Fluorescence-labeled allergen rapid detection method for hygienic products
By combining fluorescently labeled allergen-specific antibodies with microfluidic chips, the problems of long detection time, complex operation and inability to conduct multiple tests on sanitary product allergens have been solved, achieving fast, simple and highly sensitive detection results.
Patent Information
- Application Number
- CN202511214145.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-10-17
AI Technical Summary
The existing methods for detecting allergens in sanitary products have the problems of long detection time, complex operation, requirement of professional equipment, cumbersome pre-treatment and inability to perform multiple tests, making it difficult to achieve fast, simple and highly sensitive detection.
The method of combining fluorescently labeled allergen-specific antibodies with microfluidic chips is adopted. Fluorescently labeled antibodies are prepared by chemical coupling and fixed on the surface of the reaction chamber of the microfluidic chip. Sanitary product samples are directly processed and the fluorescence signal is monitored in real time using a photoelectric sensor to achieve simultaneous detection of multiple allergens.
It realizes the rapid, simple and highly sensitive detection of allergens in sanitary products, and can complete the detection of multiple allergens within 10 minutes. It is suitable for production line quality inspection and family self-inspection.
Smart Images
Figure CN120801270A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of sanitary product detection, in particular to a fluorescently labeled allergen rapid detection method for sanitary products. BACKGROUND
[0002] Allergens (such as perfumes, preservatives, chemical additives, etc.) in sanitary products (such as sanitary napkins, diapers, wet wipes, etc.) can cause allergic reactions in users, so rapid and accurate allergen detection is crucial for product quality control. Existing detection methods mainly include enzyme-linked immunosorbent assay (ELISA), immunoblotting, polymerase chain reaction (PCR), etc., but these methods have the following shortcomings: 1. Long detection time: usually takes several hours to several days to obtain results.
[0003] 2. Complex operation: requires professional operation and relies on large equipment (such as centrifuge, fluorescence microscope).
[0004] 3. Complicated pretreatment: requires sample digestion, extraction, etc., increasing the risk of contamination.
[0005] 4. Cannot detect multiple allergens simultaneously: low efficiency.
[0006] For example, the existing ELISA method for detecting allergens requires more than 2 hours and requires professional laboratory equipment. Although microfluidic technology can improve detection speed, it is mainly used for serum or food detection and has not been optimized for direct detection of sanitary products. In addition, traditional fluorescence detection methods require complex sample processing and are not suitable for on-site rapid detection. SUMMARY
[0007] (I) Technical problems solved To overcome the shortcomings of the prior art, the present application provides a fluorescently labeled allergen rapid detection method for sanitary products, which can quickly, simply and sensitively detect allergens in sanitary products without complex pretreatment and can simultaneously detect multiple allergens, solving the problems raised in the background art.
[0008] (II) Technical solutions To achieve the above purpose, the present application is implemented by the following technical solutions: a fluorescently labeled allergen rapid detection method for sanitary products, comprising the following steps: Step 1: Prepare fluorescently labeled allergen-specific antibodies: Combine fluorescent labels with allergen-specific antibodies by chemical coupling to form stable fluorescently labeled antibody complexes; Step 2: Fix the antibodies on a microfluidic chip: Fixing the fluorescently labeled antibody on the surface of the reaction chamber of the microfluidic chip; Step three: sample processing and immune reaction: Introducing the sanitary product sample or its extract into the chip, combining with the fixed fluorescently labeled antibody to form an "allergen-antibody-fluorescent label" immune complex; Step four: fluorescence signal detection: Illuminating the detection area by an excitation light source, monitoring the fluorescence intensity in real time by a photosensor, and generating the detection result by combining with a data analysis module.
[0009] Preferably, in step one, the fluorescent label is quantum dots, fluorescein or rhodamine, wherein the emission wavelength range of quantum dots is 525-620 nm, the excitation wavelength of fluorescein is 488 nm, and the emission wavelength is 520 nm.
[0010] Preferably, in the preparation process of the fluorescently labeled antibody, the molar ratio of the antibody to the fluorescent label is 1:5-20, and unbound labels are removed by gel filtration chromatography or centrifugal purification.
[0011] Preferably, in step two, the microfluidic chip contains independent reaction chambers and multi-channel detection areas, each reaction chamber is fixed with fluorescently labeled antibodies against different allergens, and at least two allergens can be detected simultaneously.
