CN116183890A

CN116183890A - Airway hydrogen peroxide detection method and kit

Info

Publication number: CN116183890A
Application number: CN202211539935.8A
Authority: CN
Inventors: 徐逸丽; 陈燃
Original assignee: Zhejiang Jfk Biological Technology Co ltd
Current assignee: Zhejiang Jfk Biological Technology Co ltd
Priority date: 2022-12-02
Filing date: 2022-12-02
Publication date: 2023-05-30

Abstract

The invention provides an airway hydrogen peroxide detection method and a kit, which are used for qualitatively detecting hydrogen peroxide in airway mucus samples. The method comprises the steps of collecting liquid in an oral cavity in a sampling tube after deep cough is rinsed with clear water, adding sample diluent, mixing, standing, and performing suction filtration to measure hydrogen peroxide released by a sample by using a test strip fixed with an indicator, wherein the result shows that the black brown color is positive, and the hydrogen peroxide content in the sample is indicated to reach or exceed a detection threshold value, so that a subject is in an abnormal state of hydrogen peroxide metabolism of an airway. The kit based on the method provided by the invention consists of a sampling tube, a tube cover, a sample diluent and a test strip. The sample diluent and the test strip are respectively sealed in the tube cover and the sampling tube. The kit integrates sampling, quality control and detection, the used reagents are conventional reagents, the assembly production and the use are convenient, a user can complete the detection within 5 minutes, the result is visual and easy to interpret, the sensitivity is high, the specificity is good, and the kit is suitable for rapidly detecting hydrogen peroxide in airway mucus samples.

Description

Airway hydrogen peroxide detection method and kit

Technical Field

The invention relates to the technical field of biochemistry, in particular to a safe, convenient, sensitive and instant airway hydrogen peroxide detection method and a kit. The kit is a disposable sealing device, and can effectively avoid pollution and cross infection.

Background

Hydrogen peroxide (H) ₂ O ₂ ) Is a small molecular compound with strong oxidability, is easy to dissolve in water and is relatively stable under weak acid alkalinity and physiological or lower temperature conditions. Hydrogen peroxide in the body is produced during cellular oxygen metabolism and can diffuse across the membrane into tissues and body fluids. The specific generation process is superoxide anion (O) generated in the oxidative phosphorylation process of cell mitochondria ₂ ^- ) Hydrogen peroxide is produced by the conversion of superoxide dismutase (SOD). Under normal conditions, hydrogen peroxide produced in human cells is converted to water under the catalysis of Catalase (CAT) and rapidly cleared, so that the hydrogen peroxide is controlled at a very low level. A great deal of researches show that CAT has reduced expression and activity in various tumor tissues such as lung cancer, esophageal cancer, rectal cancer, liver cancer and the like, the reduced expression and activity of CAT lead to the increase of hydrogen peroxide concentration in cells and tissues, oxidative stress is formed, oxidation damage and genomic instability of biomacromolecule substances such as DNA, protein, lipid and the like are caused to be increased, cell malignant transformation is directly or indirectly induced, and tumorigenesis is promoted. Meanwhile, hydrogen peroxide is also an important signal molecule of tumor cells, and can directly or indirectly activate EGFR, PKC, MAPK, CDK, JAK, ras, NF-kappa B, c-Myc, AP-1 and other signal transduction proteins with close relation to tumor development and development through oxidation of cysteine sulfhydryl in protein, and can inhibit activities of PTEN, PTPs and other phosphatases with cancer inhibiting functions, so that cancer promotion signal paths of EGFR and the like are more continuous, and the tumor cells have resistance to chemotherapeutic drugs and EGFR targeting drugs. Therefore, the detection and timely removal of the hydrogen peroxide level in the environment of the human body are beneficial to the health of the human body.

Airway hydrogen peroxide is released by intracellular production of airway tissue, mostly dissolved in airway mucus, and a small fraction is released into the environment in the form of exhaled air. In a healthy state, airway hydrogen peroxide levels are very low. Upon inflammation, neutrophils become cohesively activated in the airways to produce large amounts of reactive oxygen species with consequent increase in airway hydrogen peroxide levels. The lung cancer is usually not inflamed in early stages and asymptomatic or symptoms are not obvious, but hydrogen peroxide can still accumulate to higher levels due to the down regulation of CAT expression and the like. The airway adhesive (mucic) liquid is a kind of body fluid and gelatinous protective layer which exist on the inner surface of the airway, and mainly consists of airway adhesive (mucic) protein, moisture, inorganic salt and other components. In the course of airway canceration, factors such as the Warburg effect (Waboge effect) of cancer cells and the upregulation of vacuole ATPase (V-ATPase), the downregulation of cystic fibrosis transmembrane transduction regulator (CFTR) and the like can cause airway mucus acidity and CAT expression to be reflected in airway mucus along with the accumulation change of hydrogen peroxide, and can be detected before symptoms are obvious or expectoration is formed; meanwhile, airway mucus samples can be obtained through the continuous deep breathing-active deep cough means, the process is safe, convenient and harmless, and invasive means are not needed, so that the airway mucus hydrogen peroxide detection is expected to achieve noninvasive early screening of lung cancer when no lower respiratory tract symptoms or very slight symptoms exist, and the method has great application value.

Inflammation and infection assays are common applications for hydrogen peroxide assays, for example, vaginal secretion hydrogen peroxide assays and exhaled breath hydrogen peroxide assays. The detection method is mainly based on the strong oxidizing property of hydrogen peroxide, and concretely comprises a colorimetric method, a fluorescence method, an electrochemical sensor method and the like. When the methods are used for detecting hydrogen peroxide in airway mucus samples, a series of professional operation treatments such as sample collection, liquefaction and cleavage, mixed reaction and the like are required, degradation and loss of hydrogen peroxide are easy to cause, and as the property of hydrogen peroxide is similar to that of common inflammation markers such as various active oxygen which is not hydrogen peroxide and comprises Nitric Oxide (NO), the interference of other active oxygen is not usually excluded, which causes insensitive and inaccurate results for lung cancer related detection, and the methods require professional operation and use such as: professional precision instruments such as pipettes, spectrophotometers and the like cannot meet the convenience requirement of point of care testing (POCT). The exhaled air detection has the advantages of multiple interference factors, large performance fluctuation, complicated quality control depending on professional equipment and facilities and professional operation guidance in the using process, and special disinfection steps to avoid cross contamination; the factors such as the understanding fit degree, the oral pressure, the lung volume, the fluctuation of the flow velocity of the exhaled air, the model of the instrument and the like of the testers can cause great difference, and are not beneficial to popularization and application. For the popularization and application scenes such as early screening of lung cancer, home/community monitoring of patients and the like, development of a convenient and easy-to-use airway hydrogen peroxide detection technology and product is required.

