Hydrogen Peroxide Rapid Test Kit and Method of Use

Abstract

The present invention discloses a rapid hydrogen peroxide test kit and a method of using the same, the rapid hydrogen peroxide test kit comprising a sample diluent and a test strip containing a chromogen for detection and a chromogen for quality control in a dry environment. The sample diluent comprises 100-500 mM ammonium oxalate, 0.3-2.0 mM 8-hydroxyquinoline, 50-500 mM sodium chloride, and 10-100 mM of a first pH adjuster for pH 4.5-7.0.

Description

TECHNICAL FIELD

[0001] The present invention belongs to the field of biological detection technology and, more particularly, relates to a hydrogen peroxide rapid test kit and a method of using the same. STATE OF THE ART

[0002] Numerous studies have shown that catalase (CAT) is downregulated and significantly reduced in activity in the tissues of various solid tumors, including lung, esophageal, colon, liver, and cervical cancer. This leads to increased accumulation of hydrogen peroxide in cells and tissues, resulting in oxidative stress, which causes oxidative damage to biological macromolecules such as DNA, proteins, and lipids, and increases genomic instability, which in turn leads to the transmission of signals that promote tumor growth. This represents an important mechanism that favors the initiation and development of tumors. When the nutrients required for the growth of solid tumors come only from the diffusion of surrounding tissue, growth is extremely slow or inactive, and the tumor size remains approximately 1–2 mm.To break through the limits of growth and development, tumor angiogenesis must occur, so the tumor continuously absorbs nutrients and removes metabolic products through the blood supply. Due to the high diffusivity of hydrogen peroxide, tumors can release hydrogen peroxide even before angiogenesis to prepare growth signals. Studies have confirmed that hydrogen peroxide can activate cellular hypoxia-inducible factor 1, which in turn triggers the upregulation of several proteins that promote angiogenesis, such as vascular endothelial growth factor, thereby introducing angiogenesis into the tumor.By oxidizing cysteine ​​sulfhydryl groups in proteins, hydrogen peroxide can also directly or indirectly activate signaling proteins such as EGFR, PKC, MAPK, CDK, JAK, Ras, NF-κB, c-Myc, and AP-1, which promote tumor initiation and development, as well as suppress the activity of phosphatases such as PTEN and PTPs with anticancer effects. As a result, cancer-promoting signaling pathways remain active for longer in tumor initiation and development and are capable of inducing tumor cell resistance to chemotherapy and targeted drugs such as EGFR.

[0003] Therefore, the detection of hydrogen peroxide in human samples can be used not only for the early detection of tumors, but also for tumor treatment and prognostic tracking and monitoring. Hydrogen peroxide detection is also a common tool in the investigation of gynecological infections. The development of a hydrogen peroxide detection method that allows for easy self-testing is beneficial for health-conscious women, allowing them to self-examine and monitor themselves. Existing common methods for hydrogen peroxide detection generally require professional handling and are not suitable for widespread use.Because early symptoms of tumors are often not obvious or even non-existent, early tumor detection requires a high degree of user-friendliness and cost-effectiveness to meet common application scenarios such as home monitoring or community hospitals. There is a need to develop technologies and products for hydrogen peroxide detection that ensure high user-friendliness, simple assembly and manufacturing, longevity, and good cost-effectiveness. CONTENT OF THE PRESENT INVENTION

[0004] In some embodiments, a hydrogen peroxide test kit is proposed that includes a sample diluent and a test strip containing a chromogen for detection and a chromogen for quality control in a dry environment. The sample diluent comprises 100-500 mM ammonium oxalate, 0.3-2.0 mM 8-hydroxyquinoline, 50-500 mM sodium chloride, and 10-100 mM of a first pH adjuster for pH 4.5-7.0.

[0005] In some embodiments of the hydrogen peroxide test kit, the first pH adjuster comprises at least one of sodium hydrogen phosphate, disodium hydrogen phosphate, sodium acetate, or 2-(N-morpholino)ethanesulfonic acid.

[0006] In some embodiments of the hydrogen peroxide test kit, the chromogenic agent for detection comprises 2.0% to 8.0% (w / v) starch, 0.5 - 5.0 mM acetic acid, 0.5% - 2.0% (w / v) Brij-35, 1.0 - 7.0 M potassium iodide, 2.0 - 10.0 mM 8-hydroxyquinoline, 50 - 300 mM calcium chloride, 5 - 100 mM of a second pH adjuster, and 3 - 15 mM L-ascorbic acid or sodium thiosulfate.

[0007] In some embodiments of the hydrogen peroxide test kit, the second pH regulator comprises at least one of sodium hydrogen phosphate, disodium hydrogen phosphate, sodium acetate, sodium bicarbonate, or tris(hydroxymethyl)aminomethane.

[0008] In some embodiments of the hydrogen peroxide test kit, the quality control chromogen agent comprises 5-30 mM neutral red and 10-60 mM sodium bicarbonate.

[0009] In further embodiments, it is provided that the test strip in the hydrogen peroxide test kit further comprises a centerline reagent.

[0010] In some embodiments of the hydrogen peroxide test kit, the midline reagent comprises 5% - 15% (w / v) Brij-35 and 5% - 15% (w / v) sodium dodecyl sulfate.

[0011] In some embodiments of the hydrogen peroxide test kit, the test strip is provided with dimensions of 68 mm × 6 mm × 0.7 mm, with 3 µL of chromogen for detection in a dried environment at one end of the test strip and 3 µL of chromogen for quality control in a dried environment at the other end of the test strip, and with 3 µL of midline reagent in a dried environment centrally between the chromogen for detection and the chromogen for quality control of the test strip.

[0012] In some embodiments of the hydrogen peroxide test kit, the sample diluent comprises 500 mM ammonium oxalate, 2 mM 8-hydroxyquinoline, 450 mM sodium chloride, and 20 mM sodium hydrogen phosphate disodium hydrogen phosphate at pH 6.0.

[0013] In some embodiments of the hydrogen peroxide test kit, the chromogenic agent for detection comprises 2% (w / v) starch, 1 mM acetic acid, 250 mM calcium chloride, 2% (w / v) Brij-35, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, and 10 mM sodium bicarbonate.

[0014] In some embodiments of the hydrogen peroxide test kit, the centerline reagent comprises 10% (w / v) Brij-35 and 10% (w / v) SDS.

[0015] In some embodiments of the hydrogen peroxide test kit, the quality control chromogen comprises 15 mM neutral red and 45 mM sodium bicarbonate.

[0016] In some embodiments, a detection method for hydrogen peroxide is proposed, comprising the following steps: a. adding a sample to be tested to a sample diluent of a hydrogen peroxide rapid test kit to obtain a mixture I; and b. pipetting the mixture I into a sample dosing zone of a test strip of the hydrogen peroxide rapid test kit, allowing it to stand, and waiting until the test strip develops a color.

[0017] In some embodiments of the detection method, it is provided that step a comprises: After sampling with a throat swab sampler, a sampling end is immersed in a sampling tube with integrated sample diluent, broken off along a score line, the sampling end is left in the tube, the sampling tube is tightly closed and shaken until the sampling end is completely immersed in the sample diluent to obtain mixture I.

[0018] In some embodiments of the detection method, step b comprises: the mixture I is dropped into the sample dosing zone of the test strip and left there until the quality control chromogen area has completely turned purple; then the color development in the detection chromogen area is observed.

[0019] In some embodiments, the use of a kit or a detection method according to the present invention for detecting lung cancer is described. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows schematic views of a test strip with different test results. Fig. Figure 2 shows real views of a finished test strip with different test results. Fig. Figure 3 shows a schematic view of the contents of a kit. Fig. Figure 4 shows a main view of a sample collection tube of a mixture of sample and sample diluent. Fig. Figure 5 shows real views of a finished kit with different test results. List of reference symbols: S: Sample dosing zone; 321: T zone; 322: R zone; 323: C zone; KA1: Sampler sampling end; KB1: Sample tube body; KB2: Cap; KB3: Sample diluent; KC1: Colorimetric plate cassette; KC2: Colorimetric zone; KC3: Quality control zone; KC4: Sample well; KC5: Slot. DETAILED DESCRIPTION

[0020] In some embodiments of the present invention, a hydrogen peroxide test kit is proposed, comprising a sample diluent and a test strip containing a chromogen for detection and a chromogen for quality control in a dry environment. The sample diluent comprises 100-500 mM ammonium oxalate, 0.3-2.0 mM 8-hydroxyquinoline, 50-500 mM sodium chloride, and 10-100 mM of a first pH adjuster for pH 4.5-7.0.

[0021] In some embodiments of the hydrogen peroxide test kit, the first pH adjuster comprises at least one of sodium hydrogen phosphate, disodium hydrogen phosphate, sodium acetate, or 2-(N-morpholino)ethanesulfonic acid.

