A test paper for detecting concentration of o-phthalaldehyde disinfectant

Abstract

The invention relates to a test paper for detecting concentration of o-phthalaldehyde disinfectant in the field of detection technology. The test paper comprises a bottom layer made of a hydrophobic material; a microfluidic layer arranged on the bottom layer, and a plurality of functional areas are divided by hydrophobic boundaries; an intelligent response detection layer arranged on the microfluidic layer, and comprising temperature-responsive hydrogel microspheres and a signal enhancer; a self-calibration layer arranged on the microfluidic layer; a sample input area at one end of the test paper, and provided with a water absorption layer; and a color-variable indicating element arranged above the intelligent response detection layer, the self-calibration layer and an integrated colorimetric card. When in use, a sample is added to the sample input area and activated with ultraviolet light. After the reaction is completed, a result can be obtained by visual comparison or scanning with an intelligent device. The invention has the advantages of high sensitivity, strong specificity, simple operation, rapid detection, accurate results, and the like, and is suitable for rapid detection of o-phthalaldehyde concentration in multiple fields such as medical care, food safety, and environmental monitoring.

Description

Technical Field

[0001] The invention relates to a test paper for detecting the concentration of an o-phthalaldehyde disinfectant, in particular to a test paper for detecting the concentration of an o-phthalaldehyde disinfectant applied in the technical field of detection. Background Art

[0002] O-Phthalaldehyde is a widely used and highly effective disinfectant, which is widely used in the fields of medical device disinfection. Accurate and rapid detection of its concentration is crucial to ensure the disinfection effect and safety. There are some methods for detecting the concentration of o-Phthalaldehyde in the prior art, but there are still some limitations.

[0003] Chinese invention patent CN107024473B discloses a rapid detection test paper for the concentration of o-phthalaldehyde disinfectant and its preparation method and application. The test paper is detected by immersing a solution of a specific component in filter paper. This method is simple to operate and can quickly detect the content of o-phthalaldehyde, which is beneficial for medical staff to monitor the concentration of disinfectant at any time. However, the detection sensitivity and specificity of this method still have room for improvement, and it is difficult to achieve accurate quantitative analysis.

[0004] Chinese invention patent CN106970075B discloses another o-phthalaldehyde concentration detection test paper and its preparation method. The test paper uses the reaction principle of sodium bisulfite and o-phthalaldehyde to judge whether the concentration reaches the specified threshold by color change. This method is simple and convenient to use, and the results are accurate and reliable. However, it is mainly limited to the judgment of a specific threshold (0.35%), which is difficult to meet the detection needs of different concentration ranges.

[0005] The above design realizes the rapid detection of o-phthalaldehyde concentration through the principle of chemical reaction, but it still has certain limitations, such as insufficient detection sensitivity, limited specificity, difficulty in achieving accurate quantification, and narrow detection range. In addition, the existing technology lacks intelligence and data management functions, and it is difficult to meet the needs of modern medical and industrial fields for data tracking and analysis.

[0006] In practical applications, different scenarios may require different detection sensitivities and ranges. For example, in the process of medical device disinfection, it is necessary to accurately determine whether the concentration of o-phthalaldehyde has reached an effective disinfection level; while in environmental monitoring, it may be necessary to detect lower concentrations of o-phthalaldehyde residues. Therefore, it is of great practical significance to develop a detection method with high sensitivity, wide detection range, quantitative analysis and simple operation. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is how to develop a test strip for detecting the concentration of o-phthalaldehyde disinfectant with high sensitivity, high specificity, wide detection range, quantitative analysis and easy operation.

[0008] In order to solve the above problems, the present invention provides a test paper for detecting the concentration of o-phthalaldehyde disinfectant, comprising a bottom layer made of a hydrophobic material;

[0009] A microfluidic layer disposed on the bottom layer, wherein the microfluidic layer is divided into a plurality of functional areas by hydrophobic boundaries;

[0010] A smart response detection layer disposed on the microfluidic layer, wherein the smart response detection layer comprises temperature-responsive hydrogel microspheres and a signal enhancer;

[0011] A self-calibration layer disposed on the microfluidic layer;

[0012] A sample input area located at one end of the test paper, wherein the sample input area is provided with a water absorbing layer;

[0013] A result display area located at the other end of the test paper, wherein the result display area is provided with an integrated colorimetric card;

[0014] A color-variable indicating element is arranged above the intelligent response detection layer, the self-calibration layer and the integrated colorimetric card.