Disclosure of Invention

Aiming at the problem of insufficient convenience and usability of the conventional airway hydrogen peroxide detection method, the invention provides a method and a kit capable of being used by a portable and self-use way and rapidly detecting airway hydrogen peroxide. The method and the kit can rapidly dissolve and detect hydrogen peroxide in airway mucus samples in the using process, can eliminate the interference of common metal ions and nitrite which is a nitric oxide derivative, and can intuitively and accurately determine qualitatively that accumulated hydrogen peroxide is secreted from lung cancer lesions in airway mucus.

The technical scheme of the invention is as follows:

the invention provides a method for rapidly detecting airway hydrogen peroxide from airway mucus samples, after continuous deep cough is carried out by rinsing with clear water, liquid in an oral cavity is collected in a sampling tube, sample diluent is added for mixing, standing is carried out, hydrogen peroxide released by a sample is detected by suction filtration through a test strip fixed with upper and lower 2 area indicators, a lower area is a quality control area, and a brown indication result is effectively displayed by color change; the upper area is a detection area, the color change shows that the color change is a positive result, the hydrogen peroxide content in the sample reaches or exceeds a detection threshold value, and the detected person is in an abnormal state of hydrogen peroxide metabolism of the airway. For people with obvious respiratory symptoms, if people feel obvious expectoration, the sputum should be expectorated, and the sputum should be sampled again according to the deep cough method.

The preparation process of the test strip comprises the following steps: dripping 50-100mM copper sulfate aqueous solution with the width of 1-2 mu l/mM at a proper position at the lower end of a filter paper strip with proper size according to the width of the filter paper strip; 1-2 mu l/mM of 2-8% (w/v) starch, 1-5% (w/v) chitosan quaternary ammonium salt, 2-10mM oxalic acid, 2-5M potassium iodide, 2-5mM 8-hydroxyquinoline and 1.0-1.5mM cysteine mixed aqueous solution are dropwise added at the position 7-25mM above the area; then, the mixture was dried at 60℃for 30 minutes.

The sample diluent comprises 10-20mM potassium ferrocyanide, 2-5mM ammonium oxalate and 120-150mM sodium chloride mixed water solution, can be quickly mixed with airway mucus sample, dissolves hydrogen peroxide, provides a proper weak acid alkaline pH environment, and complexes and precipitates metal ions possibly existing in the sample so as to avoid nitrite and metal ions with active oxidation-reduction properties such as Fe ³⁺ 、Fe ²⁺ 、Cu ²⁺ Etc. to interfere with the detection process. The sodium chloride has the function of balancing osmotic pressure, so that the exfoliated cells in the sample are kept intact to avoid releasing O2 ^- 、OH ^- And lipid peroxides and other non-freely diffusing active oxygen, and reduces interference to hydrogen peroxide detection. Potassium ferrocyanide and Cu of the quality control area of the test strip ²⁺ The complex reaction, which appears tan, indicates that the detection procedure is effective.

The invention also provides a kit for detecting the airway hydrogen peroxide from the airway mucus, which consists of a sampling tube integrated with an indication groove, a tube cover, a sample diluent, a test strip and a label. Wherein, the sampling tube is a sealable transparent frosted ABS tube, one side is provided with an indication groove, and a test strip is arranged in the sampling tube; the test strip is a core component, and the preparation process comprises the following steps: 1 μl of 60mM copper sulfate aqueous solution was added dropwise to a filter paper strip of size 38mm×5mm×0.7mM (length×width×thickness) 22mM from the lower end; 3 μl of 4% (w/v) starch, 1% (w/v) chitosan quaternary ammonium salt, 2mM oxalic acid, 2.5M potassium iodide, 2.5mM 8-hydroxyquinoline, and 1.0mM cysteine mixed water solution are added dropwise at a position 33mM from the lower end of the filter paper strip; then, the mixture was dried at 60℃for 30 minutes. The tube cover is matched with the sampling tube, the inside of the tube cover is hollow, and the sample diluent is pre-stored in the tube cover. When the mouth-cleaning and deep-cough-relieving oral cavity liquid is used, after continuous deep cough is carried out by rinsing with clean water, liquid in the oral cavity is poured into the sampling tube, the tube cover is covered and screwed up, and the sharp protrusions on the inner wall of the sampling tube puncture the sealing film of the tube cover so as to enable sample diluent to fall into the sampling tube; shake the pipe shaft and make sample diluent and sample mixing that flows in the tube cap, after 2 minutes of standing, observe the instruction groove: the lower window is a quality control area (C), and the brown indication result is effectively displayed in a color-changing manner; the upper window is a detection area (T), the color change display is a positive result, the hydrogen peroxide content in the sample reaches or exceeds a detection threshold value, and the detected person is in an abnormal state of hydrogen peroxide metabolism of the airway; after the detection is finished, the whole sealing device can be treated according to medical wastes, so that pollution and cross infection are avoided.

Further, the detection kit has 3 windows

Further, the detection limit of the kit is 0.5mM, which is at least 20% higher than the upper limit of the hydrogen peroxide level of the airway of 100 healthy volunteers, so that the false positive rate of the detection of the healthy volunteers is less than 1%.