[0022] In the formulation of the sample diluent of the present invention, 100 - 500 mM ammonium oxalate can not only precipitate various oxidizable metal ions as a complex, but also precipitate the oxidizable metal ions induced by metal ions such as Fe 3+ and Fe 2+Inhibit the catalyzed decomposition of hydrogen peroxide and thus contribute to the stabilization of hydrogen peroxide in the sample. 8-Hydroxyquinoline further increases the stability of hydrogen peroxide. The pH regulator maintains the pH of the system between 4.5 and 7.0, while a pH of ≤ 4.0 is required for the oxidation of nitrite. Thus, interference from nitrite can be excluded, and sodium chloride provides a suitable ionic strength for the reaction. Using the sample diluent according to the invention, it is not only possible to stabilize the hydrogen peroxide dissolved and released from the sample, but also to prevent the decomposition-promoting effect of plastic materials such as polypropylene and polyethylene on hydrogen peroxide without impairing the rapid oxidation of iodide ions by hydrogen peroxide.

[0023] In some embodiments of the hydrogen peroxide test kit, it is provided that a quality control zone is located at a lower end of the test strip, wherein a chromogen for quality control is added in the quality control zone, wherein the amount of the added chromogen for quality control is 1 - 2 µL / mm; and that a colorimetric zone is located 9 - 11 mm below the quality control zone, wherein a chromogen for detection is added in the colorimetric zone, wherein the amount of the added chromogen for detection is 1 - 2 µL / mm, and wherein the test strip is dried for 5 min at 50 °C.

[0024] In some embodiments of the hydrogen peroxide test kit, the chromogenic agent for detection comprises 2.0% to 8.0% (w / v) starch, 0.5 - 5.0 mM acetic acid, 0.5% - 2.0% (w / v) polyoxyethylene lauryl ether (Brij-35), 1.0 - 7.0 M potassium iodide, 2.0 - 10.0 mM 8-hydroxyquinoline, 50 - 300 mM calcium chloride, 5 - 100 mM of a second pH adjuster, and 3 - 15 mM L-ascorbic acid or sodium thiosulfate.

[0025] In some embodiments of the hydrogen peroxide test kit, the second pH regulator comprises at least one of sodium hydrogen phosphate, disodium hydrogen phosphate, sodium acetate, sodium bicarbonate, or tris(hydroxymethyl)aminomethane.

[0026] In some embodiments of the hydrogen peroxide test kit, the quality control chromogen agent comprises 5-30 mM neutral red and 10-60 mM sodium bicarbonate.

[0027] In the formulation of the chromogen for detection in this application, 2.0%–8.0% w / v starch and 1.0–7.0 M potassium iodide form the basis for color development. 0.5–5.0 mM acetic acid and 0.5%–2.0% w / v Brij-35 can promote the dissolution of the starch mother liquor during preparation and support dry adhesion of the chromogen for detection to the test strip. The local application of Brij-35 at a high concentration slows the diffusion of the starch solution and makes color development clearer and more concentrated. 2.0 - 10.0 mM 8-hydroxyquinoline, 3 - 15 mM vitamin C or sodium thiosulfate can remove the background from iodine molecules and, in combination with 5 - 100 mM pH adjuster, adjust the system to an alkaline pH, so that the test strip can be stored at room temperature or even under dry and warm conditions at 40 - 45 °C for a long period of time.

[0028] In further embodiments, it is provided that the test strip in the hydrogen peroxide test kit further comprises a centerline reagent.

[0029] In some embodiments of the hydrogen peroxide test kit, the midline reagent comprises 5% - 15% (w / v) Brij-35 and 5% - 15% (w / v) sodium dodecyl sulfate.

[0030] In some embodiments of the hydrogen peroxide test kit, the test strip is provided with dimensions of 68 mm × 6 mm × 0.7 mm, with 3 µL of chromogen for detection in a dried environment at one end of the test strip and 3 µL of chromogen for quality control in a dried environment at the other end of the test strip, and with 3 µL of midline reagent in a dried environment centrally between the chromogen for detection and the chromogen for quality control of the test strip.

[0031] In some embodiments, it is provided that the test strip of the hydrogen peroxide test kit, as in Fig. 1, an S-zone for sample dosing 320 is located 13 mm from a bottom surface, the S-zone for sample dosing 320 corresponding to a sample well into which a solution of a sample to be tested can be added or immersed; and that a colorimetric T-zone 321 is located 12 mm above the S-zone, a chromogen agent being added to the colorimetric zone for detection. 5 mm above the S-zone is the centerline zone, namely an R-zone 322. 5 mm above the R-zone is a C-zone for quality control 323, into which a chromogen agent is added for quality control. After drying at 50°C for 5 minutes, the test strip can be used directly for sample testing or dried at room temperature and stored capped for later use.

[0032] In some embodiments of the hydrogen peroxide test kit, the sample diluent comprises 500 mM ammonium oxalate, 2 mM 8-hydroxyquinoline, 450 mM sodium chloride, and 20 mM sodium hydrogen phosphate disodium hydrogen phosphate at pH 6.0.

[0033] In some embodiments of the hydrogen peroxide test kit, the chromogenic agent for detection comprises 2% (w / v) starch, 1 mM acetic acid, 250 mM calcium chloride, 2% (w / v) Brij-35, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, and 10 mM sodium bicarbonate.

[0034] In some embodiments of the hydrogen peroxide test kit, the centerline reagent comprises 10% (w / v) Brij-35 and 10% (w / v) SDS.

[0035] In some embodiments of the hydrogen peroxide test kit, the quality control chromogen comprises 15 mM neutral red and 45 mM sodium bicarbonate.

[0036] In some embodiments, a detection method for hydrogen peroxide is proposed, comprising the following steps: a. adding a sample to be tested to a sample diluent of a hydrogen peroxide rapid test kit to obtain a mixture I; and b. pipetting the mixture I into a sample dosing zone of a test strip of the hydrogen peroxide rapid test kit, allowing it to stand, and waiting until the test strip develops a color.

[0037] In some embodiments of the detection method, it is provided that step a comprises: After sampling with a throat swab sampler, a sampling end is immersed in a sampling tube with integrated sample diluent, broken off along a score line, the sampling end is left in the tube, the sampling tube is tightly closed and shaken until the sampling end is completely immersed in the sample diluent to obtain mixture I.

[0038] In some embodiments of the detection method, step b comprises: the mixture I is dropped into the sample dosing zone of the test strip and left there until the quality control chromogen area has completely turned purple; then the color development in the detection chromogen area is observed.

[0039] As in Fig.As shown in Figure 2, in some embodiments of the detection method, when testing a sample to be tested with the hydrogen peroxide test strip according to the invention, the sample is mixed with a sample diluent in a mixed aqueous solution of 500 mM ammonium oxalate, 2.0 mM 8-hydroxyquinoline, 450 mM sodium chloride, and 20 mM sodium hydrogen phosphate disodium hydrogen phosphate at pH 6.0 in a volume ratio of 1:4. Subsequently, the lower end of the test strip is immersed in the sample diluent without immersing the colorimetric zone. After a waiting time of approximately 10 seconds, the C zone 323 and the T zone 321 are observed.The C zone 323 appears purple-red, indicating correct handling; the T zone 321 appears distinctly blue-black and the result is therefore positive, indicating that the sample contains ≥ 1.0 mM hydrogen peroxide; the colorimetric zone shows only the ground color or appears light gray and the result is therefore negative, indicating that the sample contains < 1.0 mM hydrogen peroxide.

[0040] Using purified water as the control sample and 1.5 mM hydrogen peroxide as the positive control sample, the hydrogen peroxide test strip according to the invention is used for testing as described above. Note that the quality control zone of the sample appears purple-red, indicating proper handling; that the colorimetric zone of the negative control sample shows only the base color, indicating a negative result; and that the colorimetric zone of the positive control sample appears distinctly blue-black, indicating a positive result.

[0041] Hydrogen peroxide test kits and their methods of use in some embodiments are described in the Fig.3 to 5. The hydrogen peroxide test kit includes one cassette of the colorimetric plate KC1, one throat swab sampler KA, one sample collection tube KB with integrated sample diluent, and one colorimetric plate KC with integrated test strip. The colorimetric plate KC contains one sample well KC4 (the cassette for the colorimetric plate KC is a colloidal gold cassette, the plate specifications are 70 × 20 mm, the sample well measures 6 × 3 mm, and the evaluation window measures 16 × 3.5 mm). Inside, there is a slot KC5 for attaching the test strip measuring 60 × 4 mm. The test strip is firmly attached to the slot and consists of a 60 mm long and 4 mm wide filter paper.The reagents for the quality control zone KC3 and the colorimetric zone KC2 are applied dried 38 mm and 24 mm from the bottom edge of the filter paper of the test strip.

