Method for detecting airway reactive oxygen species and detection kit

Abstract

This invention provides a method and kit for detecting reactive oxygen species (ROS) in the airways. The method involves gargling and deep coughing to collect a sample, adding a sample release agent to the airway mucus sample, mixing, and then filtering the sample using a test strip immobilized with an indicator to measure the released ROS. A brownish color in the detection area indicates a positive result, indicating that the ROS content in the sample has reached or exceeded the detection threshold. This method can complete the detection within 5 minutes. The operation process and reagents used are conventional, safe, non-invasive, convenient, and easy to use. The kit based on this method has a simple manufacturing process, the product can be stored for a long time and is easy to carry and use. After use, it can be sealed to protect the environment and is suitable for point-of-care testing (POCT) of ROS in the airways.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology and relates to a simple, easy-to-use, and rapid method and reagent kit for detecting reactive oxygen species in the airways. Background Technology

[0002] Reactive oxygen species (ROS), also known as oxygen free radicals, are a class of oxygen-containing substances with high reactivity and strong oxidizing properties. They are typically produced within human cells during the oxidative phosphorylation process associated with respiration. Examples include hydrogen peroxide (H₂O₂) and superoxide radicals (O₂). - ), hydroxyl radicals (OH) - ), lipid peroxides (ROOH) and peroxynitrites (ONOO) - Nitric oxide (NO) can also be considered a reactive oxygen species, but its synthesis, metabolic pathways, and physiological functions differ from those of typical reactive oxygen species. Under physiological conditions, intracellular ROS levels are regulated to lower levels by mechanisms such as superoxide dismutase (SOD), catalase (CAT), and the glutathione-ascorbic acid reduction system. ROS acts as a messenger in normal cell signal transduction, participating in various physiological processes such as cell growth and proliferation, development and differentiation, aging, and apoptosis. Various risk factors associated with chronic diseases, including cancer, such as smoking, environmental pollutants, radiation, infection, and allergies, can stimulate macrophages and neutrophils to overproduce ROS using NADPH oxidase. This ROS then activates various transcription factors, such as NF-κB, AP-1, HIF-1α, and STAT3, inducing the expression of proteins that regulate inflammation, cell transformation, tumor cell survival, tumor cell proliferation and invasion, angiogenesis, and metastasis. ROS can also regulate the expression of various tumor suppressor genes such as p53, Rb, and PTEN. Excessive accumulation of ROS can damage the normal structure of proteins, lipids, and DNA, leading to cellular senescence and death, and tissue damage. Recent studies have found that ROS are involved in the pathogenesis of more than 100 human diseases, such as cardiovascular diseases like hypertension and atherosclerosis, diabetes, chronic inflammation, neurodegenerative diseases, and cancer, as well as immune and aging processes.

[0003] Chronic obstructive pulmonary disease (COPD) is a group of progressive airway diseases characterized by incompletely reversible airway limitation. Essentially, it is a chronic airway inflammatory disease primarily characterized by neutrophil infiltration. Inflammatory cells such as macrophages and T lymphocytes also participate in the pathogenesis of COPD. Alveolar macrophages are widely distributed within the alveoli and on the bronchial surface. Under the stimulation of factors such as heavy smoking or infection, they produce and release reactive oxygen species (ROS) and various inflammatory factors, inducing the aggregation and activation of T lymphocytes and neutrophils, which is a major mechanism in the early initiation of COPD. The accumulation and activation of neutrophils in the COPD airways, continuously producing and releasing ROS, is a crucial mechanism causing irreversible lung tissue damage during COPD. T lymphocytes secrete interleukin (IL)-13, activating aldose reductase (AR), which promotes excessive ROS production by airway epithelial cells. This excessive increase in intracellular ROS leads to mitochondrial dysfunction, which in turn affects the respiratory chain, causing even more ROS production, creating a vicious cycle that exacerbates airway damage. The persistently elevated levels of airway ROS are a core mechanism in the pathogenesis and progression of COPD. While pulmonary function testing is the standard method for COPD diagnosis, it is highly specialized, inefficient, and not easily widely adopted. Developing rapid and convenient airway ROS detection methods, and creating user-friendly and widely applicable screening and monitoring methods and products, will greatly benefit the improvement and advancement of COPD prevention, control, and treatment.

[0004] Existing methods for ROS detection mainly include chemiluminescence, fluorescent probes, spectrophotometry, and electron paramagnetic resonance. These methods are all limited by their high level of specialization and inability to be used for point-of-care testing (POCT), hindering widespread application in health checkups and screenings. While absorbance has the lowest requirements for equipment, it still necessitates the use of specialized instruments such as spectrophotometers and pipettes. For example, patent applications 202010705059.6 ("A Method for Detecting Reactive Oxygen Levels in Morel Mushroom Cells") and 201611056540.7 ("A Method for Detecting the Effect of E-cigarette Products on Cellular Reactive Oxygen Secretion") both require specialized equipment and complex procedures, and neither provides a method for detecting ROS in the human airway. Summary of the Invention

[0005] In summary, there is currently a lack of airway ROS detection methods and products that can be used in non-professional settings such as homes or communities. Therefore, this invention provides a safe, non-invasive, convenient, reliable, and readily available airway ROS detection method and kit.