The design and reaction principle of the invention comprises 4 layers which are related to each other: 1) Hydrogen peroxide has good solubility and diffusivity at normal temperature, and can stably exist in weak acidity and the water solution in which metal ions actively participating in oxidation-reduction reaction are shielded. With these characteristics, the present invention uses a sample diluent mixed with ammonium oxalate and potassium ferrocyanide to release dissolved and stabilized hydrogen peroxide from airway mucus samples. The sample diluent not only provides weak acid to weak alkaline pH conditions, but also is a good metal ion complexing agent for oxalate and ferricyanide, and can complex and shield various metal ions capable of actively participating in oxidation-reduction reaction under wide pH conditions from acid to alkaline, such as: cu (Cu) ²⁺ 、Fe ³⁺ 、Fe ²⁺ 、Mn ²⁺ Etc., so that the hydrogen peroxide released from the sample can exist stably in the solution system; 2) The test strip detection area contains KI, starch, cysteine and 8-hydroxyquinoline, and the reducibility and complexation of cysteine sulfhydryl reagent and 8-hydroxyquinoline can not only stabilize I of the detection area ^- And is capable of complexing Cu migrating from the quality control zone without hydrogen peroxide at a sufficient concentration ²⁺ And possibly contaminated trace Fe in the sample ³⁺ So that if the sample does not contain hydrogen peroxide in sufficient concentration, I which can discolor starch is not produced ₂ . This is important to eliminate background and ensure specificity of detection. 2 auxiliary reagents of chitosan quaternary ammonium salt and oxalic acid are added in the detection area of the test strip to strengthen I ^- Stability on mother liquor and test strip, and enhanced color development effect. 3) The hydrogen peroxide can be used for rapidly oxidizing sulfhydryl and Cu under the condition of weak acid to weak alkalinity ²⁺ Can oxidize I rapidly ^- Is a characteristic of (a). In experiments, the inventor finds that under the condition of stronger acidity (pH is less than or equal to 4.0), acidic substances with stronger oxidability such as nitrite and the like and dissolved oxygen can also quickly oxidize mercapto and I ^- The method has the advantages that strong background interference is caused, repeated tests prove that under the weak acid to weak alkaline condition (pH is 6.0-7.5) provided by the system, the interference substances such as nitrite, dissolved oxygen and the like do not generate background interference any more, and the hydrogen peroxide can still oxidize sulfhydryl rapidly so that a quality control area of the test strip moves to Cu of a detection area along with the ascending of the solution ²⁺ Can oxidize I rapidly ^- The reaction equation is: 2Cu ²⁺ +4I ^- ＝2CuI↓+I ₂ The reaction is very fast and sensitive due to the formation of CuI precipitate. 4) I ₂ The color reaction in the presence of starch is very rapid and sensitive, indicating the presence of hydrogen peroxide in the sample. In addition, the potassium ferrocyanide in the sample diluent can also be matched with Cu in a quality control area on the test strip ²⁺ The reaction is colored, the indication detection is effective, the real-time quality control along with the whole detection process is provided, and the method can be further used for color comparison and help to judge the positive degree.

Based on the difference of the nature and the source of the hydrogen peroxide and other oxidizing substances, the invention can primarily distinguish the abnormal rise of the airway oxidizing related to lung cancer from the airway inflammation manifestation caused by external stimulus factors such as infection allergy and the like. The airway oxidative rise is typically due to reactive oxygen species, including hydrogen peroxide and O2 ^- 、OH ^- Lipid peroxides, etc., wherein only hydrogen peroxide can diffuse directly into airway mucus. O2 ^- And OH (OH) ^- The half-life of (c) is short, and is only transiently present in cells to be rapidly converted into hydrogen peroxide and lipid peroxide, and the hydrogen peroxide is efficiently and rapidly cleared by the CAT with high activity of the cells, so that the oxidative rise of the airway associated with inflammation mainly exists in the cells in the form of lipid peroxide. Lung cancer usually does not have typical airway inflammation, and the abnormal rise of related airway oxidability mainly results from the accumulation and increase of airway hydrogen peroxide caused by CAT down-regulation and airway environment acidification, and can be directly diffusedEmbodied in airway mucus. Nitric Oxide (NO) can also be regarded as an active oxygen species, but its anabolic and metabolic pathways and physiological functions differ from those of normal active oxygen. Airway NO can rise in some airway inflammations or lung cancers, mainly by nitrite (NO ₂ ^- ) In the airway mucus and exhibits oxidative properties under more acidic conditions. Dissolved oxygen and metal ions are also relatively common sources of oxidizing properties. The invention uses mild sample dilution dissolution conditions, avoiding the use of surfactants to avoid the release of intracellular lipid peroxides by lysis; weak acid alkalinity and metal ion complexing conditions with pH of 6.0-7.5 are used, strong acidic conditions are avoided to avoid nitrite or dissolved oxygen from obtaining strong oxidability, and metal ions which can actively participate in oxidation-reduction reaction and possibly exist in a passivated sample are complexed, so that detected oxidability rise can specifically indicate abnormal rise of hydrogen peroxide of the air passage closely related to lung cancer, and confusion with common airway inflammation is avoided.

Compared with the prior art, the invention has the following advantages:

(1) The invention establishes an internationally initiated suction filtration chromogenic reaction system and an integrated design, integrates sampling, quality control and detection, and can qualitatively judge the hydrogen peroxide metabolic state of the airway within a few minutes through simple and continuous operations of gargling, deep cough sampling, pipe cover screwing, pipe body shaking, standing observation and the like.

(2) Compared to conventional exhaled breath detection. Based on the solubility and diffusivity of hydrogen peroxide, most of hydrogen peroxide in an airway is dissolved in airway mucus, only a trace amount can be distributed in exhaled air, and the airway mucus sampling can exclude airflow interference compared with the conventional exhaled air sampling, so that the airway mucus sampling device has better interference resistance, is more sensitive, more specific and easier to use.

(3) Compared with conventional colorimetric or fluorescent methods. These conventional methods require specialized pipetting operations that require drawing a standard curve and diluting the sample to a linear range for re-measurement. The invention has the advantages that quantitative integrated operation is not needed in the use process, extra equipment is not needed, background interference is eliminated by the color development threshold value of the detection area, the color brown developed by the quality control area is generally weaker than the positive color black brown developed by the detection area, and the positive result is convenient to compare and determine, so that the invention is favorable for qualitative and visual judgment, is simpler, faster and more suitable for popularization.