[0042] It is further provided that the sample diluent KB3 in the body KB1 of the sample collection tube comprises a mixed aqueous solution of 500 mM ammonium oxalate, 2.0 mM 8-hydroxyquinoline, 450 mM sodium chloride and 20 mM sodium hydrogen phosphate-disodium hydrogen phosphate with pH 6.0, wherein the sample diluent has a volume of 300 µL.

[0043] Note that a slot KC5 is provided inside the colorimetric plate KC for attaching the test strip. Slot KC5 eliminates the need for the centerline zone (R zone) 322. After completing the sampling and testing procedure according to the previous steps, a distinct purple-red coloration can be observed in the quality control zones, indicating that the sample and handling were satisfactory and the result is reliable. If the hydrogen peroxide content in the sample is ≥ 1.0 mM, a distinct blue-black coloration can be observed in the colorimetric zone at the end of the test.

[0044] In some embodiments, the hydrogen peroxide test kit and method of use thereof are intended to be used for the early detection of lung cancer.

[0045] The present invention initially enables a distinction between the abnormal hydrogen peroxide accumulation in the airways associated with lung cancer and the inflammatory phenomena in the airways triggered by external stimuli such as infections and allergies. Reactive oxygen species in the airways include hydrogen peroxide and O 2- , OH - and lipid peroxides, of which only hydrogen peroxide can diffuse directly into the respiratory mucus. The half-life of O 2- and OH -are very short, and they exist in cells only for a short time before being rapidly converted into hydrogen peroxide and lipid or protein peroxides. Hydrogen peroxide is usually efficiently and rapidly removed by CAT, so that the inflammation-associated accumulation of reactive oxygen species in the airways occurs mainly in the form of lipid or protein peroxides within the cell. Lung cancer usually does not show typical signs of inflammation in the airways. The associated abnormal accumulation of reactive oxygen species in the airways is mainly due to the downregulation of CAT and the accumulation of hydrogen peroxide, which is caused by the acidification of the airway environment and can diffuse directly into the airway mucus.

[0046] Nitric oxide (NO) in the airways can be increased in certain inflammations of the airways or in lung cancer, especially in the form of nitrite (NO2- ) in respiratory mucus, and has oxidizing properties under more acidic conditions. Dissolved oxygen and oxidizing metal ions are also relatively common oxidizing substances in the respiratory tract.

[0047] According to the invention, the sample is diluted and dissolved under mild conditions, avoiding the use of surfactants to reduce the lysis and release of lipid or protein peroxides dissolved in the cells; and that weakly acidic conditions of pH 6.0 and complexing conditions for metal ions are used, avoiding the use of more acidic conditions to prevent nitrite or dissolved oxygen from acquiring strong oxidizing properties and to complex and passivate metal ions that may be present in the sample and can actively participate in redox reactions, so that the observed increase in oxidizing properties can specifically indicate the abnormal accumulation of hydrogen peroxide in the airways, which is closely related to lung cancer, and to avoid confusion with common inflammations of the airways.

[0048] Unless otherwise stated, all raw material components of the present invention are commercially available products known to those skilled in the art. The technical solutions of the embodiments of the present invention will be described clearly and completely below with reference to the embodiments of the present invention. Naturally, the described embodiments represent only a portion of the embodiments of the present invention and do not encompass all embodiments. Example 1

[0049] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 2 mM 8-hydroxyquinoline, 450 mM sodium chloride and 20 mM pH adjuster for pH 6.0.

[0050] The test strip is 68 mm long and 6 mm wide. The T-zone is located 27 mm from the bottom of the test strip. The R-zone is located 5 mm above the T-zone, and the C-zone is located 5 mm above the R-zone.

[0051] Chromogenic agent for quality control in the C zone: 5 mM neutral red, 10 mM sodium bicarbonate.

[0052] Chromogenic agent for detection in the T-zone: 2% starch, 0.5% Brij-35, 0.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0053] Centerline reagent in the R zone: 5% SDS, 5% Brij-35. Example 2

[0054] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 35 mM sodium bicarbonate.

[0055] Chromogenic agent for detection in the T-zone: 2% starch, 0.5% Brij-35, 0.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0056] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 3

[0057] Chromogenic agent for quality control in the C zone: 30 mM neutral red, 60 mM sodium bicarbonate.

[0058] Chromogenic agent for detection in the T-zone: 2% starch, 0.5% Brij-35, 0.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0059] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 4

[0060] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0061] Chromogenic agent for detection in the T-zone: 4% starch, 0.5% Brij-35, 0.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0062] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 5

[0063] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0064] Chromogenic agent for detection in the T-zone: 6% starch, 0.5% Brij-35, 0.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0065] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 6

[0066] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0067] Chromogenic agent for detection in the T-zone: 8% starch, 0.5% Brij-35, 0.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0068] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 7

[0069] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0070] Chromogenic agent for detection in the T-zone: 2% starch, 1% Brij-35, 0.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0071] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 8

[0072] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0073] Chromogenic agent for detection in the T-zone: 2% starch, 2% Brij-35, 0.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0074] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 9

[0075] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0076] Chromogenic agent for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0077] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 10

[0078] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0079] Chromogenic agent for detection in the T-zone: 2% starch, 2% Brij-35, 2.5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0080] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 11

[0081] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0082] Chromogenic agent for detection in the T-zone: 2% starch, 2% Brij-35, 5 mM acetic acid, 1 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0083] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 12

[0084] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0085] Chromogenic agent for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0086] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 13

[0087] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0088] Chromogenic agent for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 4 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0089] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 14

[0090] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0091] Chromogenic agent for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 7 M potassium iodide, 2 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0092] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 15

[0093] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0094] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0095] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 16

[0096] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0097] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 8 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0098] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 17

[0099] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0100] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 10 mM 8-hydroxyquinoline, 3 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0101] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 18

[0102] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0103] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 7 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0104] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 19

[0105] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0106] Chromogenic agent for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 11 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0107] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 20

[0108] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0109] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 5 mM sodium bicarbonate, 50 mM calcium chloride.

[0110] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 21

[0111] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0112] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 35 mM sodium bicarbonate, 50 mM calcium chloride.

[0113] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 22

[0114] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0115] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 65 mM sodium bicarbonate, 50 mM calcium chloride.

[0116] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 23

[0117] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0118] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 100 mM sodium bicarbonate, 50 mM calcium chloride.

[0119] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 24

[0120] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0121] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 5 mM sodium bicarbonate, 100 mM calcium chloride.

[0122] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 25

[0123] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0124] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 5 mM sodium bicarbonate, 200 mM calcium chloride.

[0125] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 26

[0126] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0127] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 5 mM sodium bicarbonate, 300 mM calcium chloride.

[0128] Centerline reagent in the R-zone: 5% SDS, 5% Brij-35. Otherwise, everything remains as in Example 1. Example 27

[0129] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0130] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 5 mM sodium bicarbonate, 300 mM calcium chloride.

[0131] Centerline reagent in the R-zone: 10% SDS, 10% Brij-35. Otherwise, everything remains as in Example 1. Example 28

[0132] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0133] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 5 mM sodium bicarbonate, 300 mM calcium chloride.

[0134] Centerline reagent in the R-zone: 15% SDS, 15% Brij-35. Otherwise, everything remains as in Example 1. Example 29

[0135] Chromogenic agent for quality control in the C zone: 15 mM neutral red, 45 mM sodium bicarbonate.

[0136] Chromogenic agents for detection in the T-zone: 2% starch, 2% Brij-35, 1 mM acetic acid, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, 10 mM sodium bicarbonate, 250 mM calcium chloride.

[0137] Centerline reagent in the R-zone: 10% SDS, 10% Brij-35. Otherwise, everything remains as in Example 1. Example 30

[0138] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 100 mM ammonium oxalate, 50 mM sodium chloride, 0.3 mM 8-hydroxyquinoline and 10 mM pH adjuster for pH 4.5.

[0139] The test strip is 68 mm long and 6 mm wide. The T-zone is located 27 mm from the bottom of the test strip. The R-zone is located 5 mm above the T-zone, and the C-zone is located 5 mm above the R-zone.

[0140] Chromogen for quality control in the C zone: 3 µL solution of 15 mM neutral red and 45 mM sodium bicarbonate.

[0141] Chromogenic agent for detection in the T-zone: 3 µL mixed aqueous solution of 2% (w / v) starch, 2% (w / v) Brij-35, 1 mM acetic acid, 250 mM calcium chloride, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate and 10 mM sodium bicarbonate.