[0006] The technical solution of this invention:

[0007] This invention provides a method for detecting airway ROS. The process is as follows: After rinsing their mouth, the user pats their chest or back and takes deep breaths and coughs forcefully to accelerate airway activity. The user then spits out accumulated fluid from their mouth into a collection tube, adds a sample release agent containing sodium dodecyl sulfate, sodium ferrocyanide, sodium chloride, and sodium phosphate buffer, and mixes the mixture. A test strip with a detection area fixed to an appropriate amount of starch, acetic acid, chitosan quaternary ammonium salt, potassium iodide, and cysteine ​​is then used for aspiration and detection. The color change in the detection area of ​​the test strip is observed; a brownish-red color indicates a positive result, suggesting an abnormally high ROS content in the sample. The subject should then seek professional medical attention for COPD-related examinations and diagnosis. (See attached diagram for the design of this invention.) Figure 1 .

[0008] Specifically, the sample release agent provided by the present invention consists of 5-30 mM sodium ferrocyanide, 0.5%-2.5% (w / v) sodium dodecyl sulfate, 50-200 mM sodium chloride, and 5-50 mM sodium phosphate buffer solution with pH 5.8-7.4.

[0009] Specifically, the preparation process of the test strip provided by this invention is as follows: Mark the detection area at a suitable position at the lower end of a filter paper strip of appropriate size; add a mixed aqueous solution of 1-2 μl / mm of 2%-8% (w / v) starch, 100-500 mM acetic acid, 0.5%-2% (w / v) chitosan quaternary ammonium salt, 2.0-7.0 M potassium iodide, 0.2-1.0 mM cysteine, and 10-80 mM potassium ferrocyanide; then dry at 60°C for 30 minutes. A preferred solution is a mixed aqueous solution of 4% (w / v) starch, 350 mM acetic acid, 1% (w / v) chitosan quaternary ammonium salt, 2.5 M potassium iodide, 0.5 mM cysteine, and 40 mM potassium ferrocyanide.

[0010] Furthermore, a control zone can be added 7-20 mm above the detection zone of the test strip. A 40-80 mM copper sulfate solution (1-2 μl / mm) is added here, followed by drying at 60°C for 30 minutes. The preferred solution is 60 mM copper sulfate.

[0011] Specifically, the operating method provided by this invention is as follows: After rinsing your mouth, pat your chest or back and take deep breaths and cough forcefully to accelerate the activity of the lung airways. Then, spit the liquid accumulated in your mouth into the collection tube, add 4 ml of sample release agent, shake and vibrate for about 10 seconds to mix, immerse the lower end of the test strip in the mixture, close to but not exceeding the indicator line of the detection area at the lower end of the test strip, let it stand for 10s-90s, and observe the results. If it shows a brownish color, it indicates that the airway ROS has reached or exceeded the detection threshold.

[0012] Based on the above-described method, the main components of the airway reactive oxygen species detection kit provided by this invention include: a sampling tube with an integrated indicator groove, a test strip with an indicator fixed inside, a tube cap, a sample release agent, and a label. The test strip is fixed inside the indicator groove, and the sample release agent is sealed inside the tube cap.

[0013] Specifically, the sample release agent of the kit provided by the present invention consists of: 20mM sodium ferrocyanide, 2% (v / v) sodium dodecyl sulfate, 140mM sodium chloride, and 40mM pH7.0 sodium phosphate buffer solution.

[0014] Specifically, the test strip of the reagent kit provided by the present invention consists of a 52mm×5mm (length×width) filter paper strip and a 44mm×5mm (length×width) back support plastic strip. The upper end of the plastic strip is flush with the upper end of the filter paper strip. The detection zone is marked 29mm from the bottom of the filter paper strip. 3μl of a mixed aqueous solution of 4% (w / v) starch, 350mM acetic acid, 1% (w / v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, and 40mM potassium ferrocyanide is added and dried therein. The control zone is marked 12mm above the detection zone. 3μl of 60mM copper sulfate solution is added and dried therein.

[0015] Furthermore, the test strip is pasted and fixed inside the indicator groove of the sampling tube, with the lower end of the filter paper of the test strip close to the bottom of the sampling tube. The detection area and quality control area on the test strip correspond to the detection window (D) and quality control window (Q) on the label outside the indicator groove, respectively.

[0016] Specifically, the operating steps of the reagent kit provided by this invention are as follows: 1. Rinse your mouth with water, pat your chest 10 times, take 5 deep breaths, and cough forcefully 6 times; 2. Spit the liquid accumulated in your mouth into the sampling tube; 3. Cover the tube and tighten it, shake for about 10 seconds to mix the liquid in the tube evenly; 4. Let it stand for about 2-3 minutes, observe and compare the results. A brownish-red Q window indicates that the operation is effective; a brownish-red D window indicates a positive result (+), meaning that the ROS content in the sample is abnormally high, and the subject should go to a professional institution for COPD-related examinations and diagnosis.

[0017] Furthermore, the detection limit of the above kit is 1.0 mM (calculated as an equivalent amount of hydrogen peroxide). This value is at least 50% higher than the upper limit of the normal reference range for airway reactive oxygen species measured using conventional absorbance methods on airway mucus samples from 250 healthy volunteers. Therefore, it can be guaranteed that the false positive rate for testing healthy volunteers is ≤0.4%.