(4) Compared to various blood detection methods. Many blood detection methods related to tumor use different blood amounts and performances, but the popularization and application are limited by blood drawing requirements. The invention does not need blood drawing, has excellent safe and noninvasive characteristics, and can directly indicate the hydrogen peroxide metabolism condition of the lung more sensitively than blood detection.

(5) Compared to various DNA and protein detection methods. Because DNA has good stability, not only has chemical stability, but also has genetic stability, so the detection of tumor DNA is usually a relatively long-term correlation detection, and the occurrence and development of tumors cannot be timely fed back. The stability of the protein is also relatively good, different proteins have different half-lives, but often can reach several months or even longer, and thus, the tumor-related dynamic changes cannot be fed back very timely. The invention is based on the characteristic of short hydrogen peroxide half-life, has good dynamic property, is suitable for indicating the dynamic change related to the tumor in time, and is hopeful to develop into a powerful means for screening and monitoring various related to lung cancer. The invention can help potential patients to find lung cancer risk as early as possible and receive relevant examinations such as CT/PET-CT and the like in time, thereby being beneficial to early diagnosis and early treatment and recovery of health.

In a word, the method solves the problem of insufficient convenience and usability of the airway hydrogen peroxide examination in a targeted manner, has the advantages of safety, no wound, convenience, easiness in use, reliable performance, moderate price and the like, is favorable for popularization and application, and is suitable for early screening and dynamic monitoring of lung cancer risk of lung nodules, lung cancer and people concerned with lung cancer.

Drawings

The invention is further described below with reference to the accompanying drawings:

FIG. 1 is a schematic diagram of test strips and test results.

FIG. 2 is a photograph of the finished test strip and the detected fruit.

Fig. 3 is a top view of the tube cap.

FIG. 4 is a schematic diagram of an integrated design.

FIG. 5 is a photograph of the finished product of the kit and the detected fruit.

1: a sampling tube; 2: a tube cover; 3: an indication groove; 4: a base; 6: a sharp protrusion; 32: a test strip; 33: a quality control indication groove (C); 34: a result indicating groove (T); 41: sample and sample diluent mixture.

321: a quality control region; 322: and a detection area.

Detailed Description

The present invention will now be described more fully hereinafter with reference to the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.

The means used in the examples, unless otherwise specified, are all conventional in the art.

The reagents used in the examples were all commercially available unless otherwise specified.

FIG. 1 is a schematic diagram of a test strip. The test strip 32 of this embodiment is set as a quality control area 321 at a distance of 22mM from the bottom, and 60mM CuSO is added dropwise ₄ An aqueous solution. A detection area 322 is arranged at the position 11mM above the area, and a mixed aqueous solution of 4% (w/v) starch, 1% (w/v) chitosan quaternary ammonium salt, 2mM oxalic acid, 2.5M potassium iodide, 2.5mM 8-hydroxyquinoline and 1.0mM cysteine is dripped. After drying treatment at 60 ℃ for 30 minutes, the test strip can be directly used for sample detection, and can also be sealed, dried and stored at normal temperature for standby.

Fig. 2 is a schematic diagram of a test strip finished product and a detection result, 321: a quality control region; 322: and a detection area.

When a test strip is used for testing a sample to be tested, the sample is tested according to the following ratio of 1:4 volume ratio with a sample dilution of a mixed aqueous solution of 10mM potassium ferrocyanide, 3mM ammonium oxalate, 140mM sodium chloride, and immersing the lower end of the test strip in the sample solution without immersing a quality control zone (the limit thereof is a short line of about 1mM in width at the lower end of the test stripLabel) and then allowed to stand for about 90 seconds to observe the quality control zone and the detection zone. The quality control area is brown and indicates that the operation is effective; the detection area is obviously black brown; the result is positive, indicating H in the sample ₂ O ₂ The content is more than or equal to 0.5mM; the detection zone is only light in ground color or light gray, and the result is negative, indicating H in the sample ₂ O ₂ The content is less than 0.5mM.

To purify water or 25mM NaNO ₂ Is a negative control sample, 1.0mM H ₂ O ₂ The quality control areas of the 3 samples observed after the positive control samples are tested according to the above are brown, and the operation is indicated to be effective; the detection areas of the 2 negative control samples are all light ground color, and the indication result is negative; the detection area of the positive control sample is obviously black brown; the indication result is positive.

Further, calibrating a quality control area at a proper position at the lower end of a filter paper strip with proper size, and dripping 50-100mM copper sulfate aqueous solution with the width of 1-2 μl/mM according to the width of the filter paper strip; 1-2 mu l/mM of 2-8% (w/v) starch, 1-5% (w/v) chitosan quaternary ammonium salt, 2-10mM oxalic acid, 2-5M potassium iodide, 2-5mM 8-hydroxyquinoline and 1.0-1.5mM cysteine mixed aqueous solution are added dropwise above the solution at the position of 7-25mM, and then the solution is dried at 60 ℃ for 30 minutes; the composition of the sample diluent is 10-20mM potassium ferrocyanide, 2-5mM ammonium oxalate and 120-150mM sodium chloride mixed water solution, and the negative or positive control sample can be processed according to the steps to obtain the expected result.

As shown in fig. 3 to 5, an airway hydrogen peroxide rapid detection kit established according to the method of the present invention mainly includes a sampling tube 1 for integrating a test strip placed in an indication tank and a tube cover 2 for placing a sample diluent.

In one embodiment of the invention, the sampling tube 1 is a frosted ABS tube, and has the caliber: 25mm height: 90mm can stand, and the side is fixed with the instruction groove 3 that is used for holding the test strip, bore: height of 10 mm: 70mm, which has a passage with a caliber of 5mm only at the bottom and communicates with the inside of the sampling tube. The collecting pipe is provided with a pipe cover, and the collecting pipe can be screwed and sealed. After the sample detection is completed, the whole kit and the sample solution are in a tightly covered and sealed state, so that environmental pollution and cross infection are avoided.

