukocyte esterase (LE) was adsorbed by the fibers of the toilet paper, or because liquid penetration required additional time; however, over time, all test strips eventually reached the expected color level. More importantly, at medium to high concentrations (>75 cells / pL), due to the high sensitivity of the test strip itself to LE, even with toilet paper covering, the test strips were able to reach the highest color development level (+3) in a very short time.

[0049] This experimental result strongly supports the design concept of the present disclosure. Even if there is an ab sorb ent / fi Iter layer above the detection layer, the timeliness and accuracy of detection for clinically meaningful medium to high concentration inflammation indicators will not be significantly affected. Although there is a slight delay at very low concentrations, it can still react correctly, confirming that the structure proposed by the present disclosure has good feasibility in detecting WBC / LE in body fluids.

[0050] Example 4: Image Analysis and Standard Curve Establishment.

[0051] To elevate the detection results of the present disclosure from semi-quantitative to quantitative, analysis can be combined with image processing technology. Please refer to FIG. 9, which is a schematic diagram of a standard curve for different leukocyte concentrations established using the test paper of the present disclosure in conjunction with image analysis software.

[0052] The basic principle of image analysis is as follows: First, the tested paper is photographed using a handheld device 170 (e.g., a smartphone). Then, image processing software (e.g., open-source ImageJ software) is used to analyze the reaction area. The software converts the color image into a grayscale image and calculates the average grayscale value of the reaction area. Since the reaction produces a dark substance, the higher the fecal leukocyte esterase (FLE) concentration, the darker the color, and the lower the corresponding grayscale value. By plotting different known concentrations of leukocyte standards against their corresponding grayscale values, a standard curve can be established.

[0053] As shown in FIG. 9, there is a good negative correlation between leukocyte values and grayscale values. In the future, when a user photographs the tested paper with the handheld device 170 and uploads it to an APP, the backend algorithm can automatically extract the grayscale value of the reaction area. This value can then be interpolated using the standard curve to derive a quantitative FLE concentration value. This method eliminates the subjective error of visual interpretation and provides more objective and accurate detection results.

[0054] In summary, the dissolvable fecal leukocyte esterase test paper provided by the present disclosure, through its unique three-layer structural design, cleverly utilizes the buoyancy and barrier properties of the substrate layer and the selective absorption function of the detection layer, successfully overcomes the technical bottleneck of interference from fecal samples in the prior art. At the same time, its double-sided symmetrical design, flushable and degradable environmental characteristics, and the potential for combining colorimetric reference areas with smart image analysis make it a home self-testing tool with significant clinical application value. The present disclosure not only allows IBD patients to monitor their intestinal inflammation in a non-invasive, low-cost, and extremely convenient manner but also provides objective, recordable data to facilitate communication between doctors and patients and adjustment of treatment plans, which is of great significance for achieving the "treat-to-target" strategy for IBD.

Claims

WHAT IS CLAIMED IS:

1. A dissolvable fecal leukocyte esterase test paper, comprising:a substrate layer formed of a material degradable in an aqueous environment, and having hydrophilic and hydrophobic properties configured to float the dissolvable fecal leukocyte esterase test paper on a surface of a test environmental liquid;a front detection layer disposed on one side of the substrate layer; and a back detection layer disposed on an opposite side of the substrate layer; wherein the front detection layer and the back detection layer are each independently coated with a chemical reagent, the chemical reagent being configured to react with a fecal leukocyte esterase and produce a color change; anda colorimetric reference area is further disposed on the substrate layer.

2. The dissolvable fecal leukocyte esterase test paper according to claim 1, wherein the substrate layer comprises an opacifier to block color interference from the test environmental liquid.

3. The dissolvable fecal leukocyte esterase test paper according to claim 1, wherein the colorimetric reference area is disposed on the front side and the back side of the substrate layer.

4. The dissolvable fecal leukocyte esterase test paper according to claim 1, wherein the front detection layer and the back detection layer are formed of a cellulose material or a glass fiber material.

5. The dissolvable fecal leukocyte esterase test paper according to claim 1, further comprising at least one filter layer disposed on a surface of the front detection layer or a surface of the back detection layer.

6. The dissolvable fecal leukocyte esterase test paper according to claim 1, wherein the chemical reagent comprises a substrate and a chromogenic agent, the substratebeing hydrolyzed by the fecal leukocyte esterase to generate a reactant, the reactant then reacting with the chromogenic agent to produce the color change.

7. The dissolvable fecal leukocyte esterase test paper according to claim 6, wherein the substrate is an indoxyl ester derivative, and the chromogenic agent is a diazonium salt compound.

8. The dissolvable fecal leukocyte esterase test paper according to claim 6, wherein the substrate is alpha-naphthyl acetate, and the chromogenic agent is a diazonium salt compound.

9. The dissolvable fecal leukocyte esterase test paper according to claim 1, wherein the chemical reagent is a self-chromogenic substrate, a product of which, after being hydrolyzed by the fecal leukocyte esterase, inherently has color.

10. The dissolvable fecal leukocyte esterase test paper according to claim 9, wherein the self-chromogenic substrate is p-nitrophenyl acetate.

Images