[0018] The basic principle of this invention is to utilize the significantly elevated airway ROS in COPD patients. By collecting and simultaneously detecting the strong oxidizing properties of airway mucus samples, and using a weakly acidic pH (>4.0) and metal ion complexation conditions to exclude interference from highly oxidizing substances such as nitric oxide (in the form of its derivative nitrite), it specifically indicates whether there is a significant abnormal increase in airway ROS levels. Airway mucus is a fluid and gel-like protective layer existing on the inner surface of the airways, mainly composed of airway mucin, water, and inorganic salts. In a healthy state, airway reactive oxygen species are mainly O2. - In the form of oxygen, reactive oxygen species (ROS) are produced within airway epithelial cells and rapidly converted into hydrogen peroxide by superoxide dismutase (SOD), which is then quickly cleared by highly active catalase (CAT), resulting in low airway ROS levels. In COPD, macrophages and neutrophils aggregate and activate in the airways, producing and releasing large amounts of ROS, causing airway ROS levels to be several times higher than in a healthy state, leading to cell and tissue damage. This significantly elevated airway ROS can be relatively stably reflected in airway mucus samples through exfoliated cells and lipid peroxides. The sampling and detection method of this invention can detect ROS before symptoms become apparent or before sputum production. The process is safe, convenient, and non-invasive, requiring no invasive procedures. Therefore, airway mucus ROS detection when lower respiratory tract symptoms are mild or very mild holds promise for non-invasive early screening of COPD, demonstrating significant application value.

[0019] The specific detection reaction principle of this invention is as follows: 1) A sample release agent containing sodium dodecyl sulfate, sodium phosphate buffer, sodium ferrocyanide, and sodium chloride is used to release, dissolve, and stabilize ROS from airway mucus samples, while simultaneously eliminating interference from nitrite and metal ions. Sodium dodecyl sulfate is a commonly used nonionic surfactant that can rupture cells, promote the dissolution of lipids, including lipid peroxides, without interfering with the pH, ionic strength, or redox reactions of the system. Sodium phosphate buffer provides suitable weakly acidic pH conditions, ensuring that nitrite does not have strong oxidizing properties and thus can eliminate its interference. Sodium ferrocyanide is a good metal ion complexing agent, capable of complexing and shielding various metal ions that can actively participate in redox reactions, such as Cu. 2+ Fe 3+ Fe 2+ Mn 2+ This ensures that the ROS released from the sample can remain stable in the solution system. Sodium chloride supplements the ionic strength. 2) The test strip detection area contains KI, starch, cysteine, chitosan quaternary ammonium salt, potassium ferrocyanide, and acetic acid as a solvent. This combination utilizes the reducing property of the cysteine ​​thiol reagent to stabilize the mother liquor and the I in the detection area. -This process eliminates interference from dissolved oxygen, ensuring that if the sample does not contain sufficient ROS concentration, I2, which can cause starch discoloration, will not be generated. This is crucial for eliminating background noise and ensuring the specificity of the detection. Chitosan quaternary ammonium salt and potassium ferrocyanide can further enhance the I2 concentration. - The stability of the mother liquor and test strips is improved, and the color development effect is enhanced. 3) Under the system conditions provided by this invention, the ROS released from the sample can rise with the solution filtration and rapidly oxidize the thiol groups and I groups in the detection area of ​​the test strip. - This generates I2, which then binds to starch to produce a color change. The reaction is very rapid and sensitive, indicating the presence of excess ROS in the sample. Simultaneously, during the detection process, sodium ferrocyanide rises with the liquid to the quality control area and reacts with Cu. 2+ The reaction produces a brownish-brown precipitate, indicating that the operation is effective.

[0020] Compared with other existing technologies, the inventive points and advantages of this invention are as follows:

[0021] (1) This invention establishes an internationally pioneering aspiration and filtration colorimetric reaction system and an integrated design that integrates sampling, quality control and detection. Users can qualitatively determine whether there is a significant abnormality in the airway ROS level within minutes by performing simple and continuous operations such as gargling and deep coughing to collect samples, closing the tube cap and tightening it, shaking the tube body and observing it.

[0022] (2) Compared with traditional exhaled breath testing, COPD-related airway ROS mainly exist in the form of lipid peroxides in cells, with a small portion dissolved in airway mucus as hydrogen peroxide. Only a trace amount can be distributed in exhaled breath. Furthermore, the airway mucus sampling of this invention can eliminate airflow interference compared to conventional exhaled breath sampling. Therefore, this invention has better anti-interference properties, is more sensitive, more specific, and easier to use.

[0023] (3) Compared with conventional colorimetric or fluorescence methods. These conventional methods require professional pipetting operations, the plotting of standard curves, and the dilution of samples to the linear range before measurement. The process of using this invention is an integrated operation that does not require quantitative analysis or additional equipment. The colorimetric threshold of the detection area has eliminated background interference. Therefore, this invention is conducive to qualitative and intuitive judgment, and is simpler, faster, and more suitable for widespread use.

[0024] (4) Compared with various blood testing methods. Blood tests related to COPD mainly count inflammatory cells such as neutrophils, which can help determine the inflammatory status, but are not specific to the airways. Moreover, the inflammation in the early stages of COPD is relatively mild and may not yet be reflected in the blood. This invention does not require blood draws, has excellent safety and non-invasive characteristics, and can more sensitively and directly indicate abnormal airway ROS conditions than blood tests.