The test strip 32 is fixedly arranged in the indication groove 3 and consists of 38mm multiplied by 5m (m length multiplied by width) filter paper and 50mm multiplied by 5mm back support plastic strips. The reagent in the quality control zone and the detection zone as described above is dried and fixed on the filter paper of the test strip at a distance of 22mm and 33mm from the lower end.

In one embodiment, a cylindrical sealing tube is fixed in the tube cover 2 for sealing the sample diluent, and the sealing film can be pierced by the sharp protrusion 12 on the upper edge of the sampling tube 1 to enable the diluent to fall into the tube bottom during the screwing process. In this example, the sample diluent volume was 4ml, and the composition was 10-20mM potassium ferrocyanide, 2-5mM ammonium oxalate, 140mM sodium chloride mixed aqueous solution.

In this embodiment, the test strip 32 is fixed in the indication groove 3 by adhesive, and the sample control line may not be provided.

The indication groove 3 is externally provided with a label, and a quality control indication groove (C) and a result indication groove (T) are arranged at the corresponding positions of the detection area and the quality control area of the test strip 32. After the sampling detection operation is finished according to the using steps, obvious tan can be observed in a window C, the sample is indicated to be qualified and the operation is qualified, and the result is reliable; if H in the sample ₂ O ₂ If the content is more than or equal to 0.5mM, obvious blackish brown color is observed in a T window after detection is completed.

The dimensions of the various structures in the above embodiments do not constitute a unique limitation of the present invention, and suitable dimensions may be selected according to the detection needs of the sample, the shape performance of the detection product, and the like.

The kit is mainly used for detecting whether an airway mucus sample provider contains trace airway hydrogen peroxide or not, so as to detect whether the risk of lung cancer is high or not. The specific using steps are as follows: (1) After rinsing with clear water, drinking a mouthful of water, beating the chest and back for 10 times, deeply breathing for 5 times, and forcefully cough for 6 times; (2) spitting the oral liquid into a collection tube; (3) Covering and screwing the tube cover, and shaking left and right for about 10s to fully mix the sample and the sample diluent; (4) standing for about 90s, and observing the result. The window C displays obvious brown color, indicates that the sample and operation are qualified, and the result is reliable; the T window shows obvious black brown positive, indicating H in the sample ₂ O ₂ The content is more than or equal to 0.5mM, has higher risk of lung cancer, and is suggested to go toHospitals and other specialized institutions conduct deep and relevant lung cancer examination.

The present invention will be described in further detail with reference to examples.

Example 1: preferred confirmation of test strip composition

The test strip consists of a filter strip of 38mm by 5mm in size (length by width) and a back support plastic strip of 50mm by 5mm. A quality control area (C) is marked at the position 22mm away from the bottom end of the filter paper strip, and 1 mul of copper sulfate aqueous solution is evenly dripped into the pipette tip; a detection area (T) is marked at the position 11mm above the detection area, and 3 mu l of starch, chitosan quaternary ammonium salt, oxalic acid, potassium iodide, 8-hydroxyquinoline and cysteine mixed water-soluble solution are uniformly dripped into the pipette tip. The detection zone and quality control zone reagent formulations are shown in table 3. The composition of the sample dilutions was: 15mM potassium ferrocyanide, 3mM ammonium oxalate, 100mM sodium chloride. The negative reference substance is purified water, and the positive reference substance is 1.0mM H ₂ O ₂ . The detection method is carried out according to instructions. After the operation is finished, the C area can be observed to be obvious brown, and the correct operation is indicated; no obvious color development is observed in the detection area of the negative control substance, and the detection result is negative; the positive control detection areas can all observe obvious blackish brown, and the positive detection results are indicated, so that the test strips prepared in the concentration range of the tested reagent can all reach the expected detection results (table 1).

Table 1 control test and statistics of test strip composition

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Example 2: sample diluent composition test

Test strip specification and preparation are described in the text of the specification and in example 1. 1 μl of 60mM copper sulfate solution was added dropwise to the quality control zone; the detection area was constituted by dropping 3. Mu.l of a mixed aqueous solution of 4% (w/v) starch, 1% (w/v) chitosan quaternary ammonium salt, 2mM oxalic acid, 2.5M potassium iodide, 2.5mM 8-hydroxyquinoline, and 1.0mM cysteine. The sample diluent formulations are shown in table 2. The negative reference substance is purified water, and the positive reference substance is 1.0mM H ₂ O ₂ . The detection method is carried out according to instructions. After the operation is finished, the C area can be observed to be obvious brown, and the correct operation is indicated; no obvious color development is observed in the detection area of the negative control substance, and the detection result is negative; the positive control detection areas can all observe obvious blackish brown, and the positive detection results are indicated, so that the expected detection results can be achieved in the tested sample diluent (table 2).

TABLE 2 composition of sample dilutions

Numbering device	Sample dilution composition	Remarks
			F1	10mM potassium ferrocyanide, 3mM ammonium oxalate, 140mM sodium chloride
F2	15mM potassium ferrocyanide, 3mM ammonium oxalate, 140mM sodium chloride
			F3	20mM potassium ferrocyanide, 3mM ammonium oxalate, 140mM sodium chloride
G1	10mM potassium ferrocyanide, 2mM ammonium oxalate, 140mM sodium chloride
			G2	10mM potassium ferrocyanide, 3.5mM ammonium oxalate, 140mM sodium chloride
G3	10mM potassium ferrocyanide, 5mM ammonium oxalate, 140mM sodium chloride
			H1	10mM potassium ferrocyanide, 3mM ammonium oxalate, 120mM sodium chloride
H2	10mM potassium ferrocyanide, 3mM ammonium oxalate, 130mM sodium chloride
			H3	10mM potassium ferrocyanide, 3mM ammonium oxalate, 150mM sodium chloride

Example 3: the influence of the common beverage and medicine remained in the sample on the judgment of the result