[0012] Preferably, the surface of the reaction chamber of the microfluidic chip is fixed with fluorescently labeled antibodies by physical adsorption or chemical coupling, wherein the chemical coupling method includes EDC / NHS coupling or silanization treatment.
[0013] Preferably, the volume of the reaction chamber of the microfluidic chip is 10-50 μL, and the flow rate is controlled at 5-20 μL / min, ensuring that the immune reaction is completed within 5 minutes.
[0014] Preferably, in step three, the sanitary product sample does not need to be digested or extracted, and can be directly processed by the following methods: (1) Cutting the sample (such as sanitary napkin, paper diaper) and directly putting it into the chip sample channel; (2) Soaking the sample in physiological saline and taking the supernatant as the detection liquid.
[0015] Preferably, in step four, the excitation light source is an LED light source with a wavelength of 405 nm, 460 nm or 530 nm, corresponding to the excitation spectrum of different fluorescent labels.
[0016] Preferably, in step four, the data analysis module has a built-in standard curve, and whether the allergen exists is determined by a fluorescence intensity threshold, and the threshold is determined by calibrating the known concentration of the allergen standard.
[0017] (Three) beneficial effects The present application provides a fluorescently labeled allergen rapid detection method for sanitary products, with the following beneficial effects: 1. By combining fluorescently labeled antibodies with microfluidic technology, rapid and high-sensitivity detection of allergens in sanitary products is achieved.
[0018] 2. This method does not require complex pretreatment and can complete detection within 10 minutes, making it suitable for production line quality inspection and home self-checking, with the advantages of being fast, simple, and multi-detection. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 The present application provides a fluorescently labeled allergen rapid detection method for sanitary products, with the following beneficial effects: Figure 2 The present application provides a fluorescently labeled allergen rapid detection method for sanitary products, with the following beneficial effects: DETAILED DESCRIPTION
[0020] The technical solutions in the embodiments of the present application will be described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.
[0021] Embodiment one
[0022] As shown in the present application, a fluorescently labeled allergen rapid detection method for sanitary products is provided, including the following steps: Figure 1 Step one: preparation of fluorescently labeled allergen-specific antibodies: The fluorescent label is combined with the allergen-specific antibody by chemical coupling method to form a stable fluorescently labeled antibody complex. The fluorescent label is quantum dots, fluorescein or rhodamine, with the emission wavelength range of quantum dots being 620 nm, the excitation wavelength of fluorescein being 488 nm, and the emission wavelength being 520 nm. In addition, the molar ratio of the antibody to the fluorescent label in the preparation process of the fluorescently labeled antibody is 1:20, and the unbound label is removed by gel filtration chromatography or centrifugal purification. Step two: immobilization of antibodies on microfluidic chip: Step three: sample preparation: The fluorescently labeled antibody is fixed on the surface of the reaction chamber of the microfluidic chip; wherein the microfluidic chip comprises independent reaction chambers and a multi-channel detection area, each reaction chamber fixes a fluorescently labeled antibody specific to a different allergen, enabling simultaneous detection of at least two allergens; secondly, the surface of the reaction chamber of the microfluidic chip fixes the fluorescently labeled antibody by physical adsorption or chemical coupling, wherein the chemical coupling method includes EDC / NHS coupling or silanization treatment; and the volume of the reaction chamber of the microfluidic chip is 50 μL, the flow rate is controlled at 20 μL / min, ensuring that the immune reaction is completed within 5 minutes; Step three: sample processing and immune reaction: The sanitary product sample or its extract is introduced into the chip, combined with the fixed fluorescently labeled antibody, and forms an "allergen-antibody-fluorescent label" immune complex; wherein the sanitary product sample does not need to be digested or extracted, and is directly processed by the following methods: (1) The sample (such as sanitary napkin, paper diaper) is cut and directly placed into the chip sample channel; (2) The sample is soaked in physiological saline, and the supernatant is taken as the detection liquid; Step four: fluorescence signal detection: The detection area is irradiated by an excitation light source, and the fluorescence intensity is monitored in real time by a photoelectric sensor, and the detection result is generated by combining a data analysis module; wherein the excitation light source is an LED light source with a wavelength of 405 nm, 460 nm or 530 nm, corresponding to the excitation spectrum of different fluorescent labels; secondly, the data analysis module has a built-in standard curve, which determines whether the allergen exists by the fluorescence intensity threshold, and the threshold is determined by the known concentration of the allergen standard.