[0015] In the above-mentioned test paper for detecting the concentration of o-phthalaldehyde disinfectant, the detection sensitivity and specificity are improved, the detection range is expanded, the needs of different application scenarios are met, the self-calibration function is integrated, the reliability and accuracy of the detection results are improved, and the visualization and intelligent detection methods are combined. It can be quickly judged by the naked eye and accurate quantitative analysis can be achieved through intelligent devices. It provides data management functions to facilitate the recording, storage and analysis of test results.

[0016] As a further improvement of the present application, the multiple functional areas include a sample input area, a reaction area, a self-calibration area and a result display area.

[0017] As a further improvement of the present application, the bottom layer is made of at least one material selected from polyethylene terephthalate (PET), polypropylene (PP) or polyethylene (PE);

[0018] The microfluidic layer is made of at least one hydrophilic porous membrane material selected from polyvinylidene fluoride (PVDF) membrane, nitrocellulose membrane or cellulose acetate membrane;

[0019] The hydrophobic boundary is formed of at least one material selected from the group consisting of wax, polydimethylsiloxane (PDMS), and photocurable resin.

[0020] As a further improvement of the present application, the temperature-responsive hydrogel microspheres are prepared by copolymerization of N-isopropylacrylamide (NIPAM) and at least one crosslinking agent selected from N,N'-methylenebisacrylamide or N,N'-ethylenebisacrylamide.

[0021] As another improvement of the present application, the temperature-responsive hydrogel microspheres encapsulate functionalized nanogold particles with a particle size of 5-100 nm, the surface of which is modified with thiol compounds; an enzyme system comprising aldehyde dehydrogenase, a coenzyme and an electron acceptor; and a photosensitizer.

[0022] As another improved supplement of the present application, the aldehyde dehydrogenase is selected from aldehyde dehydrogenases derived from bacteria or fungi;

[0023] The coenzyme is nicotinamide adenine dinucleotide (NAD+) or its reduced form (NADH);

[0024] The electron acceptor is selected from at least one of tetrazolium blue (NBT), dichloroindophenol (DCIP) or methylene blue;

[0025] The photosensitizer is selected from at least one of azobenzene derivatives, spiropyran derivatives, and diarylethene derivatives.

[0026] As another improved supplement of the present application, the signal enhancer is selected from at least one of graphene quantum dots, carbon quantum dots or semiconductor quantum dots;

[0027] When the signal enhancer is graphene quantum dots, its particle size is 1-20nm and the emission wavelength is 400-700nm;

[0028] The self-calibration layer contains a preset o-phthalaldehyde solution of known concentration, the concentration range of which is 0.1%-1.0%;

[0029] The water-absorbing layer is made of at least one of modified cellulose, glass fiber or polyester fiber.

[0030] As another improvement of the present application, the integrated colorimetric card comprises a plurality of color regions, each color region corresponding to a predetermined o-phthalaldehyde concentration range;

[0031] The color-variable indicator element is a two-dimensional code or a bar code printed with ink that changes with the concentration of o-phthalaldehyde;

[0032] It also includes a peelable transparent protective layer covering the surface of the test paper, and the protective layer is made of at least one material selected from polyethylene, polypropylene or polyethylene terephthalate.

[0033] A method for preparing a test paper for detecting the concentration of o-phthalaldehyde disinfectant comprises the following steps:

[0034] S1. Creating microfluidic channels using hydrophobic material printing technology on hydrophilic porous membranes;

[0035] S2, preparing temperature-responsive hydrogel microspheres comprising functionalized gold nanoparticles, an enzyme system and a photosensitized enzyme inhibitor;

[0036] S3, applying the hydrogel microsphere suspension to the reaction zone;

[0037] S4, applying a signal enhancer on the hydrogel microsphere layer;

[0038] S5 adds a known concentration of o-phthalaldehyde standard solution to the self-calibration area;

[0039] S6. Setting an integrated colorimetric card and a color-changing indicator element;

[0040] S7, assembling the bottom layer, the treated hydrophilic porous membrane and the water absorbing layer;

[0041] S8, covering with a protective layer;

[0042] S9, treating the coated test paper using freeze drying technology, wherein the freeze drying is performed at -40°C to -60°C, 0.01-0.1 mbar, for 6-24 hours;

[0043] The method for preparing the hydrogel microspheres in step S2 comprises:

[0044] M1, in the presence of a water-oil surfactant, dispersing N-isopropylacrylamide monomer, a cross-linking agent, functionalized nano-gold particles, an enzyme system and a photosensitizer in an oil phase;

[0045] M2. Under nitrogen protection, add initiator to carry out polymerization reaction;

[0046] M3. Centrifuge to obtain hydrogel microspheres, and wash with deionized water.