Test strip specification and preparation are described in the text of the specification and in example 1. 1 μl of 60mM copper sulfate solution was added dropwise to the quality control zone; the detection area was constituted by dropping 3. Mu.l of a mixed aqueous solution of 4% (w/v) starch, 1% (w/v) chitosan quaternary ammonium salt, 2mM oxalic acid, 2.5M potassium iodide, 2.5mM 8-hydroxyquinoline, and 1.0mM cysteine. The composition of the sample dilutions was: 10mM potassium ferrocyanide, 3mM ammonium oxalate, 140mM sodium chloride. The negative reference substance is purified water, and the positive reference substance is 1.0mM H ₂ O ₂ . The detection method is carried out according to instructions. The interfering substances tested are shown in Table 3. The content of the interfering substance in the sample is unified to be that the conventional dosage of the substance accounts for 2% of the volume of the sample, which is higher than the possible volume ratio of the residual quantity of the substance after the conventional use and the gargling in the sampling. 3 replicates were performed for each sample, for a total of 3 runs. The result observation statistics show that after the operation is finished, the C region can be observed to be obviously brown, and the correct operation is indicated; no obvious color development is observed in the detection area of the negative control substance, and the detection result is negative; the positive reference substance detection areas can all observe obvious blackish brown, and the positive detection result is indicated, so that the tested interfering substances do not influence the detection result in the tested concentration range, namely, the detection result is not interfered.

TABLE 3 interfering substances

Example 4: mass production of the kit of the invention

In this example 1200 sets of planned kit productions were performed. First, a lot number and a production schedule were prepared (table 4), and material preparation was performed (table 5).

Table 4.1200 set of schedule component tables

Numbering device	Component (A)	Specification of specification	1200 sets of schedule	Remarks
					KKG121	Sampling tube	Personal (S)	1200
KKG122	Pipe cover	Personal (S)	1200
					KKG123	Sample diluent	4.0mL	4800mL
KKG124	Test paper strip	Personal (S)	1200
					KKG125	Label (Label)	Personal (S)	1200

TABLE 5 bill of materials

Key procedure-test strip preparation: taking a pencil marking line as a quality control area from about 22mM at the bottom of 270g of common qualitative filter paper cut into a specification of 300mM multiplied by 60mM, taking a pencil marking line as a detection area from 11mM above the common qualitative filter paper, taking a pencil marking line as a detection area, marking a line by a sample application pen according to the text method of the specification, dropwise adding 60mM copper sulfate aqueous solution into the quality control area, and dropwise adding 4% (w/v) starch, 1% (w/v) chitosan quaternary ammonium salt, 2mM oxalic acid, 2.5M potassium iodide, 2.5mM 8-hydroxyquinoline and 1.0mM cysteine mixed aqueous solution into the detection area by the sample application pen marking line; after drying at 60 ℃ for 30 minutes, the filter paper strip is stuck and fixed on the PVC back plate, the lower end of the filter paper strip is 18mm longer than the lower end of the PVC back plate, and the filter paper strip is vertically cut into strips of 5mm. After the test strip is prepared, performing spot check of key working procedures to determine that the test strip quality is qualified.

Preparing and subpackaging a sample diluent: 4000ml of the sample diluent of the kit shown in the text of the specification is prepared, and the sample diluent is subpackaged into tube covers according to 4 ml/part and sealed by sealing films.

Assembling a sampling tube: sticking double-sided foam rubber on the back of the test strip, placing and sticking the test strip in a sampling tube indication groove, enabling the lower end of the test strip to be just close to the bottom of the tube, and sealing and fixing the sampling tube base on the tube body; and (3) reserving labels of a C window and a T window outside the indication groove, so that the C window and the T window respectively correspond to a quality control area and a detection area on the test strip. And after the workshop inspector checks the assembly to be qualified on site, attaching a qualification mark on the top of the indication groove. And (3) placing the tube cover and the sampling tube into a self-sealing bag provided with a drying agent, sealing, temporarily storing at room temperature to obtain a semi-finished product, and carrying out external packaging after the sampling inspection is qualified.

And (3) external packing: and (5) filling the semi-finished products and specifications which are subjected to the sampling inspection into an external packaging box, attaching a qualification certificate, and warehousing for later use.

Example 5: quality inspection of the kit of the invention

1. Key procedure spot check

And performing spot check on key procedures after the test strip is prepared, so as to determine that the test strip quality is qualified.

(1) Appearance of

The appearance size of the test strip is in accordance with the setting.

(2) Negative reference compliance

And (5) taking 10 enterprise negative reference products for detection, wherein the detection results are negative.

(3) Compliance rate of positive reference

And (5) taking 10 enterprise positive reference products for detection, wherein the detection results are positive.

(4) Detection limit

And (3) taking the enterprise detection limit reference to carry out repeated detection for 20 times, wherein the detection result of at least 19 times is positive.

2. Inspection of semifinished products

(1) Appearance of

The components are complete, the self-sealing bag and the drying agent are well packaged, no leakage, no damage and no moisture regain are caused; the label writing is clear, the top of the indication groove is stuck with a qualified mark, and the position of the window is matched with the corresponding area on the test strip.

(2) Negative reference compliance

And directly adopting a key procedure spot check result.

(3) Compliance rate of positive reference

And directly adopting a key procedure spot check result.

(4) Detection limit

And directly adopting a key procedure spot check result.

(5) Repeatability of

And (3) taking enterprise repetitive reference, and repeatedly detecting for 10 times, wherein the detection results are positive and the color development is uniform.

(6) Sample processing efficiency

Sampling according to the airway mucus sampling method disclosed by the invention, respectively detecting 5 negative samples obtained by mixing and sub-packaging airway mucus samples of 10 healthy volunteers and 5 positive samples obtained by mixing and sub-packaging airway mucus samples of 10 lung cancer patients, wherein the negative sample detection results are negative, and the positive sample detection results are positive.

3. Inspection of finished products

(1) Appearance of

The components are complete, the self-sealing bag and the drying agent are well packaged, no leakage, no damage and no moisture regain are caused; the label writing is clear, the top of the indication groove is stuck with a qualified mark, and the position of the window is matched with the corresponding area on the test strip. The total weight of the finished product should be more than or equal to 24.90g, which indicates that the sufficient amount of the reagent is stored.

(2) Performance of

After the appearance check is correct, the performance indexes of the key process spot check and the semi-finished product check are directly adopted.