[0023] Example two
[0024] As shown in the Figure 2 embodiments of the present application provide a fluorescently labeled allergen rapid detection method for sanitary products, comprising the following steps: 1. Preparation of fluorescently labeled antibody The fluorescent dye (such as quantum dots, fluorescein) is labeled on the allergen-specific antibody by chemical coupling method to prepare the fluorescently labeled antibody. For example, 1-ethyl-3-(3-dimethylaminopropyl) carbonyl diimide (EDC) and N-hydroxysuccinimide (NHS) are used to couple quantum dots with anti-histamine antibodies to form a stable fluorescently labeled antibody complex.
[0025] 2. Design of microfluidic chip The chip comprises a sample channel, a reaction chamber and a detection area. The sample channel is used to introduce the sanitary product extract or directly contact the sample surface; the reaction chamber pre-fixes the fluorescently labeled antibody, which combines with the allergen to form an immune complex; the detection area integrates a fluorescence detector to monitor the fluorescence signal in real time.
[0026] 3. Detection steps (1) Sample preparation: Put the sanitary product sample directly into the sample channel of the chip, or soak it in physiological saline and take the supernatant. No digestion or extraction is needed to avoid the loss of allergens.
[0027] (2) Immune reaction: The sample flows through the reaction chamber, and the allergen binds with the fluorescently labeled antibody to form an "allergen-antibody-fluorescent marker" complex. The unbound antibody is removed by the flushing liquid.
[0028] (3) Fluorescence detection: The excitation light source (such as LED) irradiates the detection area, and the fluorescent marker emits fluorescence, which is detected by the photosensor. The signal is processed by the data analysis module to generate the detection result.
[0029] 4. Multi-detection expansion Multiple independent reaction chambers are set up on the chip, each chamber fixing fluorescently labeled antibodies of different allergens, allowing simultaneous detection of multiple allergens (such as fragrances, preservatives, latex proteins, etc.).
[0030] Example Three: Fragrance allergen detection in sanitary napkins 1. Preparation of fluorescently labeled antibodies Quantum dots (emission wavelength 620 nm) are labeled with anti-coumarin antibodies by EDC / NHS coupling method to prepare fluorescently labeled antibodies.
[0031] 2. Assembly of microfluidic chip Fluorescently labeled antibodies are fixed on the surface of the chip reaction chamber, and blue LED (excitation wavelength 460 nm) and photosensor are integrated in the detection area.
[0032] 3. Detection steps: (1) Take 1 cm² of sanitary napkin sample and put it directly into the sample channel.
[0033] (2) Inject physiological saline (containing 0.1% Tween 20) to push the sample extract into the reaction chamber and incubate for 5 minutes.
[0034] (3) Flush the unbound antibody with PBS, and irradiate the detection area with the excitation light source to detect the fluorescence intensity.
[0035] (4) If the fluorescence intensity exceeds the threshold value (determined by the standard curve), it is determined to be positive.
[0036] Example Four: Multi-detection of preservative allergens in paper diapers 1. Preparation of fluorescently labeled antibodies Quantum dot-labeled anti-methylparaben antibody (emission wavelength 525 nm) and anti-phenoxyethanol antibody (emission wavelength 620 nm) were prepared, respectively.
[0037] 2. Microfluidic chip design The chip contains two independent reaction chambers, respectively fixing two kinds of fluorescently labeled antibodies. The detection area is set with a double-channel filter, corresponding to two kinds of fluorescent wavelengths.
[0038] 3. Detection steps (1) After the diaper sample was soaked in physiological saline, the supernatant was injected into the sample channel.
[0039] (2) The extract flowed through the two reaction chambers at the same time, and incubated for 8 minutes.
[0040] (3) After washing, the light source emitted 405 nm and 460 nm light in turn, and the two kinds of fluorescent signals were detected.
[0041] (4) According to the fluorescence intensity ratio, the type and concentration of preservative were determined.
[0042] Example Five: Detection of Latex Protein Allergen in Wet Wipes 1. Preparation of fluorescently labeled antibodies FITC-labeled anti-latex protein monoclonal antibody was prepared: FITC was dissolved in dimethyl sulfoxide (DMSO), mixed according to the molar ratio of antibody to FITC 1:10, reacted in 0.1 mol / L carbonate buffer (pH 9.0) at 4℃ for 8 hours, and the unbound FITC was removed by dialysis to obtain fluorescently labeled antibodies (excitation wavelength 490 nm, emission wavelength 520 nm).