[0142] Centerline reagent in the R-zone: 3 µL mixed solution of 10% (w / v) SDS and 10% (w / v) Brij-35. Example 31

[0143] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 300 mM ammonium oxalate, 50 mM sodium chloride, 0.3 mM 8-hydroxyquinoline, and 10 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 32

[0144] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 50 mM sodium chloride, 0.3 mM 8-hydroxyquinoline, and 10 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 33

[0145] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 200 mM sodium chloride, 0.3 mM 8-hydroxyquinoline, and 10 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 34

[0146] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 350 mM sodium chloride, 0.3 mM 8-hydroxyquinoline, and 10 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 35

[0147] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 0.3 mM 8-hydroxyquinoline, and 10 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 36

[0148] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 1 mM 8-hydroxyquinoline, and 10 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 37

[0149] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 1.5 mM 8-hydroxyquinoline, and 10 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 38

[0150] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 2 mM 8-hydroxyquinoline, and 10 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 39

[0151] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 2 mM 8-hydroxyquinoline, and 25 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 40

[0152] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 2 mM 8-hydroxyquinoline, and 50 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 41

[0153] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 2 mM 8-hydroxyquinoline, and 100 mM pH regulator for pH 4.5. Otherwise, everything remains as in Example 30. Example 42

[0154] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 2 mM 8-hydroxyquinoline, and 25 mM pH adjuster for pH 6.0. Otherwise, everything remains as in Example 30. Example 43

[0155] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 500 mM sodium chloride, 2 mM 8-hydroxyquinoline, and 25 mM pH adjuster for pH 7.0. Otherwise, everything remains as in Example 30. Example 44

[0156] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 450 mM sodium chloride, 2 mM 8-hydroxyquinoline, and 25 mM sodium hydrogen phosphate disodium hydrogen phosphate at pH 6.0. Otherwise, everything remains as in Example 30. Example 45

[0157] Hydrogen peroxide test kit with sample diluent composition: mixed aqueous solution of 500 mM ammonium oxalate, 450 mM sodium chloride, 2 mM 8-hydroxyquinoline, and 20 mM sodium hydrogen phosphate disodium hydrogen phosphate at pH 6.0. Otherwise, everything remains as in Example 30. Experimental example 1

[0158] The present invention compares throat swab sampling with expectoration sampling as disclosed in the patent application with application number CN 2022115399358. Previous experimental studies have shown that after rinsing the mouth, users take a sip of water, pat their chest or back 10 times, inhale deeply, and cough forcefully 6 times, so that a small amount of respiratory mucus is expelled from the respiratory tract and collects in the mouth. The mucus in the mouth is then coughed out and collected for testing. Using this method, MUC5AC or its mRNA, which is specific to the respiratory tract, can be detected 100%, suggesting that a sample of respiratory mucus can be collected. However, upon extension, it was found that some people, especially young children, had difficulty spitting out the liquid in their mouth, resulting in sampling failure.To ensure the sampling success rate, both throat swabs and self-induced expectorations are used in the patent application with application number CN 2022115399358.

[0159] Sampling by throat swab: After completing the preceding steps so that a small amount of respiratory mucus from the respiratory tract enters and collects in the mouth, the user does not need to cough up this mucus. Instead, the user wipes the mouth with a throat swab sampler, dips the sampling end of the sampler into the sample diluent, breaks it off along the score line, and completes the sampling by leaving the sampling end and the sample in the sample diluent. Sampling by self-induced expectoration in CN 2022115399358: After completing the preceding steps so that a small amount of respiratory mucus from the respiratory tract enters and collects in the mouth, the user coughs up this mucus.

[0160] Thirty healthy volunteers, including 20 adults and 10 infants aged 1 to 5 years, were selected to compare the two sampling methods once a day at different times for three consecutive days. The adults collected the samples themselves throughout the process. The infants were instructed by their parents to cough 6 times, after which the parents collected a sample from the infants using a throat swab. After sampling, the sample weight is calculated by comparing the weight of the sampling tube before and after sampling. The sample volume is measured using a pipette. It is found that throat swab sampling according to the present invention can be successfully carried out on adult and infant volunteers, and the fluctuations in sampling are small, with a single sample amount being approximately 100±15 µL (volume) and 100±15 µL (volume), respectively.100±15 mg (weight). In contrast, in the expectoration sampling method in patent application number CN 2022115399358, it was found that in adults, sampling was successful in both the morning and evening, but the sample volume was approximately 850±185 µL (volume) and 850±150 mg (weight), respectively. In the afternoon, sampling was unsuccessful in five cases for adults; and in infants, there were three cases of unsuccessful sampling across all time periods. The specific statistics are shown in the table below. Table 1: Statistics of sampling by throat swab and expectoration of volunteers Volunteer Sampling period Sampling by throat swab according to the present invention Sampling by expectoration inCN 2022115399358 Volume range of sampling Weight range of sampling Volume range of sampling Weight range of sampling adult 8:30 - 9:00 a.m. 110 - 115 µL 110 - 115 mg 503 - 1035 µL 503 - 1035 mg adult 1:30 pm - 2:00 pm 103 - 110 µL 103 - 110 mg 0 - 686 µL 0 - 686 mg adult 6:30 pm - 7:00 pm 107 - 113 µL 107 - 113 mg 363 - 897 µL 363 - 897 mg toddler 8:30 - 9:00 a.m. 95 - 103 µL 95 - 103 mg 0 - 835 µL 0 - 835 mg toddler 1:30 pm - 2:00 pm 85 - 97 µL 85 - 97 mg 0 - 760 µL 0 - 760 mg toddler 6:30 pm - 7:00 pm 89 - 100 µL 89 - 100 mg 0 - 695 µL 0 - 695 mg

[0161] The hydrogen peroxide test kits described in Examples 1 to 29 were used to determine whether the respiratory mucus sample donor contained traces of hydrogen peroxide and thus a higher risk of developing lung cancer. The negative control was purified water, and the positive control was 1.5 mM hydrogen peroxide.

[0162] The specific process steps are as follows: 1. When collecting samples using the KA throat swab sampler, the KA throat swab sampler can be inserted directly into the back of the tongue in the mouth and held there for approximately 3 seconds with the mouth closed. For the detection of respiratory mucus, the user should rinse their mouth, drink a sip of water, pat their chest or back 10 times, take a deep breath, and then cough forcefully 6 times to expel a small amount of respiratory mucus from the respiratory tract and collect in the mouth. The KA throat swab sampler is then inserted into the back of the tongue in the mouth and held there for approximately 3 seconds with the mouth closed. 2. After sampling, unscrew the cap KB2 of the sample collection tube KB, dip the sampling end KA1 of the throat swab sampler KA containing the sample into the sample diluent in the tube, and snap off along the score line on the swab. Then, screw the cap tightly shut, shake the tube vigorously for approximately 5 seconds, tap the bottom of the tube on the table 5 times, unscrew the cap, and pour all the liquid in the tube into the sample well of the colorimetric plate KC. 3. Then, it is allowed to stand until the quality control zone C displays a distinct purple color, indicating that the sample and handling are satisfactory and the result is reliable. If the T window appears distinctly blue-black, this is a positive result, indicating that the sample contains ≥ 1.0 mM hydrogen peroxide, which is associated with a higher risk of lung cancer. It is recommended to consult a hospital or other professional institution for further investigation related to lung cancer.

[0163] The results showed that the negative coincidence rate of the test strips prepared according to Working Examples 1 to 29 was 100%, the positive coincidence rate was also 100%, and a distinct purple color was observed in the C zones, indicating proper handling. No significant color development was observed in the colorimetric zone of the negative control, indicating a negative test result. A distinct blue-black color was observed in the colorimetric zone of the positive control, indicating a positive test result. Therefore, the test strips prepared according to Working Examples 1 to 29 can all achieve the expected test results. Experimental example 2

[0164] Reference is made to the working steps in Working Example 1. The kits described in Working Examples 30 to 45 should be used to conduct the hydrogen peroxide test. The negative control is purified water, and the positive control is 1.5 mM hydrogen peroxide. It was found that a distinct purple-red color was observed in the C zones of the test strips of the kits prepared according to Working Examples 30 to 45, indicating correct handling. No distinct color development was observed in the colorimetric zone of the negative control, indicating a negative test result. A distinct blue-black color was observed in the colorimetric zone of the positive control, indicating a positive test result. Therefore, the kits prepared according to Working Examples 30 to 45 can all achieve the expected test results. Experimental example 3

[0165] The specifications and preparation of the test strip can be found in Example 45. Reference is made to the steps in Example 1. 100 mg of bovine serum albumin (BSA) / skimmed milk powder solution and 100 µL of 3.0 mM FeCl3 / CuSO4 / NaNO2 solution are added to 100 µL of 2.0 mM H2O2 solution, and the experiment is repeated three times. The test results are listed in Table 2. In the hydrogen peroxide detection method, no discoloration was observed for any of the other substances except hydrogen peroxide, indicating good specificity of the method. Table 2: Test results regarding specificity Samples Test result 1 Test result 2 Test result 3 Empty Positive Positive Positive BSA Negative Negative Negative Skimmed milk powder Negative Negative Negative FeCl3 Negative Negative Negative CuSO4 Negative Negative Negative NaNO2 Negative Negative Negative Experimental example 4