[0025] (5) Compared with traditional sputum testing methods, sputum testing has the advantages of being non-invasive and low-cost. However, although COPD airway mucus secretion levels are elevated, potentially leading to sputum production, this mostly occurs in the early morning or late at night, which is not conducive to sample collection and testing. Especially in the early stages of COPD, symptoms are not obvious, and there is no significant sputum production. Therefore, traditional sputum testing is not beneficial for early screening and monitoring of COPD. This invention allows for the collection and testing of airway mucus samples during regular periods in a prescribed manner, offering better sensitivity and versatility.

[0026] (6) Compared with pulmonary function tests. Pulmonary function tests are the standard method for diagnosing COPD, but they are highly specialized, inefficient, and not easily popularized, with a consistently low adoption rate. Furthermore, they cannot detect the early stages of COPD before irreversible lung damage occurs. This method is a beneficial supplement to pulmonary function tests, enabling the identification of early-stage COPD patients and high-risk groups, encouraging them to prioritize disease prevention and timely participation in necessary pulmonary function tests. In addition, based on the limited half-life of ROS, this invention has good dynamic characteristics, suitable for timely indication of dynamic changes in COPD, and also applicable to home monitoring for patients.

[0027] In summary, this invention specifically addresses the problem of insufficient convenience and ease of use in airway ROS detection. It has advantages such as safety, non-invasiveness, ease of use, reliable performance, and moderate price, which are conducive to its widespread application. It is suitable for COPD patients and high-risk groups for early screening and dynamic monitoring of COPD, which will greatly help improve and promote the prevention, control, and treatment of COPD. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings:

[0029] Figure 1 A diagram showing the test strip and test results.

[0030] Figure 2 Photos of the finished test strips and test results.

[0031] Figure 3 Top view of the pipe cap.

[0032] Figure 4 Integrated design schematic diagram.

[0033] Figure 5 Photos of the finished reagent kit and test results.

[0034] 1: Sampling tube; 2: Tube cap; 3: Indicator groove; 4: Base; 6: Sharp protrusion; 32: Test strip; 33: Result indicator window (D window); 34: Quality control indicator window (Q window); 321: Detection area; 322: Quality control area. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments are merely illustrative of the present invention and not intended to limit the invention. The embodiments described herein are only some embodiments of the present invention, and not all embodiments.

[0036] Unless otherwise specified, the methods used in the embodiments are all conventional methods in the art.

[0037] Unless otherwise specified, all reagents used in the examples were commercially available.

[0038] Figure 1 This is a schematic diagram of the test strip. In this embodiment, the test strip 32 is positioned as the detection zone 321, 29 mm from the bottom, containing a mixed aqueous solution of 4% (w / v) starch, 350 mM acetic acid, 1% (w / v) chitosan quaternary ammonium salt, 2.5 M potassium iodide, 0.5 mM cysteine, and 40 mM potassium ferrocyanide. 12 mm above this zone is the control zone 322, containing a dried 60 mM copper sulfate aqueous solution. After drying at 60°C for 30 minutes, the test strip can be used directly for sample testing or stored in a sealed, dry container at room temperature for later use. A brownish-red color in both the detection zone 321 and the control zone 322 indicates a positive result. If the control zone 322 is already brownish-red before use, the test strip has deteriorated and should not be used. If this area does not show a brownish-red color after use, the operation was improper and the result is unreliable.

[0039] Figure 2 This is a schematic diagram of the finished test strip and the test results. 321: Testing area; 322: Quality control area.

[0040] Figure 3 - Figure 5 As shown, a test kit for detecting reactive oxygen species in the airways, developed according to the method of the present invention, mainly includes a sampling tube 1 (including a base 4), a tube cap 2, and an indicator groove 3 containing test strips.

[0041] In this embodiment of the invention, the sampling tube 1 is a frosted ABS tube with a diameter of 25mm and a height of 90mm. It can stand upright and is equipped with a tube cap 2 and a base 4. After the sample testing is completed, the entire reagent kit and the sample solution are kept in a tightly sealed state to avoid environmental contamination and cross-infection.

[0042] The sampling tube 1 is matched in length with the indicator groove 3. A test strip 32 is fixedly placed inside the indicator groove 3. The size of the test strip 32 is determined according to the size of the indicator groove. The sampling tube 1 is as follows: Figure 4 As shown, base 4 contains a mixture of sample release agent and airway mucus.

[0043] A cylindrical sealing tube is fixed in the cap 2 to secure the sample release agent. During the process of screwing in the cap, the liquid can be allowed to fall to the bottom of the tube by piercing the sealing film through the sharp protrusion 6 on the upper edge of the sampling tube 1. In this embodiment, the sample release agent has a volume of 4 ml and is composed of 20 mM sodium ferrocyanide, 2% (v / v) sodium dodecyl sulfate, 140 mM sodium chloride, and 40 mM pH 7.0 sodium phosphate buffer solution.

[0044] In this embodiment, the test strip 32 inside the indicator slot 3 consists of a 52mm × 5mm test strip and a 44mm × 5mm back support plastic strip. At a distance of 29mm from the bottom of the test strip, a mixed aqueous solution of 4% (w / v) starch, 350mM acetic acid, 1% (w / v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, and 40mM potassium ferrocyanide (3μL) is added. At a distance of 12mm above this, a mixed aqueous solution of 60mM copper sulfate (3μL) is added. A result indicator slot (D) 33 with a diameter of approximately 2mm is located approximately 29mm from the top of the bottom of the indicator slot 3. When the ROS content is greater than 1.0mM, a distinct brownish-red band will appear. A quality control indicator slot (Q) 34 with a diameter of approximately 2mm is located approximately 41mm from the top of the bottom of the indicator slot 3. If this window is already brownish-red before use, this kit is not recommended for further use. If the window does not turn brown after testing, it indicates that the sample or operation is unqualified and the results are unreliable.