[0043] 2. Optimization of microfluidic chip The surface of the chip reaction chamber was modified with aminopropyl triethoxysilane (APTES) to covalently fix the fluorescently labeled antibodies, enhancing the stability of the antibodies; the sample channel inlet was designed as a "sponge adsorption head" that could directly adhere to the surface of the wet wipe to adsorb the sample liquid (without cutting the sample).
[0044] 3. Detection steps (1) The surface of the wet wipe sample was contacted with the chip sponge adsorption head for 30 seconds, and the sample liquid entered the channel by capillary action.
[0045] (2) The sample liquid was driven to flow through the reaction chamber, and the latex protein combined with the fluorescently labeled antibody, incubated for 4 minutes.
[0046] (3) Wash with PBS containing 0.05% Tween 20 for 2 minutes to remove unbound antibodies.
[0047] (4) Adopt 490nm LED excitation, detect 520nm place fluorescence intensity, when intensity≥1500 count unit, judge as latex protein positive (threshold value passes 0.1ng / mL latex protein standard calibration).
[0048] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely divergences of the principles and spirit of the application and that numerous modifications, changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the application as defined by the appended claims and their equivalents.
Claims
1. A method for rapid detection of fluorescently labeled allergens in sanitary products, characterized by: The following steps are involved: Step 1: Preparation of fluorescently labeled allergen-specific antibodies: The fluorescent marker is combined with the allergen-specific antibody through chemical coupling to form a stable fluorescent-labeled antibody complex; Step 2: Immobilize antibodies on the microfluidic chip: Immobilizing the fluorescently labeled antibody on the surface of the reaction chamber of the microfluidic chip; Step 3: Sample processing and immune response: The sanitary product sample or its extract is introduced into the chip and combined with the fixed fluorescent-labeled antibody to form an "allergen-antibody-fluorescent marker" immune complex; Step 4: Fluorescence signal detection: The detection area is illuminated by an excitation light source, the fluorescence intensity is monitored in real time using a photoelectric sensor, and the detection results are generated in combination with a data analysis module.
2. The method for rapid detection of fluorescently labeled allergens in sanitary products according to claim 1, characterized in that: In step 1, the fluorescent marker is quantum dots, fluorescein or rhodamine, wherein the emission wavelength range of quantum dots is 525-620 nm, the excitation wavelength of fluorescein is 488 nm, and the emission wavelength is 520 nm.
3. The method for rapid detection of fluorescently labeled allergens in sanitary products according to claim 2, characterized in that: During the preparation of the fluorescent-labeled antibody, the molar ratio of the antibody to the fluorescent marker is 1:5-20, and unbound markers are removed by gel filtration chromatography or centrifugal purification.
4. The method for rapid detection of fluorescently labeled allergens in sanitary products according to claim 1, characterized in that: In step 2, the microfluidic chip comprises independent reaction chambers and multi-channel detection areas. Each reaction chamber is fixed with fluorescently labeled antibodies targeting different allergens, thereby achieving simultaneous detection of at least two allergens.
5. The method for rapid detection of fluorescently labeled allergens in sanitary products according to claim 4, characterized in that: The surface of the reaction chamber of the microfluidic chip is fixed with fluorescent-labeled antibodies by physical adsorption or chemical coupling, wherein the chemical coupling method includes EDC / NHS coupling or silanization treatment.
6. The method for rapid detection of fluorescently labeled allergens in sanitary products according to claim 5, characterized in that: The reaction chamber volume of the microfluidic chip is 10-50 μL, and the flow rate is controlled at 5-20 μL / min, ensuring that the immune reaction is completed within 5 minutes.
7. The method for rapid detection of fluorescently labeled allergens in sanitary products according to claim 1, characterized in that: In step 3, the sanitary product samples are processed directly by the following methods without digestion or extraction: (1) Cut the sample (such as sanitary napkins, diapers) and place it directly into the chip sample channel; (2) Soak the sample in physiological saline and take the supernatant as the test solution.
8. The method for rapid detection of fluorescently labeled allergens in sanitary products according to claim 1, characterized in that: In step 4, the excitation light source is an LED light source with a wavelength of 405 nm, 460 nm or 530 nm, corresponding to the excitation spectra of different fluorescent markers.
9. The method for rapid detection of fluorescently labeled allergens in sanitary products according to claim 1, characterized in that: In step 4, the data analysis module has a built-in standard curve, and determines whether the allergen is present based on a fluorescence intensity threshold, which is determined by calibration with an allergen standard of known concentration.