[0166] The specifications and preparation of the test strip can be found in Working Example 45. Reference is made to the working steps in Working Example 1. To conduct a gradient test, hydrogen peroxide solutions with concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 mM were prepared. Each sample was tested 20 times in duplicate, i.e., a total of 3 batches. The results are shown in Table 3, where samples with hydrogen peroxide concentrations as low as 0.8 to 1.5 mM can be clearly colored, and samples with hydrogen peroxide concentrations greater than 1.5 mM can be clearly colored 100%, while the color development rate at 0.7 mM hydrogen peroxide is 0, i.e., 100% no color development occurs. This sample diluent not only provides good sensitivity for the detection of hydrogen peroxide but also ensures good specificity. Table 3: Test results for hydrogen peroxide in different concentrations Samples Positive rate of the first batch Positive rate of the second batch Positive rate of the third batch 0.6 mMH2O2 0% 0% 0% 0.7 mMH2O2 0% 0% 0% 0.8 mMH2O2 35% 40% 40% 0.9 mMH2O2 85% 90% 85% 1.0 mMH2O2 95% 100% 100% 1.1 mMH2O2 100% 100% 100% 1.2 mMH2O2 100% 100% 100% 1.3 mMH2O2 100% 100% 100% 1.4 mMH2O2 100% 100% 100% 1.5 mMH2O2 100% 100% 100% 1.6 mMH2O2 100% 100% 100%

[0167] At the same time, further extended tests were conducted, and the method in the patent application with application number CN 2022115399358 was also tested with 0.6 mM / 0.7 mM / 1.5 mM / 1.6 mM hydrogen peroxide, as shown in the following table. The results are shown in Table 4. It can be seen that with the present invention, the background of 0.7 mM hydrogen peroxide can be better controlled, and the difference between 1.5 mM and 0.7 mM hydrogen peroxide samples is more clearly discernible. Table 4: Test results of sampling by expectoration in CN 2022115399358 Sample concentration Positive rate of the first batch Positive rate of the second batch Positive rate of the third batch 0.6 mM H2O2 10% 15% 10% 0.7 mM H2O2 20% 25% 15% 1.5 mM H2O2 100% 100% 100% 1.6 mM H2O2 100% 100% 100% Experimental example 5

[0168] The specifications and preparation of the test strip can be found in Working Example 45. Reference is made to the working steps in Working Example 1. 3 µL of a solution containing 15 mM neutral red and 45 mM sodium bicarbonate was added to the quality control zone. 3 µL of a mixed solution containing 10% w / v SDS and 10% w / v Brij-35 was added to the midline zone. 3 µL of a mixed aqueous solution containing 2% w / v starch, 2% w / v Brij-35, 1 mM acetic acid, 250 mM calcium chloride, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, and 10 mM sodium bicarbonate was added to the detection zone. Composition of the sample diluent: a mixed aqueous solution of 500 mM ammonium oxalate, 2 mM 8-hydroxyquinoline, 450 mM sodium chloride, and 20 mM pH adjuster for pH 6.0. The negative control is purified water, and the positive control is 1.5 mM H2O2. The tested interfering substances are listed in Table 5.The content of interfering substances in the samples is uniformly set at 2% of the sample volume, which is above the possible volume fraction of the residue after routine use and rinsing of the substance during sampling. Each sample was tested 3 times in duplicate, i.e., a total of 3 batches. Statistical analysis of the results showed that after completion of the procedure, a distinct purple-red color was observed in the C zones, indicating correct handling. No distinct color development was observed in the detection zone of the negative control, indicating a negative test result. A distinct blue-black color was observed in the detection zone of the positive control, indicating a positive test result. Therefore, within the concentration range of the tests, the tested interfering substances do not affect the test results, i.e., they do not interfere with the test results. Table 5: Interfering substances Nr. Interfering substance Nr. Interfering substance Nr. Interfering substance GRWZ01 13% (v / v) milk GRWZ08 13% (v / v) schnapps GRWZ15 150 µg / ml budesonide GRWZ02 13% (v / v) Cola GRWZ09 13% (v / v) cough syrup GRWZ16 5 µg / ml salmeterol GRWZ03 13% (v / v) soy milk GRWZ10 180 µg / ml ibuprofen GRWZ17 3 µg / ml tiotropium bromide GRWZ04 13% (v / v) tea GRWZ11 130 µg / ml aspirin GRWZ18 3 mM ferric chloride GRWZ05 13% (v / v) coffee GRWZ12 120 µg / ml cephalosporins GRWZ19 3 mM copper sulfate GRWZ06 13% (v / v) fruit juice GRWZ13 60 µg / ml Paracetamol GRWZ20 3 mM sodium nitrite GRWZ07 13% (v / v) electrolyte drink GRWZ14 100 µg / ml salbutamol Experimental example 6

[0169] In this example, a planned quantity of 1200 sets was produced. First, the production batch number and production plan were created (Table 6), and material preparation was performed (Table 7). Table 6: Details of the planned quantity of 1200 sets Nr. Detail specification Planned quantity of 1200 sets KKG121 Cassette with colloidal gold set 1200 KKG 122 Sample collection tubes Piece 1200 KKG123 Sample diluent 300 µL 360 ml KKG 124 test strips Piece 1200 KKG125 swab Piece 1200 Table 7: Material list Nr. Designation specification Characteristic Number Dilution factor KKGM12001 cassette set Raw material, clean 1200 - KKGM12002 Sample collection tubes Piece Raw material, clean 1200 - KKGM12003 270 g qualitative filter paper 30 × 30 cm Raw material, clean 20 - KKGM12004 Ammonium oxalate Analytically pure Raw material, clean 33,5 g - KKGM12005 Sodium dodecyl sulfate (SDS) Analytically pure Raw material, clean 0,5 g KKGM12006 Sodium chloride Analytically pure Raw material, clean 14,2 g - KKGM12007 Sodium hydrogen phosphate Analytically pure Raw material, clean 1,3 g - KKGM12008 Disodium hydrogen phosphate Analytically pure Raw material, clean 1,5 g - KKGM12009 Strength Raw material, clean 2,2 g - KKGM12010 Acetic acid mother liquor 100 mM intermediate product, temporary 54 µL 100 x KKGM12011 Calcium chloride Analytically pure Raw material, clean 0,15 g - KKGM12012 Polyoxyethylene lauryl ether (Brij-35) Analytically pure Raw material, clean 0,55 g - KKGM12013 Neutral red mother liquor 150 mM intermediate product, temporary 0.5mL 10 x KKGM12014 Potassium iodide Analytically pure Raw material, clean 1,8 g - KKGM12015 8-Hydroxyquinoline Analytically pure Raw material, clean 0,08 g - KKGM12016 Sodium thiosulfate mother liquor 150 mM intermediate product, temporary 54 µL 10 x KKGM12017 Sodium bicarbonate mother liquor 1M intermediate product, temporary 54 µL 100 x KKGM12018 etiquette Piece Raw material, clean 1200 - KKGM12019 Aluminum composite foil bags and desiccants set Raw material, clean 1200 - KKGM12020 Instructions for use Piece Raw material, clean 1200 - KKGM12021 Outer packaging box Piece Raw material, clean 1200 - KKGM12022 Self-sealing bag Piece Raw material, clean 1200 -

[0170] Critical process - test strip preparation: On an ordinary 270 g filter paper cut to 300 mm × 68 mm, the detection area is marked along a pencil line approximately 27 mm from the bottom, the center line area 5 mm above it along a pencil line, and the quality control area 5 mm above it along a pencil line. A solution of 15 mM neutral red and 45 mM sodium bicarbonate is added to the quality control region, a mixed solution of 10% (w / v) SDS and 10% (w / v) Brij-35 is added to the midline region, and a mixed aqueous solution of 2% (w / v) starch, 2% (w / v) Brij-35, 1 mM acetic acid, 250 mM calcium chloride, 2M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate, and 10 mM sodium bicarbonate is added to the detection region line by line using an autosampler according to the procedure described, followed by drying at 50 °C for 5 minutes.After test strip production is completed, critical sample tests should be performed to determine the quality of the test strips.

[0171] Preparation and sub-packaging of the sample diluent: Prepare 360 ​​ml of the sample diluent according to the recipe in the kit of Example 45 and divide it into sample collection tubes containing 300 µL each. The sample collection tubes are tightly closed, placed in self-sealing bags containing desiccant, sealed, and temporarily stored at room temperature to form semi-finished products. After the sample has been qualified, they are further processed with the outer packaging.