Example 6: healthy volunteer test

The finished kit is shown in example 4. 50 healthy volunteers are collected on site in a park, the age is 20-40 years, the occupation is high-tech industry practitioners, the proportion of men and women is about 50%, the patients take comprehensive physical examination in one year, the patients have no abnormal conditions, the patients have no medical history in the severe smoking or severe pollution industry, the patients have no lung medical history such as lung cancer or asthma, and the patients have no related disease family history. Some women are in gestation or lactation period and feel good. The staff hosts the registration, dispenses the finished product of the kit, and carries out operation explanation demonstration. The test operation was then performed by the volunteer as described in the text of the present specification, and the result was reported to the staff. And after comparison and confirmation, carrying out result statistical analysis on the staff. The results show that 50 cases of detection windows C all show obvious tan, and the operation accuracy is 100%;50 cases of detection T windows have no obvious color development, are negative, and have low lung cancer risk.

Example 7: lung cancer patient testing

The finished kit is shown in example 4. The 20 volunteers of the lung cancer patients are collected, the ages are 40-70, the occupation is unlimited, the proportion of men and women is about 50%, and the lung cancer patients are comprehensively diagnosed and have not been subjected to operation treatment. Women are not in gestation or lactation. The staff hosts the registration, dispenses the finished product of the kit, and carries out operation explanation demonstration. If the throat is felt to have foreign bodies, after the throat is lightly cough and clear and expectoration is eliminated, the detection operation is carried out by volunteers according to the method described in the text of the specification, and the result is reported to staff. And after comparison and confirmation, carrying out result statistical analysis on the staff. The results show that 20 cases of detection windows C all show obvious tan, and the operation accuracy is 100%;20 cases of detection T windows all show obvious blackish brown, are positive and indicate an air passage H ₂ O ₂ Abnormally elevated levels have a higher risk of lung cancer.

Example 8: pulmonary nodule patient testing

The finished kit is shown in example 4. 50 volunteers with lung nodule collecting patients are aged 20-70 years, have unlimited occupation, have a proportion of about 50% of men and women, have nodules in the lung through low-dose spiral CT examination in the year and are comprehensively diagnosed as non-lung cancer. Women are not in gestation or lactation. The staff hosts the registration, dispenses the finished product of the kit, and carries out operation explanation demonstration. If the throat is felt to have foreign bodies, after the throat is lightly cough and clear and expectoration is eliminated, the detection operation is carried out by volunteers according to the method described in the text of the specification, and the result is reported to staff. And after comparison and confirmation, carrying out result statistical analysis on the staff. The results show that 50 cases of detection windows C all show obvious tan, and the operation accuracy is 100%;50 cases of test T windows have no obvious color development, are negative, and have low lung cancer risk.

Example 9: upper respiratory tract inflammation patient test

The finished kit is shown in example 4. 30 patients suffering from common cold, cough, rhinitis and sphagitis are respectively collected, the ages are 20-40, the occupation is unlimited, the proportion of men and women is about 50%, the patients take comprehensive physical examination within one year, no other abnormal conditions, no medical history in heavy smoking or severe pollution industries, no pulmonary medical history such as lung cancer or asthma, and no family history of related diseases. Women are not in gestation or lactation. The staff hosts the registration, dispenses the finished product of the kit, and carries out operation explanation demonstration. After volunteers cleaned the nasal discharge, and were lightly couched to clear the throat and eliminate the expectoration, the test procedure was performed as described in the text of this specification and the results reported to the staff. And after comparison and confirmation, carrying out result statistical analysis on the staff. The results show that 90 cases of detection windows C all show obvious tan, and the operation accuracy is 100%; the 90 cases of detection T windows have no obvious color development, are all negative, and have low lung cancer risk.

Example 10: lung cancer examination screening application

The finished kit is shown in example 4. A staff is arranged in the physical examination center and is responsible for registering forms and issuing and recycling the reagent kit, necessary answering guidance is carried out when a subject is in question, and propaganda explanation videos are rolled and played. The using method of the kit is described in the text of the specification. Physical examination subjects can follow up, taking an average of about 5 minutes. Multiple persons can be self-tested in parallel when the persons arrive at the scene, and 30 persons can only take about 10 minutes for detection. From this estimate, detection was completed about 1440 times for 1 day (8 hours). Low-dose spiral CT generally requires 3-5 minutes, but subjects typically need to wait in line, 30 human tests take about 150 minutes, and 96 human tests can be completed in 8 hours. The invention can improve the screening efficiency of physical examination by about 15 times. Furthermore, generally low dose spiral CT results take 1-3 days, whereas the present invention can result in 5 minutes.

Example 11: community application

The finished kit is shown in example 4. Putting 100 sets of finished products of the kit in a test point community supermarket, pasting propaganda posters with two-dimension codes, enabling a user to scan the codes by a mobile phone to watch video explanation, acquiring the kit on site to automatically detect after online registration, performing detection operation according to the method disclosed by the specification of the invention, and updating the detection result online. Summarizing and counting detection results, counting positive according to the reported results of users, and counting negative according to the reported results of users and not reported results; the logical basis is positive, so that the user's communication desire can be stimulated, the report missing rate is very low, and negative can reduce the user's communication desire, and the report missing rate is high. Statistics show that 2 cases of positive cases are provided, the positive rate is 2%, and the lung cancer incidence rate is higher than that of the common people (0.08%). Considering that the trial users 70% are the lung nodule population, a lung nodule positive rate of 3.6% and a total positive rate of 2% is reasonable. The community hospitals are important construction contents of the health of the whole people, and at present, lung cancer screening cannot be performed, and the community hospitals need to go to a central hospital for examination, so that lung cancer screening is not facilitated. The kit is convenient for residents to participate in lung cancer screening in community hospitals, and is hopeful to develop into a powerful supplement for the health construction of the residents.