[0045] Example 1: Optimal Confirmation of Test Strip Components

[0046] The test strip consists of a 52mm × 5mm filter paper strip and a 44mm × 5mm plastic backing strip. The filter paper strip and the plastic backing strip are aligned at the top. The detection zone (D) is marked 29mm from the bottom of the filter paper strip. A mixed solution of starch, acetic acid, chitosan quaternary ammonium salt, potassium iodide, cysteine, and potassium ferrocyanide is evenly added using a pipette tip. The control zone (Q) is marked 12mm above this zone. Copper sulfate solution is evenly added using a pipette tip. The reagent formulations for both the detection and control zones are shown in Table 3, with a volume of 3μL for each. The sample release agent consists of 20mM sodium ferrocyanide, 2% (v / v) sodium dodecyl sulfate, 140mM sodium chloride, and 40mM pH 7.0 sodium phosphate buffer solution. The negative control is purified water, and the positive control is 1.5mM H₂O₂. The detection method is performed according to the instructions. After the operation was completed, obvious dark brown color was observed in the Q area, indicating that the operation was correct; no obvious color was observed in the D area of ​​the negative control, indicating that the test result was negative; obvious brown color was observed in the detection area of ​​the positive control, indicating that the test result was positive. Therefore, the test strips prepared within the range of reagent concentrations tested could all achieve the expected test results (Table 1).

[0047] Table 1. Test results and statistics of control samples composed of test strips.

[0048]

[0049]

[0050] Example 2: Sample release agent composition test

[0051] The specifications and preparation of the test strips are described in the main text of the instruction manual and Example 1. The detection zone consists of 3 μl of a mixed solution of 4% (w / v) starch, 350 mM acetic acid, 1% (w / v) chitosan quaternary ammonium salt, 2.5 M potassium iodide, 0.5 mM cysteine, and 40 mM potassium ferrocyanide; the control zone consists of 3 μl of 60 mM copper sulfate solution. The sample release agent formulation is shown in Table 2. The negative control is purified water, and the positive control is 1.5 mM H₂O₂. The detection method is performed according to the instruction manual. After the operation, a distinct brown color was observed in all control zones, indicating correct operation; no obvious color development was observed in the negative control detection zones, indicating a negative result; a distinct brown color was observed in all positive control detection zones, indicating a positive result. Therefore, the sample release agents prepared within the tested reagent concentration range all achieved the expected detection results.

[0052] Table 2 Composition of the sample release agent

[0053]

[0054] Example 3: The impact of common beverage and drug residues in samples on result interpretation

[0055] The specifications and preparation of the test strips are detailed in the main text of the instruction manual and Example 1. The detection zone consists of 3 μl of a mixed aqueous solution containing 4% (w / v) starch, 350 mM acetic acid, 1% (w / v) chitosan quaternary ammonium salt, 2.5 M potassium iodide, 0.5 mM cysteine, and 40 mM potassium ferrocyanide; the control zone consists of 3 μl of copper sulfate solution. The negative control is purified water, and the positive control is 1.5 mM H₂O₂. The detection method is performed according to the instruction manual. The interfering substances tested are listed in Table 3. The concentration of interfering substances in the sample is uniformly set at 2% of the sample volume (the usual dosage of the substance), higher than the possible volume ratio of the residual amount after routine use and rinsing. Each sample is tested three times, for a total of three batches of tests. The results showed that after the operation was completed, obvious dark brown color was observed in the quality control area, indicating that the operation was correct; no obvious color was observed in the negative control detection area, indicating that the test result was negative; obvious brown color was observed in the positive control detection area, indicating that the test result was positive. Therefore, within the tested concentration range, the interfering substances did not affect the test results, that is, they did not interfere with the test results.

[0056] Table 3 Interfering Substances

[0057]

[0058]

[0059] Example 4: Production of the rapid detection kit for reactive oxygen species in the airways according to the present invention

[0060] This example demonstrates the production of 1000 kits. First, production batch numbers and a production plan are established (Table 4), and material preparation is carried out (Table 5).

[0061] Table 4. Quantity Components of 1000 Sets

[0062] serial number Components Specification 1000 sets planned quantity Remark KKG121 Sampling tube indivual 1000 KKG122 pipe cover indivual 1000 KKG123 Sample release agent 4.0mL 4000mL KKG124 test strips indivual 1000 KKG125 Label indivual 1000

[0063] Table 5. Bill of Materials

[0064]

[0065]

[0066] Key Step - Test Strip Preparation: On a 270g ordinary qualitative filter paper cut to 300mm × 60mm, mark the detection area approximately 29mm from the bottom with a pencil line. Mark the detection area 12mm above the bottom line with another pencil line. Following the instructions, use a spotting pen to add a mixed aqueous solution of 4% (w / v) starch, 350mM acetic acid, 1% (w / v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, and 40mM potassium ferrocyanide to the detection area. Add a 60mM copper sulfate solution to the 12mm quality control area above the filter paper. After drying at 60℃ for 30 minutes, glue the filter paper strip to a PVC backing board, ensuring the bottom of the filter paper strip is 26mm longer than the bottom of the PVC backing board. Cut the strip vertically into 5mm strips. After test strip preparation, a key step sampling inspection should be performed to confirm the quality of the test strips.