[0172] Detection plate assembly: The test strip is inserted into a slot in the lower part of the cassette so that the lower end of the test strip just fits into the slot. Then, the upper part of the cassette is placed on the lower part so that the positions of C and T correspond to the quality control area and the detection area of ​​the test strip, respectively. After the on-site inspector checks the assembly and finds it satisfactory, a registration mark is affixed to the top of the detection plate. The detection plate is placed in an aluminum foil bag with desiccant, sealed, and temporarily stored at room temperature to obtain a semi-finished product. After the sample is qualified, they are further processed with the outer packaging.

[0173] Outer packaging: The semi-finished products that have passed the sample inspection and the instructions for use are placed in outer packaging boxes, the certificate of conformity is attached, and the products are stored for future use.

[0174] Quality Control: After test strip production is complete, critical sample checks should be performed to determine the quality of the test strips.

[0175] Appearance: The appearance and dimensions of the test strip should comply with the specifications.

[0176] Coincidence rate of negative reference products: 10 commercially available negative reference products are tested, and all test results should be negative. Coincidence rate of positive reference products: 10 commercially available positive reference products are tested, and all test results should be positive.

[0177] Limit of detection: Commercially available reference products for the limit of detection are tested 20 times, with test results being positive in at least 19 cases. Control of semi-finished products

[0178] Visual appearance: The details are complete, the aluminum foil pouch and desiccant are well packaged, without leaks, damage, or moisture accumulation; the label is clearly legible, the registration mark is affixed to the top of the indicator window, and the positions of the windows correspond to the corresponding areas on the test strip.

[0179] Coincidence rate of negative reference products: The results of the critical sampling test are simply used. Coincidence rate of positive reference products: The results of the critical sampling test are simply used. Detection limit: The results of the critical sampling test are simply used. Reproducibility: Commercially available reference products for reproducibility are tested 10 times, with the test results being positive in all cases and the color development being uniform.

[0180] Sample processing efficiency: Samples are collected according to the respiratory mucus collection method described in the working examples. Ten samples are collected for testing: five negative samples, which are mixed and subpackaged from respiratory mucus samples of ten healthy volunteers, and five positive samples, which are mixed and subpackaged from respiratory mucus samples of ten lung cancer patients. The test results of the negative samples should all be negative, and the test results of the positive samples should all be positive. Control of finished products

[0181] Visual inspection: The details are complete. The aluminum foil pouch and desiccant are well packaged, without leaks, damage, or moisture accumulation. The label is clearly legible, the registration mark is affixed to the top of the indicator window, and the window positions correspond to the corresponding areas on the test strip. The finished product should weigh ≥ 9.0 g, indicating that sufficient reagent is contained. Performance: After the visual inspection, the performance indicators are used for critical sampling and the control of semi-finished products.

[0182] Healthy volunteers for testing the hydrogen peroxide test kit according to the invention: 50 healthy volunteers aged 2 to 40 were selected. They worked in the high-tech industry, with a male to female ratio of approximately 50%. They had undergone a comprehensive medical examination within one year, had no abnormal findings, had no history of heavy smoking or work in highly polluting industries, did not suffer from lung diseases such as lung cancer or asthma, and had no family history of such diseases. Some women were pregnant or breastfeeding and felt comfortable doing so. The staff registered the volunteers, distributed the completed test kits, and demonstrated their use. The volunteers then performed the test according to the procedure described in this application and reported the results to the staff.After staff confirmed the results, they were statistically analyzed. The results showed that C-test windows displayed a distinct purple color in 50 cases, corresponding to a handling accuracy of 100%; and that T-test windows showed no distinct color in 50 cases and were all negative, indicating that the tested volunteers had a low risk of lung cancer.

[0183] Lung cancer patients for testing the hydrogen peroxide test kit according to the invention: 24 lung cancer patients between the ages of 40 and 70, with no occupational restrictions, were selected, with a male to female ratio of approximately 50%. They had been diagnosed with lung cancer but had not undergone surgical treatment. None of the women were pregnant or breastfeeding. The staff registered the volunteers, distributed the completed test kits, and demonstrated their use. If the volunteers felt a foreign body in their throat, they were instructed to cough gently to clear the throat and expel mucus. The volunteers then performed the test according to the procedure described in the description and reported the results to the staff. After the results were confirmed by the staff, they were statistically analyzed.The results showed that C-test windows displayed a distinct purple-red color in 24 cases, corresponding to a handling accuracy of 100%; and that T-test windows displayed a distinct blue-black color in 24 cases, and all were positive, suggesting that the concentration of H2O2 in the airways is abnormally elevated, which is associated with a higher risk of lung cancer.

[0184] Patients with lung nodules for testing the hydrogen peroxide test kit according to the invention: 80 patients with lung nodules, aged 20 to 70, with no work restrictions, were selected, with a male to female ratio of approximately 50%. These patients had lung nodules detected in the same year by low-dose spiral computed tomography and were diagnosed with non-lung cancer. None of the women were pregnant or breastfeeding. The staff registered the volunteers, distributed the completed test kits, and demonstrated their use. If the volunteers felt they had a foreign body in their throat, they were instructed to cough gently to clear the throat and get rid of mucus. The volunteers then performed the test according to the procedure described in the description and reported the results to the staff. After the results were confirmed by the staff, they were statistically analyzed.The results showed that C-test windows displayed a distinct purple color in 80 cases, corresponding to a handling accuracy of 100%; and that T-test windows showed no distinct coloration in 80 cases and were all negative, suggesting that the tested volunteers had a low risk of lung cancer.

[0185] Patients with upper respiratory tract inflammation for testing the hydrogen peroxide test kit according to the invention: 30 patients each with a cold cough, rhinitis, and pharyngitis, aged 20 to 40 years, were selected without any occupational restrictions, with a male to female ratio of approximately 50%. They had undergone a comprehensive medical examination within one year, had no other abnormal findings, had no history of heavy smoking or work in highly polluting industries, did not suffer from lung diseases such as lung cancer or asthma, and had no family history of such diseases. No woman was pregnant or breastfeeding. The staff registered the volunteers, distributed the completed test kits, and demonstrated their use.After clearing nasal mucus and coughing gently to clear the throat and get rid of mucus, they performed the test according to the procedure described in the manual and reported the results to the staff. After confirmation by the staff, the results were statistically analyzed. The results showed that C test windows displayed a clear purple color in 90 cases, corresponding to a handling accuracy of 100%; and that T test windows showed no clear color in 90 cases and all were negative, suggesting that the tested volunteers had a low risk of lung cancer.

[0186] Use of the hydrogen peroxide test kit for lung cancer screening during medical examinations: A staff member at the medical examination center should be responsible for on-site registration, distribution and collection of the kits, as well as for providing instructions and answering questions from the participants. In addition, relevant educational videos should be played repeatedly. The method of using the kit is the same as described. Participants for medical examinations can be tested immediately upon arrival, which takes an average of approximately 5 minutes. For multiple participants, self-tests can be conducted in parallel, with a total testing time of only approximately 10 minutes for 30 participants. An estimated 1,440 participants can be tested in one day (8 hours).Low-dose spiral computed tomography typically takes 3 to 5 minutes, and subjects typically have to wait in line. Examining 30 people takes approximately 150 minutes, and up to 96 people can be examined in 8 hours. With this invention, the efficiency of medical screening examinations can be increased by approximately 15 times. Furthermore, low-dose spiral computed tomography results typically take 1 to 3 days to obtain, whereas the present invention can deliver results within 5 minutes.

[0187] Use of the hydrogen peroxide test kit according to the invention in the community: 100 sets of ready-made kits according to the present invention were delivered to the supermarket in the pilot community, and promotional posters with QR codes were displayed. Users scanned the QR codes with their mobile phones to watch video instructions, register online, and then pick up the kit on site to conduct the self-tests. The tests were conducted according to the method described in the description of the present invention, and the test results were subsequently updated online. The test results were summarized and counted, with positive results counted according to the results reported by users and negative results counted according to the results reported by users and unreported results.The logic behind this is that positive results are more likely to stimulate users' reporting and the omission rate is very low, while negative results tend to dampen users' reporting and the omission rate is higher. Statistically, there were 2 positive cases in total, corresponding to a positive rate of 2%, which is higher than the incidence rate of lung cancer in the general population (0.08%). Since 70% of the subjects had lung nodules, the lung nodule positive rate of 3.6% and the overall positive rate of 2% are reasonable. Community hospitals are an important part of the national health system, but lung cancer screening is not yet offered there. Patients must go to a central hospital for examination, which is not conducive to lung cancer screening.The kit according to the invention makes it easier for residents to participate in lung cancer screenings in community hospitals and is likely to become an effective complement to national efforts to develop the health care system.