[0047] A method for using a test paper for detecting concentration of o-phthalaldehyde disinfectant, comprising the following steps:

[0048] B1. Add the o-phthalaldehyde sample to be tested to the sample input area;

[0049] B2. Irradiate the test paper with light of wavelength 350-400nm for 5-15 seconds to activate the detection reaction;

[0050] B3. Wait for 30 seconds to 5 minutes at 15-30°C to allow the reaction to complete;

[0051] B4. Observe the color changes of the reaction area and the self-calibration area, and compare the colors with the integrated colorimetric card;

[0052] B5. Use smart devices to scan the color-changing indicator element to obtain quantitative results

[0053] B6. Use a dedicated application on a smart device to analyze the color change of the color-changing indicator element, calculate the precise concentration of o-phthalaldehyde, and record, store and transmit the test results.

[0054] In summary, this application has the following beneficial effects:

[0055] 1. High sensitivity and specificity; the temperature-responsive hydrogel microspheres combined with the specific enzyme system achieve highly selective detection of o-phthalaldehyde, with a high theoretical detection limit that meets strict detection requirements and a wide linear detection range that covers the common concentration ranges in practical applications.

[0056] 2. Quick and easy operation: The detection process is short, which improves the detection efficiency. The operation steps are simple. Only the sample needs to be added and simple illumination is required. No complicated sample pre-treatment is required. It is suitable for non-professionals and is conducive to promotion and application.

[0057] 3. Combination of visualization and intelligence; the integrated colorimetric card supports rapid visual semi-quantitative analysis to meet the needs of rapid on-site detection. The color-changing QR code cooperates with smart devices to achieve precise quantitative analysis and provide professional-level test results. It can flexibly meet different usage scenarios and accuracy requirements and enhance the applicability of the product.

[0058] 4. Self-calibration and anti-interference; The built-in self-calibration area significantly improves the reliability of the test results. The design of temperature-responsive hydrogel microspheres and functionalized nano-gold particles enhances the anti-interference ability, effectively reduces the impact of environmental factors (such as temperature and humidity), and improves the detection accuracy.

[0059] 5. Stability and portability: Freeze-drying and a peelable protective layer improve the stability and shelf life of the test paper. It is small and light, easy to carry and store, suitable for rapid on-site testing, and expands its application scenarios.

[0060] 6. Data management and scalability; through scanning with smart devices, the test results can be easily recorded, stored and transmitted. Dedicated applications support long-term data management and analysis, which helps to establish a test database. The technical platform has good scalability and can be used for other types of aldehyde detection by replacing the specific enzyme system. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is an overall exploded view of this application;

[0062] Figure 2 A top view of the present application;

[0063] Figure 3 For this application Figure 2 Middle AA section view;

[0064] Figure 4For this application Figure 3 Middle BB section view;

[0065] Figure 5 This is the appearance structure diagram of this application.

[0066] Description of the numbers in the figure:

[0067] 1. Bottom layer; 2. Microfluidic layer; 3. Intelligent response detection layer; 4. Self-calibration layer; 5. Water absorption layer; 6. Integrated colorimetric card. DETAILED DESCRIPTION

[0068] Three implementation modes of the present application are described in detail below with reference to the accompanying drawings.

[0069] Embodiment 1:

[0070] Figure 1-Figure 5 As shown, this embodiment provides a test paper for detecting the concentration of o-phthalaldehyde disinfectant, and its structure includes a bottom layer 1, a microfluidic layer 2, an intelligent response detection layer 3, a self-calibration layer 4 and a color-changing indicator element from bottom to top. A sample input area is provided at one end of the test paper, and a result display area is provided at the other end. The detailed description is as follows:

[0071] The bottom layer 1 is made of a hydrophobic material, which mainly plays a supporting and waterproofing role. In this embodiment, the bottom layer 1 is made of polyethylene terephthalate (PET) material with a thickness of 0.1 mm. PET has excellent mechanical strength and chemical stability, and can provide good support for the entire test paper system. In addition, polypropylene (PP) or polyethylene (PE) can also be used as alternative materials, which have good hydrophobicity and chemical resistance.