[0067] Sample release agent preparation and aliquoting: Prepare 4000 ml of the sample release agent according to the formula of the kit as shown in the main text of this instruction manual, aliquot 4 ml / part into the tube cap, and seal with sealing film.

[0068] Sampling tube assembly: Apply double-sided foam adhesive to the back of the test strip, place the test strip into and attach it to the indicator groove of the sampling tube, ensuring the lower end of the test strip is just close to the bottom of the tube. Then, seal and fix the sampling tube base to the tube body. Affix labels for the pre-reserved D and Q windows to the outside of the indicator groove, ensuring the D and Q windows correspond to the detection and quality control areas on the test strip, respectively. After the workshop inspector checks the assembly and confirms it is qualified, affix a qualification mark to the top of the indicator groove. Place the tube cap and sampling tube into a self-sealing bag containing desiccant, seal it tightly, and temporarily store it at room temperature to obtain a semi-finished product. After passing random inspection, proceed with outer packaging.

[0069] Outer packaging: After the semi-finished products that have passed the random inspection are placed into the outer packaging box, a certificate of conformity is affixed, and the product is put into storage for future use.

[0070] Example 5: Quality control of the rapid detection kit for reactive oxygen species in the airway of the present invention

[0071] I. Spot checks of key processes

[0072] After the test strips are prepared, key processes are sampled and inspected to ensure that the test strips are of acceptable quality.

[0073] (1) Appearance

[0074] The dimensions of the test strip should conform to the specifications.

[0075] (2) Negative reference sample compliance rate

[0076] Take 10 negative reference samples from the enterprise for testing, and all test results should be negative.

[0077] (3) Positive reference sample compliance rate

[0078] Take 10 positive reference samples from the enterprise for testing, and all test results should be positive.

[0079] (4) Detection limit

[0080] The enterprise's testing limit reference sample should be repeated 20 times, and at least 19 of the test results should be positive.

[0081] II. Inspection of Semi-finished Products

[0082] (1) Appearance

[0083] The components are complete, the self-sealing bag and desiccant packaging are intact, with no leakage, damage, or moisture; the label is clear, the top of the indicator slot has a qualified mark, and the window position matches the corresponding area on the test strip.

[0084] (2) Negative reference sample compliance rate

[0085] The results of sampling inspections of key processes are used directly.

[0086] (3) Positive reference sample compliance rate

[0087] The results of sampling inspections of key processes are used directly.

[0088] (4) Detection limit

[0089] The results of sampling inspections of key processes are used directly.

[0090] (5) Repeatability

[0091] Take a repeatable reference sample from the enterprise and repeat the test 10 times. The test results should all be positive and the color development should be uniform.

[0092] (6) Sample processing efficiency

[0093] According to the airway mucus sampling method of the present invention, five negative samples obtained by mixing and aliquoting airway mucus samples from 10 healthy volunteers and five positive samples obtained by mixing and aliquoting airway mucus samples from 10 COPD patients are tested separately. The test results of negative samples should all be negative and the test results of positive samples should all be positive.

[0094] III. Finished Product Inspection

[0095] (1) Appearance

[0096] The components are complete, and the self-sealing bag and desiccant packaging are intact, with no leakage, damage, or moisture. The label is clearly legible, and a qualified mark is affixed to the top of the indicator slot. The window position matches the corresponding area on the test strip. The total weight of the finished product should be ≥24.70g, indicating that the amount of reagent sealed inside is sufficient.

[0097] (2) Performance

[0098] After verifying that the appearance is correct, the performance indicators of key process sampling and semi-finished product inspection are directly adopted.

[0099] Example 6: Testing on Healthy Volunteers

[0100] The finished reagent kit is described in Example 4. Fifty healthy volunteers, aged 20-50, working in high-tech industries, with a male-to-female ratio of approximately 50%, were recruited on-site within the park. They had undergone a comprehensive physical examination within the past year with no abnormalities, no history of heavy smoking or working in heavily polluting industries, no history of lung cancer or COPD, and no family history of related diseases. Some women were pregnant or breastfeeding and reported feeling well. Staff members oversaw registration, distributed the finished reagent kits, and provided operational demonstrations. Volunteers then performed the tests according to the methods described in the instruction manual and reported the results to staff. After verification, staff conducted statistical analysis of the results. The results showed that all 50 tests showed a clear dark brown color in the Q window, indicating a 100% accuracy rate; all 50 tests showed no significant color development in the D window, indicating negative results. The risk of COPD in the tested volunteers was very low.

[0101] Example 7: Test in COPD patients

[0102] The finished kit is described in Example 4. Thirty volunteers aged 40-70 years with no occupational restrictions were recruited. They were diagnosed with COPD by pulmonary function tests or reported experiencing symptoms such as shortness of breath, worsening wheezing, increased sputum production (purulent or mucopurulent sputum), and chest tightness within the past year, which had resolved spontaneously. They had not used bronchodilators, corticosteroids, or phosphodiesterase-4 inhibitors. Female volunteers were not pregnant or breastfeeding. Staff members conducted registration, distributed the finished kits, and provided operational instructions and demonstrations. Volunteers then performed the tests according to the methods described in the instructions and reported the results to the staff. After verification, the results were statistically analyzed. The results showed that all 30 tests showed a distinct dark brown Q-window, indicating a 100% accuracy rate. All 30 tests showed a distinct brown D-window, indicating a positive result, suggesting an abnormally high level of reactive oxygen species in the airways and a risk of COPD-related airway inflammation. Further medical examination at a hospital was recommended.