[0188] Use of the hydrogen peroxide self-test kit according to the invention: 30 volunteers with lung nodules were selected to test samples at home once a month using this kit and undergo regular hospital examinations as prescribed by a doctor. The volunteers were between 20 and 70 years old, with a gender ratio of 50%. Users returned the test results via the internet, or staff collected usage information and recorded the test results through follow-up phone calls or WeChat. Statistical results showed that two individuals tested positive, including a 28-year-old woman who had been diagnosed with a 4 mm lung nodule during a medical examination earlier that year. The first three test kits were negative, but the fourth was positive. When she returned to the hospital for follow-up, the nodule in her right upper lung had grown to 6.5 mm.The doctor recommended surgery, which included wedge resection of the nodule in the right lung under general anesthesia with thoracoscopy. The pathological diagnosis was microinvasive adenocarcinoma. The other patient was a 35-year-old man who had been smoking for 12 years. A medical examination the previous year revealed a 3 mm lung nodule. The first 10 test kits were negative, and the 11th test kit was positive. When she returned to the hospital for follow-up, the nodule in the upper lobe of the left lung had grown to 5 mm. The doctor recommended surgery, and the pathological diagnosis was microinvasive adenocarcinoma. Further CT examinations are complicated and multifaceted, and the cycle is relatively long, which is not conducive to the early detection of lung nodules and the early diagnosis of lung cancer.The present invention facilitates self-monitoring and tracking at home and helps patients arrive at the hospital for timely follow-up examinations.

[0189] Storage and stability testing of the hydrogen peroxide test kit according to the invention: The detection plate (including the test strip) was sealed, dried, and assembled in the final product packaging of the kit. The storage conditions for the kit are room temperature 25°C ± 5°C or refrigerated and humidity ≤ 60%. The negative reference product was prepared by mixing respiratory mucus samples from healthy volunteers and subpackages of 1 ml each. Using standard absorptiometry, an H2O2 content of < 0.5 mM was determined. The positive reference product was prepared by mixing respiratory mucus samples from volunteers with lung cancer and subpackages of 1 ml each. Using standard absorptiometry, an H2O2 content of ≥ 1.0 mM was determined. The reference product for reproducibility is 1.5 mM H2O2 and the reference product for the limit of detection is 1.0 mM H2O2.Testing was performed as described in the description. Each batch was tested three times for each of the three parameters (negative, positive, and reproducibility), with the detection limit being five times. One batch was tested each month. After six consecutive months and six tests, preliminary statistics showed that the coincidence rates for negative / positive / reproducibility / detection limit all reached 100%, thus meeting the quality requirements.

[0190] Accelerated stability of the hydrogen peroxide test kit according to the invention: Three batches each of the kit according to the invention and the kit in CN 2022115399358 were stored in the dark for one week at 45°C and 50°C, respectively. Testing was performed as described in the description for the reference product test. Each batch was tested three times for each of the three parameters (negative, positive, and reproducibility), with the detection limit being five times higher. The statistical results are shown in Table 8. It can be seen that the coincidence rates for negative / positive / reproducibility / detection limit of the reference product all reached 100%, thus meeting the quality requirements. However, the coincidence rate of the reference kit according to CN 2022115399358 decreased. It can be seen that the accelerated stability of the kit according to the invention is better than that in CN 2022115399358. Table 8: Accelerated stability test results Kit in the present invention Kit in CN 2022115399358 1st batch 2nd batch 3.Charge 1st batch 2nd batch 3.Charge 45°C Negative / positive coincidence rate for reproducibility testing 100% 100% 100% 100% 100% 88,9% Coincidence rate for detection limit 100% 100% 100% 80% 80% 80% 50°C Negative / positive coincidence rate for reproducibility testing 100% 100% 100% 66,7% 88,9% 66,7% Coincidence rate for detection limit 100% 100% 100% 80% 80% 60%

[0191] Transport stability and portability of the present hydrogen peroxide test kit: Transport stability and portability are important factors for POCT products. The better the transport stability and portability, the more suitable the product is for rapid on-site testing. Three batches (20 pieces per batch) of the kit of the present invention and the kit in patent CN 2022115399358 were taken and stored at a high temperature of 40°C for 7 days. The kits were then subjected to three free-fall drops from a height of 1.5 m to conduct the impact drop test. The outer and inner packaging of the kits were inspected for damage. Finally, the negative reference products, positive reference products, detection limit reference products, and reproducibility reference products were tested. The results are shown in Table 9.It is found that the outer and inner packaging of the kit of the present invention were undamaged, and the negative / positive / reproducibility / detection limit coincidence rates all reached 100%, thus meeting the quality requirements. Furthermore, the outer packaging of a kit from a batch of patent CN 2022115399358 was partially damaged, but the inner packaging was intact, and statistical verification revealed that the negative / positive / reproducibility / detection limit coincidence rates did not reach 100%. Therefore, the kit of the present invention has better transport stability and portability than patent CN 2022115399358. Table 9: Test results regarding transport stability Kit in the present invention Kit in CN2022115399358 Coincidence rate for negative / positive / reproducibility / detection limit 100% 93,3% Is the outer packaging damaged? No Yes Is the inner packaging damaged? No No