[0072] The microfluidic layer 2 is arranged on the bottom layer 1 and is made of a hydrophilic porous membrane material. In this embodiment, a polyvinylidene fluoride (PVDF) membrane is selected with a pore size of 0.45 μm and a thickness of 100 μm. The PVDF membrane has good chemical stability and mechanical strength and is suitable for use as a base material for the microfluidic layer 2. In addition, nitrocellulose membrane or cellulose acetate membrane can also be used as an alternative.

[0073] The microfluidic layer 2 is divided into multiple functional areas by hydrophobic boundaries, including the sample input area, reaction area, self-calibration area and result display area. The hydrophobic boundary is made by melt wax printing technology, using paraffin as the raw material. The melting point of wax is about 60°C. During printing, the molten wax is sprayed to the predetermined position through a precision inkjet printer, and the hydrophobic boundary is formed after cooling. The width of these boundaries is 0.5mm and the height is about 1.5 times the thickness of the PVDF membrane, that is, 150μm. In addition to wax, polydimethylsiloxane (PDMS) or photocurable resin can also be used as a hydrophobic boundary material.

[0074] The intelligent response detection layer 3 is arranged above the reaction area of ​​the microfluidic layer 2, and is mainly composed of temperature-responsive hydrogel microspheres and a signal enhancer. The detailed composition and preparation method of the hydrogel microspheres will be described in detail in the subsequent embodiments. The signal enhancer is a graphene quantum dot with a particle size of 5 nm and an emission wavelength of 520 nm.

[0075] The self-calibration layer 4 is disposed above the self-calibration area of ​​the microfluidic layer 2 and contains a preset o-phthalaldehyde solution of known concentration. In this embodiment, a 0.5% concentration o-phthalaldehyde standard solution is selected, and the volume is 2 μL.

[0076] The sample input area is located at one end of the test paper and is provided with a water absorbing layer 5. The water absorbing layer 5 is made of modified cellulose material and has a thickness of 0.5 mm, which can effectively absorb and evenly distribute the sample to be tested.

[0077] The result display area is located at the other end of the test paper and is provided with an integrated colorimetric card 6. The colorimetric card contains 6 color areas, corresponding to 0%, 0.2%, 0.4%, 0.6%, 0.8% and 1.0% o-phthalaldehyde concentrations respectively. Each color area is 3mm×3mm in size and is printed with anti-fading ink.

[0078] The color-changing indicator element is arranged above the intelligent response detection layer 3, the self-calibration layer 4 and the integrated colorimetric card 6. In this embodiment, a photosensitive color-changing ink that can change with the concentration of o-phthalaldehyde is selected to print the QR code. It is located next to the result display area.

[0079] To prevent the test paper from getting wet or contaminated, a peelable transparent protective layer is covered on the entire surface of the test paper. The protective layer is made of a polyethylene (PE) film with a thickness of 0.05mm and can be easily torn off when in use.

[0080] The o-phthalaldehyde disinfectant concentration test strip of this embodiment realizes automatic flow, accurate response and convenient reading of samples through reasonable design and material selection. The design of the microfluidic layer 2 ensures the orderly flow of samples between the functional areas, the intelligent response detection layer 3 provides a highly sensitive detection mechanism, and the self-calibration layer 4 and the integrated colorimetric card 6 further improve the accuracy of the detection. The introduction of the variable color indicator element allows the test results to be quickly read and analyzed by intelligent devices, greatly improving the convenience of use.

[0081] The following is a detailed working process of the o-phthalaldehyde disinfectant concentration detection test paper described in Example 1, sample addition; the user first tears off the PE protective layer on the surface of the test paper, and then drops about 50 μL of the o-phthalaldehyde disinfectant sample to be tested into the sample input area of ​​the test paper.

[0082] Sample flow: Due to the capillary action of the water-absorbing layer 5, the sample is quickly absorbed and begins to flow to the right along the microfluidic layer 2. The hydrophobic boundary effectively controls the flow direction and speed of the sample and prevents the sample from diffusing to unintended areas.