[0103] Example 8: Testing in patients with upper respiratory tract inflammation

[0104] The finished reagent kit is described in Example 4. Twenty patients each with colds, coughs, rhinitis, and pharyngitis were recruited, aged 20-40 years, with no occupational restrictions, a male-to-female ratio of approximately 50%, who had undergone a comprehensive physical examination within the past year, had no other abnormalities, no history of heavy smoking or working in heavily polluting industries, no history of lung cancer or COPD, and no family history of related diseases. Females were not pregnant or breastfeeding. Staff conducted registration, distributed the finished reagent kits, and provided operational instructions and demonstrations. Volunteers cleaned their noses, cleared their throats with a light cough, and expelled phlegm before performing the testing procedure as described in the main text of this instruction manual, and reported the results to the staff. After verification, the staff performed statistical analysis of the results. The results showed that all 60 tests showed a clear dark brown color in the Q window, indicating a 100% accuracy rate; all 60 tests showed no significant color development in the D window, indicating negative results, and the risk of COPD in the tested volunteers was very low.

[0105] Example 9: Application of COPD screening in physical examinations

[0106] The finished kit is shown in Example 4. A staff member is assigned to the health checkup center to handle registration forms, kit distribution and collection, and to provide necessary answers and guidance to participants with questions, while also playing promotional videos. The kit usage method is the same as described in the instruction manual. Participants can be tested immediately upon arrival, with an average testing time of approximately 5 minutes. When multiple people are present, they can perform parallel self-testing; 30 tests can be completed in approximately 10 minutes. Based on this, it is estimated that approximately 1440 tests can be completed per day (8 hours). In contrast, pulmonary function tests require one-on-one professional guidance and quality control, and participants typically have to wait in line; 30 tests take approximately 40 minutes, and only about 120 tests can be completed in 8 hours. This invention can improve the efficiency of health checkup screening by approximately 12 times.

[0107] Example 10: Screening Application for Middle-aged and Elderly People

[0108] The finished kit is described in Example 4. Thirty middle-aged and elderly individuals (aged 40-70 years, approximately 50% male and female) who experienced spontaneous remission of symptoms such as shortness of breath, worsening wheezing, increased sputum production (purulent or mucopurulent sputum), and chest tightness within the past year were recruited. These patients had not previously undergone nebulized inhalation or other treatments. Staff conducted registration, distributed the finished kits, and provided operational instructions and demonstrations. If a foreign body sensation was felt in the throat, patients were instructed to clear their throat and expel sputum before proceeding with the testing procedure described in the main text of this instruction manual and reporting the results to staff. Staff then compared and confirmed the results and performed statistical analysis. The results showed that all 30 cases showed a distinct dark brown Q-window, indicating a 100% accuracy rate; all 30 cases showed a distinct brown D-window, indicating a positive result, suggesting an abnormally elevated airway ROS level and a risk of COPD-related airway inflammation. Further medical attention was recommended. Early intervention and continuous management of COPD are beneficial in alleviating symptoms and reducing the frequency and severity of acute exacerbations. However, current auxiliary examinations that are helpful in diagnosing COPD, such as pulmonary function tests and chest X-rays, are difficult to conduct on a large scale among middle-aged and elderly people due to equipment limitations. The sampling and testing processes of this invention are convenient and easy to understand, which is beneficial for early screening, early diagnosis, and early treatment of COPD in middle-aged and elderly people.

[0109] Example 11: Community Application

[0110] The finished reagent kit is described in Example 4. Fifty sets of the finished reagent kit were distributed in pilot community supermarkets, along with promotional posters featuring QR codes. Users scanned the codes with their mobile phones to watch video explanations, registered online, and then picked up the kits on-site for self-testing. They performed the tests according to the methods described in the invention's instructions and updated the results online. Test results were compiled and statistically analyzed. Positive results were counted based on user-reported results, while negative results were counted based on both user-reported and unreported results. The rationale is that positive results are more likely to encourage user communication, resulting in a low underreporting rate, while negative results may reduce user communication and lead to a higher underreporting rate. Statistics showed 6 positive cases, a positive rate of 12%, significantly higher than the 8.6% prevalence of COPD in adults aged 20 and above. Since 40 of the 50 users were 40 years of age or older, and their COPD prevalence was 13.7%, the overall positive rate of 12% is reasonable. Primary healthcare institutions lack specialized facilities for COPD diagnosis, such as pulmonary function tests, and cannot directly diagnose COPD, requiring patients to visit secondary or higher-level hospitals for examination, which discourages residents from participating in COPD screening. The kit described in this invention facilitates residents' participation in COPD screening at primary healthcare institutions and is expected to become a powerful supplement to the development of public health.