Example 12: household self-test application

The finished kit is shown in example 4. 30 lung nodule volunteers were collected and tested by home self using the kit of the invention 1 time a month, and were sent to the hospital for review on a regular basis following the order. Volunteers age 20-70 years, with a proportion of about 50% for men and women. The user returns the detection result through the network or collects the use condition by the phone call or WeChat follow-up visit of the staff and registers the detection result. The result statistics shows that 2 persons are positive, wherein one person is a female, the person is 28 years old, the lung nodule is found to be 4mm in the current annual physical examination, the first 3 kits are negative, the fourth kit is tested to be positive, the patient goes to a hospital for rechecking, the upper right lung nodule is increased to be 6.5mm, the doctor comprehensively judges that the surgical treatment is recommended, the general anesthesia thoracoscope right lung nodule wedge excision is carried out, and the micro-infiltration gonadal cancer is diagnosed in the pathology. Another man, 35 years old, has a smoking history of 12 years, the previous annual physical examination finds out that the lung nodule is 3mm, the previous 10 times of kit is negative, the 11 th kit test is positive, the patient goes to a hospital for review, she Jiejie on the left lung is increased to 5mm, and the doctor comprehensively judges that the operation treatment is recommended, and the pathological diagnosis is micro-infiltration gonadal cancer. The CT review follow-up path is complex and various, has long period, and is unfavorable for timely diagnosis of lung nodules and timely discovery of lung cancer. The invention is convenient for home self-test tracking of patients and is beneficial to promoting the patients to go to the hospital for rechecking.

Example 13: kit preservation and stability test

The finished kit is shown in example 4. The sampling tube (containing test strip) is sealed, dried and assembled in the finished product package of the kit. The preservation condition of the kit is that the room temperature is 25+/-5 ℃ or the refrigeration temperature is low, and the humidity is less than or equal to 60%. The negative reference is prepared by mixing airway mucus samples of healthy volunteers and sub-packaging with 1 ml/part, and H is shown by standard absorbance method ₂ O ₂ The content is less than 0.5mM. The positive reference is prepared by mixing airway mucus samples of volunteers of lung cancer patients and subpackaging 1 ml/part, and H is shown by standard absorbance method ₂ O ₂ The content is more than or equal to 1.5mM. The repetitive reference was 1.5mM H ₂ O ₂ The detection limit reference is 0.5mM H ₂ O ₂ . The detection method is carried out according to instructions. Each batch was tested 3 times each, 5 times at the limit of detection, and 2 batches per month. After 4 times of detection in the 2 nd month, preliminary statistics show that the coincidence rate of the negative/positive/repeatability detection limit reaches 100%, and meets the quality requirement.

Example 14: portability and transport stability

The finished kit is shown in example 4. Kit storage conditions and references were the same as in example 13. Taking 3 batches of kits, storing at a high temperature of 40 ℃ for 3 days, and then, falling the kits from 1.5m height, repeating the three times for 3 times, performing shock drop tests, observing whether the external package, the internal package and the like of the kits are damaged, and then, testing by using a negative reference, a positive reference and a detection limit reference. Results: only one kit of one batch has damaged outer package part, no damage to inner package, and detection statistics show that the female/male/repeatability detection limit meets 100% and meets the quality requirement.

Claims

1. The method is characterized in that after continuous deep cough is carried out by rinsing with clear water, liquid in oral cavity is collected in a sampling tube, sample diluent is added for mixing, standing is carried out, hydrogen peroxide released by a sample is measured by suction filtration through a test strip fixed with upper and lower 2 area indicators, a quality control area is arranged in the lower area, and a brown indication result is effectively displayed by color change; the upper area is a detection area, and the color change shows that the black brown color is a positive result, and indicates that the hydrogen peroxide content in the sample reaches or exceeds a detection threshold.

2. The method for detecting airway mucus hydrogen peroxide according to claim 1, wherein the test strip is a filter strip with a proper size, and a 50-100mM copper sulfate aqueous solution with a proper position at the lower end is dried according to the width of the filter strip with a concentration of 1-2 μl/mM; a mixed aqueous solution of 1-2. Mu.l/mM of 2% -8% (w/v) starch, 1% -5% (w/v) chitosan quaternary ammonium salt, 2-10mM oxalic acid, 2-5M potassium iodide, 2-5mM 8-hydroxyquinoline and 1.0-1.5mM cysteine was dried 7-25mM above the region.

3. The method for detecting airway mucus hydrogen peroxide according to claim 1, wherein the sample diluent comprises a mixed aqueous solution of 10-20mM potassium ferrocyanide, 2-5mM ammonium oxalate and 120-150mM sodium chloride.

4. The airway mucus hydrogen peroxide detection kit consists of a sampling tube integrated with an indication groove, a tube cover, a sample diluent, a test paper strip, a label and the like. Wherein the test strip is a core component and comprises a filter paper strip with the size of 38mM multiplied by 5mM multiplied by 0.7mM (length multiplied by width multiplied by thickness), and 1 mul of 60mM copper sulfate aqueous solution is dried at the position 22mM away from the lower end of the filter paper strip; at 33mM from its lower end, 3. Mu.l of a mixed aqueous solution of 4% (w/v) starch, 1% (w/v) chitosan quaternary ammonium salt, 2mM oxalic acid, 2.5M potassium iodide, 2.5mM 8-hydroxyquinoline, 1.0mM cysteine was dried.

5. The airway mucus hydrogen peroxide test kit according to claim 4, wherein the sample diluent is sealed in a tube cap by a film that is pierced by a spike attached to the upper end of the inner portion of the sample tube, and comprises a mixed aqueous solution of 10mM potassium ferrocyanide, 3mM ammonium oxalate, and 140mM sodium chloride.

6. The airway mucus hydrogen peroxide detection kit according to claim 4, wherein the use process is that after continuous deep cough with clear water, liquid in the oral cavity is spitted into the sampling tube, the tube cover is covered and screwed up, the tube body is shaken to mix the sample diluent flowing out of the tube cover with the sample, and after standing for 2 minutes, the indication groove is observed: the lower window is a quality control area (C), and the brown indication result is effectively displayed in a color-changing manner; the upper window is a detection area (T), the color change shows that the black brown color is a positive result, the hydrogen peroxide content in the sample reaches or exceeds a detection threshold value, and the detected person is in an airway hydrogen peroxide metabolism abnormal state.

7. An airway mucus hydrogen peroxide assay kit according to claim 4, characterised in that the limit of detection is 0.5mM.