[0083] Reaction process: When the sample flows to the reaction zone, the temperature-responsive hydrogel microspheres in the smart response detection layer 3 begin to change. The aldehyde dehydrogenase encapsulated in the microspheres reacts specifically with o-phthalaldehyde, and the coenzyme NAD+ is reduced to NADH. NADH then reacts with an electron acceptor (such as NBT) to produce a visible blue-purple precipitate.

[0084] Signal enhancement: The light signal generated during the reaction is enhanced by the graphene quantum dots, which increases the sensitivity of the detection. The strong fluorescence emission of the graphene quantum dots at a wavelength of 520nm further enhances the visibility of the color change.

[0085] Self-calibration: The sample flows through the self-calibration area at the same time, and reacts with the preset 0.5% o-phthalaldehyde standard solution. This process provides an internal reference for each test and helps to eliminate the influence of environmental factors (such as temperature and humidity).

[0086] The self-calibration process can correct the impact of environmental factors (such as temperature and humidity) on the test results by comparing with a standard solution of known concentration. Specifically, by comparing the color change of the self-calibration area with the color change of the reaction area, a correction factor can be calculated. Applying this correction factor to the result of the reaction area can obtain a more accurate concentration value of o-phthalaldehyde.

[0087] Results display: After about 2-3 minutes, the sample reaches the result display area. The user can directly compare the color changes of the reaction area and the self-calibration area with the integrated colorimetric card 6 to estimate the concentration of o-phthalaldehyde with the naked eye.

[0088] Quantitative analysis: To obtain more accurate quantitative results, users can use a smartphone to scan the color-changing indicator element (QR code). The color change of the QR code is proportional to the concentration of o-phthalaldehyde, and the concentration of o-phthalaldehyde in the sample can be accurately calculated through a dedicated mobile phone application.

[0089] The design of the enzymatic reaction and temperature-responsive hydrogel microspheres in the smart response detection layer 3 ensures high sensitivity and specificity of the detection. The theoretical detection limit can reach 0.01% of o-phthalaldehyde concentration.

[0090] The entire detection process only takes 2-3 minutes, shortening the time required for traditional detection methods.

[0091] When using, you only need to add samples, no other complicated operations are required, which lowers the threshold for use.

[0092] Through visual comparison and intelligent device scanning, rapid qualitative judgment and accurate quantitative analysis are achieved simultaneously.

[0093] The built-in self-calibration area improves the reliability of the test results and reduces interference from environmental factors.

[0094] The color-changing indicator element (QR code) is not only used for result reading, but also serves as an anti-counterfeiting mark to prevent counterfeit products.

[0095] By scanning with smart devices, test results can be conveniently recorded, stored and transmitted, which facilitates long-term data management and analysis.

[0096] This test paper is not only suitable for the medical and health field, but can also be used for rapid detection of phthalaldehyde concentration in many fields such as food safety and environmental monitoring.

[0097] Embodiment 2:

[0098] Figure 1-Figure 5 It is shown that this embodiment mainly describes the detailed composition and preparation method of the intelligent response detection layer 3 in the o-phthalaldehyde disinfectant concentration detection test paper.

[0099] Composition and preparation of temperature-responsive hydrogel microspheres, raw material composition; main monomer: N-isopropylacrylamide (NIPAM), mass fraction 95%, cross-linker: N,N'-methylenebisacrylamide, mass fraction 5%, initiator: ammonium persulfate (APS), concentration 0.1 mol / L, surfactant: sodium dodecyl sulfate (SDS), concentration 0.2 mmol / L.

[0100] Preparation steps: dissolve 0.95g NIPAM and 0.05g N,N'-methylenebisacrylamide in 50mL deionized water; add 10mg SDS and stir evenly; pass nitrogen into the solution for 20 minutes to deoxygenate; heat the reaction mixture in a 70°C water bath; quickly add 1mL 0.1mol / LAPS solution to start the polymerization reaction; react at 70°C for 4 hours; cool to room temperature and dialyze for 48 hours to remove unreacted monomers and impurities; freeze-dry to obtain temperature-responsive hydrogel microspheres.