[0111] Example 12: Home self-testing

[0112] The finished kit is described in Example 4. Thirty COPD patients routinely using one or more bronchodilators, glucocorticoids, or phosphodiesterase-4 inhibitors were recruited as volunteers. They self-tested at home using the kit of this invention, once a week for a total of four tests. Sampling and testing were conducted before inhaled medication. Volunteers were aged 40-60 years, with a gender ratio of approximately 50%, and their medication treatment duration was no more than two years and no less than one month. The kit was used according to the instructions in this manual. Users submitted their test results online or were followed up by staff via telephone or WeChat to collect usage information and record test results. Results showed that 10 patients had four negative tests, and 7 patients had at least one negative test. The remaining 13 patients had four positive tests. The decrease or disappearance of the positive test frequency during the medication treatment may be related to the control and improvement of airway inflammation. A definitive diagnosis requires confirmation at a secondary or higher-level hospital. Maintenance therapy for COPD is a long-term process. Many patients stop taking medication after their condition improves, leading to a worsening of their condition and posing a risk to COPD control. This invention facilitates home-based self-monitoring and tracking, which helps encourage patients to adhere to their medication regimen.

[0113] Example 13: Reagent kit storage and stability test

[0114] The finished kit is described in Example 4. Sampling tubes (including test strips) are sealed, dried, and assembled within the finished kit packaging. The kit should be stored at room temperature (25℃±5℃) or refrigerated, with humidity ≤60%. The negative reference is prepared by aliquoting airway mucus samples from healthy volunteers at 1 ml / sample, with H2O2 content <1.2 mM as determined by standard absorbance. The positive reference is prepared by aliquoting airway mucus samples from COPD patients at 1 ml / sample, with H2O2 content ≥2.0 mM as determined by standard absorbance. The repeatability reference is 2.0 mM H2O2, and the limit of detection reference is 1.0 mM H2O2. The detection method is performed according to the instructions. For each batch, three negative / positive / repeatability tests and five limit of detection tests are performed, with two batches tested monthly. After four tests in the second month, preliminary statistics show that the compliance rate for negative / positive / repeatability limits of detection all reached 100%, meeting the quality requirements.

[0115] Example 14: Portability and Transportation Stability

[0116] The finished reagent kit is shown in Example 4. The storage conditions for the reagent kit and the reference materials are the same as in Example 14. Three batches of reagent kits (40 kits per batch) were taken and stored at 40°C for 3 days. Then, the kits were dropped freely from a height of 1.5m three times to perform a drop test. The outer and inner packaging of the reagent kits were observed for any damage. Negative, positive, and detection limit reference materials were then used for testing. Results: Only one batch of reagent kits had partial damage to the outer packaging, while the inner packaging remained undamaged. Statistical analysis showed that the compliance rates for negative / positive / reproducibility detection limits were all 100%, meeting the quality requirements.

Claims

1. A method for detecting reactive oxygen species in an airway, comprising, The procedure involves rinsing the mouth with water, taking a sip of water, patting the chest 10 times, taking 5 deep breaths, coughing forcefully 6 times, spitting out the liquid, collecting it, and mixing it with the sample release agent. The sample is then tested for reactive oxygen species released through a test strip with an indicator fixed to it. A brownish-red color in the detection area indicates a positive result, meaning the reactive oxygen species content in the sample has reached or exceeded the detection threshold. The sample release agent consists of 5-30 mM sodium ferrocyanide, 0.5%-2.5% w / v sodium dodecyl sulfate, 50-200 mM sodium chloride, and 5-50 mM sodium chloride. The test strip is composed of a sodium phosphate buffer solution with a pH of 5.8-7.

4. The test strip with the indicator is a filter paper strip. At the bottom of the filter paper strip, 29 mm from the bottom, there is a 1-2 μl / mm mixed aqueous solution of 2%-8%, w / v starch, 100-500 mM acetic acid, 0.5%-2%, w / v chitosan quaternary ammonium salt, 2.0-7.0 M potassium iodide, 0.2-1.0 mM cysteine, and 10-80 mM potassium ferrocyanide. At 7-20 mm above the detection area, there is a 40-80 mM copper sulfate mixed solution.

2. A kit for detecting reactive oxygen species in the airway, using the method for detecting reactive oxygen species in the airway as described in claim 1, characterized in that, The kit mainly consists of: a sampling tube with an integrated indicator slot, a test strip with an indicator fixed in place, a cap, a sample release agent, and a label. The test strip is the core component, consisting of a 52mm × 5mm (length × width) filter paper strip and a 44mm × 5mm (length × width) back support plastic strip. The tops of the filter paper strip and the plastic strip are flush. The detection zone is located 29mm from the bottom of the filter paper strip. The sample contains 3μl of 4% (w / v) starch and 350mM acetone. The sample contains a mixed aqueous solution of 1% (w / v) chitosan quaternary ammonium salt, 2.5M potassium iodide, 0.5mM cysteine, and 40mM potassium ferrocyanide; the quality control zone is located 12mm above the detection zone and contains 3μl of 60mM copper sulfate aqueous solution; the sample release agent is sealed in the tube cap by a membrane that can be punctured by the pointed object inside the sampling tube, and its composition is 20mM sodium ferrocyanide, 2% (v / v) sodium dodecyl sulfate, 140mM sodium chloride, and 40mM pH 7.0 sodium phosphate buffer aqueous solution.

3. The airway reactive oxygen species detection kit according to claim 2, characterized in that, The detection limit of the kit is 1.0 mM, calculated as H2O2.

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