[0192] The principle of the present invention is essentially based on the fact that hydrogen peroxide can rapidly oxidize iodide ions in various environments to iodine molecules (I2), which combine with starch to trigger a color reaction. However, there are three technical difficulties that must be overcome to achieve qualitative detection on a test strip: 1) The combination of iodine and starch must be agglomerated to improve the clarity and readability of the results. The color reaction of iodine in combination with starch must take place in solution. However, the dissolution of iodine and starch on the test strip can lead to diffusion dilution, which significantly impairs the readability of the test results and the sensitivity of the detection. 2) It is necessary to eliminate background interference from iodine molecules caused by dissolved oxygen and atmospheric oxygen.Potassium iodide solutions and test papers can be slowly oxidized and decomposed by dissolved oxygen and atmospheric oxygen, resulting in a small amount of iodine molecules in the background. To avoid and control this, a suitable reducing agent must be selected at the correct concentration. 3) It is necessary to eliminate interference from oxidizing substances such as oxidizing metal ions and nitrites, as well as from proteins and enzymes that may be present in the sample. In the present invention, these three problems are solved through experimental optimization. In general, the essential inventive steps and the advantageous effects of the present invention are as follows: 1. The present invention focuses on improving the versatility of sample collection and processing, as well as the anti-interference and stability properties of the color reaction system. The present invention creates an internationally pioneering system for synchronizing sample lysis, preservation, and interference removal. Upon release of hydrogen peroxide from the sample, the hydrogen peroxide is stabilized in the solution while simultaneously eliminating interference from metal ions, nitrites, and proteins, increasing the sensitivity and specificity of hydrogen peroxide detection in biological samples. Iodometry and other colorimetric methods are widely used to determine hydrogen peroxide, but they require precise measurement, and the reagents must be prepared immediately before use, making them unsuitable for rapid POCT testing.Hydrogen peroxide testing relies on a rapid redox reaction and requires the exclusion of interference from substances that can actively participate in redox reactions. Biological samples can often contain interfering substances such as metal ions and nitrites, which are strongly oxidizing under acidic conditions. In the present invention, these are removed by adjusting the pH of the system to weakly acidic conditions and adding a complexing agent that is active under acidic conditions, such as potassium ferrocyanide. Biological samples are typically rich in proteins. The sulfhydryl groups of cysteine ​​or the disulfide bonds of cystine in their composition are in a folded and protected state in the initial state of the sample.During sample lysis or detection, they may be exposed to the solution system and react with hydrogen peroxide or the reducing agent used in the detection, thereby impairing the detection. Biological samples can also be rich in catalase, peroxidase, and amylase, which can also impair the detection by degrading the hydrogen peroxide released in the sample or the starch used for color development. Another problem in the detection of hydrogen peroxide in biological samples is how to quickly and easily eliminate the interference caused by these proteins without losing hydrogen peroxide in the sample. In the present invention, 8-hydroxyquinoline is used to simultaneously knock down macromolecules such as nucleic acids and proteins and stabilize the hydrogen peroxide in the sample, thereby greatly improving the reproducibility of the detection.Furthermore, a sterilizing effect is achieved, helping to prevent the spread of pathogens that may be present in biological samples. The present invention provides a formulation of reagents for the detection of hydrogen peroxide such that the reagents used for the detection can be stably stored not only at room temperature for extended periods of time, but also under warm conditions of 40 to 45°C. This significantly improves the convenience and user-friendliness of hydrogen peroxide detection compared to the conventional method, which requires freshly prepared reagents and storage at low temperatures. 2. When applying the present invention to respiratory specimen testing, it has the advantages of Chinese Patent Application CN 2022115399358, which is advantageous for POCT testing. In addition, the following major extensions and improvements have been made compared to CN 2022115399358: A) The sampling success rate and its versatility have been improved. The method described in CN 2022115399358 is only suitable for the detection of respiratory specimens, which requires the user to cough up the sample themselves. Some users may not be good at this. However, the invention uses a throat swab, so a 100% sampling success rate is achieved. In this way, the limitations of some users who are not good at coughing up the sample are effectively overcome, and the versatility and specificity of early detection and monitoring of respiratory specimens are improved.In addition, the sample volume of the throat swab can be defined with a fluctuation range of 15%, making the sample volume variation easier to control than with expectoration. The present invention can be used not only for respiratory tract samples, but also for various sampling sites that can be contacted by throat swabs, such as gynecological examinations, which is advantageous for various daily self-tests. B) The clarity and readability of the test results have been improved. The method and kit according to the present invention are used for detection.It was shown that samples with It was shown that samples with hydrogen peroxide concentrations as low as 0.8 to 1.5 mM can be clearly colored, and samples with hydrogen peroxide concentrations greater than 1.5 mM can be clearly colored 100%, while the color development rate is 0 at 0.7 mM hydrogen peroxide, 3 mM sodium nitrite or 3 mM ferric chloride or 3 mM copper sulfate, that is, there is 100% no color development. Thus, the present invention not only provides good sensitivity for the detection of hydrogen peroxide but also ensures good specificity. Compared with CN 2022115399358, the present invention can better control the background of 0.7 mM hydrogen peroxide and more clearly display the difference between the 1.5 mM and 0.7 mM hydrogen peroxide samples. C) The cost-effectiveness has been improved. The kit provided by CN 2022115399358 requires customized sample tubes and production processes.The kit provided by the present invention uses standard colloidal gold cassettes and screw-capped polyethylene tubes, which simplifies the production process and reduces packaging, is compatible with the automated colloidal gold POCT production line, greatly improves production throughput, reduces the production, packaging and transportation costs of the kit, extends the shelf life of the product, and ensures that the product maintains stable quality under hot conditions for a long time, thus promoting the popularization and application of the product. 3. Comparison with conventional breath analysis: Due to the solubility and diffusibility of hydrogen peroxide, most of the hydrogen peroxide in the respiratory tract is dissolved in the respiratory mucus, and only trace amounts can enter the exhaled air. Furthermore, compared to conventional breath analysis, the present invention eliminates airflow disturbances when sampling respiratory mucus. Therefore, the present invention is more resistant to interference, more sensitive, more specific, and easier to handle. 4. Comparison with conventional colorimetry or fluorimetry: These conventional methods require professional pipetting, the preparation of standard curves, and dilution of samples to a linear range before measurement. The use of the present invention is a unified process with no need for quantification and no need for additional equipment and devices, in which the color development threshold of the colorimetric zone has eliminated background interference. In addition, the brownish color developed in the quality control zone is generally weaker than the light brown color in the colorimetric positive result zone, which facilitates comparison to determine positive results. The present invention thus enables qualitative and intuitive assessment and is simpler, faster, and more suitable for dissemination. 5. Comparison with various blood tests: Numerous tumor-related blood tests exist with different blood volumes and performance levels, but their widespread application is limited by the need for blood sampling. The present invention does not require blood sampling and is characterized by excellent safety and non-invasive properties. It can also indicate hydrogen peroxide metabolic status in relation to the sampling site more sensitively and directly than blood tests. 6. Comparison with various detection methods for DNA and proteins: DNA is not only chemically but also genetically stable, so the detection of tumor DNA is generally a relatively long-term correlation test that does not reflect the occurrence and development of tumors in real time. Different proteins have different half-lives, but these can usually be several months or longer, so they cannot reflect tumor-related dynamic changes in real time. Thanks to the limited half-life of hydrogen peroxide, the present invention features good dynamics and is suitable for displaying tumor-related dynamic changes, so it is expected to become a powerful tool for various screening and monitoring methods for lung cancer.The present invention can help potential patients to identify their lung cancer risk as early as possible so that they can undergo relevant examinations such as CT / PET-CT in a timely manner, which promotes early diagnosis and treatment as well as the recovery of health.

[0193] In summary, the present invention specifically solves the problem of the lack of convenience and user-friendliness in the detection of hydrogen peroxide. The advantages of this invention are its convenient and simple application, reliable performance, and moderate price, which promotes widespread use and application. It is suitable for the detection of hydrogen peroxide in various samples that can be collected with a throat swab sampler.

[0194] Only preferred embodiments of the invention have been described above. It should be noted that those skilled in the art may make further improvements and modifications without departing from the scope of the invention, and that these improvements and modifications also fall within the scope of the invention. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] CN 2022115399358 [0158, 0159, 0160, 0167, 0190, 0191, 0192]

Claims

[1] Hydrogen peroxide rapid test kit, characterized by that it includes: a sample diluent comprising 100-500 mM ammonium oxalate, 0.3-2.0 mM 8-hydroxyquinoline, 50-500 mM sodium chloride, and 10-100 mM of a first pH adjuster for pH 4.5-7.0; and a test strip containing a chromogen for detection and a chromogen for quality control in a dry environment. [2] Kit according to claim 1, characterized by that the first pH regulator comprises at least one of sodium hydrogen phosphate, disodium hydrogen phosphate, sodium acetate or 2-(N-morpholino)ethanesulfonic acid. [3] Kit according to claim 1, characterized by that the chromogenic agent for detection comprises 2.0% to 8.0% (w / v) starch, 0.5 - 5.0 mM acetic acid, 0.5% - 2.0% (w / v) Brij-35, 1.0 - 7.0 M potassium iodide, 2.0 - 10.0 mM 8-hydroxyquinoline, 50 - 300 mM calcium chloride, 5 - 100 mM of a second pH adjuster and 3 - 15 mM L-ascorbic acid or sodium thiosulfate. [4] Kit according to claim 3, characterized by that the second pH regulator comprises at least one of sodium hydrogen phosphate, disodium hydrogen phosphate, sodium acetate, sodium bicarbonate or tris(hydroxymethyl)aminomethane. [5] Kit according to claim 1, characterized by that the chromogen for quality control comprises 5 - 30 mM neutral red and 10 - 60 mM sodium bicarbonate. [6] Kit according to claim 1, characterized by that the test strip further comprises a midline reagent. [7] Kit according to claim 6, characterized by that the centerline reagent comprises 5% - 15% (w / v) Brij-35 and 5% - 15% (w / v) sodium dodecyl sulfate. [8] Kit according to any one of claims 1 to 7, characterized bythat the test strip has the dimensions 68 mm × 6 mm × 0.7 mm, with 3 µL of detection chromogen in the dried environment at one end of the test strip and 3 µL of quality control chromogen in the dried environment at the other end of the test strip, and with 3 µL of centerline reagent in the dried environment centrally between the detection chromogen and the quality control chromogen of the test strip. [9] Kit according to claim 8, characterized by that the sample diluent comprises 500 mM ammonium oxalate, 2 mM 8-hydroxyquinoline, 450 mM sodium chloride and 20 mM sodium hydrogen phosphate-disodium hydrogen phosphate at pH 6.

0. [10] Kit according to claim 8, characterized by that the chromogenic agent for detection comprises 2% (w / v) starch, 1 mM acetic acid, 250 mM calcium chloride, 2% (w / v) Brij-35, 2 M potassium iodide, 5 mM 8-hydroxyquinoline, 15 mM sodium thiosulfate and 10 mM sodium bicarbonate. [11] Kit according to claim 8, characterized by that the midline reagent comprises 10% (w / v) Brij-35 and 10% (w / v) SDS. [12] Kit according to claim 8, characterized by that the quality control chromogen comprises 15 mM neutral red and 45 mM sodium bicarbonate. [13] Detection method for hydrogen peroxide, characterized by that it includes the following steps: a. adding a sample to be tested to a sample diluent of a kit according to any one of claims 1 to 12 to obtain a mixture I; and b. Pipetting the mixture I into a sample dosing zone of a test strip of the kit according to any one of claims 1 to 12, leaving it to stand and waiting until the test strip develops a color. [14] Detection method according to claim 13, characterized bythat step a comprises: After sampling with a throat swab sampler, a sampling end is immersed in a sampling tube with integrated sample diluent, broken off along a score line, the sampling end is left in the tube, the sampling tube is tightly closed and shaken until the sampling end is completely immersed in the sample diluent to obtain Mixture I. [15] Detection method according to claim 13, characterized by that step b comprises: Mixture I is dropped into the sample dosing zone of the test strip and left there until the quality control chromogen area has completely turned purple; then the colour development in the detection chromogen area is observed. [16] Use of a kit according to any one of claims 1 to 7 or a detection method according to any one of claims 8 to 15 for detecting lung cancer.

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