[0101] Preparation of functionalized gold nanoparticles; Raw materials; chloroauric acid (HAuCl4), concentration 1mmol / L; sodium citrate, concentration 38.8mmol / L; 3-mercaptopropionic acid, concentration 1mmol / L;

[0102] Preparation steps: heat 50mL HAuCl4 solution to boiling; quickly add 5mL sodium citrate solution and continue stirring; continue boiling reaction for 15 minutes, the solution changes from light yellow to wine red; cool to room temperature, add 1mL 3-mercaptopropionic acid solution, stir at room temperature for 2 hours; centrifuge purification, and resuspend in deionized water; the functionalized nanogold particles finally obtained have an average particle size of 15nm.

[0103] Composition of the enzyme system; aldehyde dehydrogenase activity ≥ 5U / mg protein; coenzyme β-nicotinamide adenine dinucleotide (NAD+), purity ≥ 98%; electron acceptor tetrazolium blue (NBT), purity ≥ 98%;

[0104] Photosensitizer; the azobenzene derivative 4-((4-hydroxyphenyl)azo)benzoic acid is selected, with a purity of ≥97%.

[0105] Preparation of smart response detection layer 3; disperse 1 mg of temperature-responsive hydrogel microspheres in 1 mL of PBS buffer (pH 7.4); add 100 μL of functionalized gold nanoparticle solution and incubate at room temperature for 2 hours; add aldehyde dehydrogenase (final concentration 0.5 U / mL), NAD+ (final concentration 1 mmol / L) and NBT (final concentration 0.5 mmol / L); finally add photosensitizer (final concentration 0.1 mmol / L); gently apply the mixture on the reaction area of ​​the test paper and dry at room temperature.

[0106] Signal enhancer; graphene quantum dots are selected as the signal enhancer, and its characteristics are as follows; average particle size: 5nm; emission wavelength: 520nm; quantum yield: 15%; a graphene quantum dot solution with a concentration of 1mg / mL is evenly sprayed on the smart response detection layer 3 at a dosage of 5μL / cm².

[0107] Self-calibration layer 4; carrier: cellulose acetate membrane, pore size 0.22 μm; standard solution: 0.5% o-phthalaldehyde aqueous solution.

[0108] Preparation method: Add 2 μL of 0.5% o-phthalaldehyde standard solution onto a 1 cm² cellulose acetate membrane; vacuum dry at 37°C for 1 hour; fix the dried membrane on the self-calibration area of ​​the test paper.

[0109] Integrated colorimetric card 6; number of color areas: 6, corresponding concentration ranges: 0%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%.

[0110] Production method: Use color management software to design standard color blocks corresponding to each concentration; use anti-fading ink and high-precision screen printing technology to print the color blocks on a PET film; cut the printed colorimetric card and fix it in the result display area of ​​the test paper.

[0111] Method for making a color-changing indicator element; designing a QR code containing product information and a serial number; preparing a photochromic ink: mixing a photosensitive dye (such as spiropyran) with a polymer matrix (such as polymethyl methacrylate); using microjet printing technology to print the photochromic ink on a test paper in a QR code pattern; and curing under 365nm ultraviolet light for 10 seconds.

[0112] Method for making the protective layer: select food-grade PE film; cut the film into the same size as the test paper; apply low-viscosity peelable glue on one side of the film; cover the test paper surface with the glue-coated side facing down, and press lightly to ensure the fit; design an easy-tear pull tab on the exposed part of the protective layer to facilitate peeling during use.

[0113] Overall assembly; according to the basic structure described in Example 1, the bottom layer 1, the microfluidic layer 2 and the intelligent response detection layer 3 are assembled; the self-calibration layer 4 is fixed at a specified position; the integrated colorimetric card 6 is pasted on the result display area; the QR code is printed using photosensitive color-changing ink; and finally, the protective layer is covered.

[0114] When the o-phthalaldehyde disinfectant sample is added to the input end of the test paper, the sample flows toward the detection area along the microfluidic channel due to capillary action.

[0115] Temperature response and enzymatic reaction: After the sample reaches the intelligent response detection layer 3, the temperature-responsive hydrogel microspheres begin to change. At room temperature (about 25°C), the microspheres are in an expanded state, allowing the o-phthalaldehyde molecules in the sample to enter the interior of the microspheres.

[0116] Enzyme catalyzed reaction: Aldehyde dehydrogenase inside the microsphere reacts specifically with o-phthalaldehyde. In the presence of NAD+, o-phthalaldehyde is oxidized to phthalic acid, while NAD+ is reduced to NADH.

[0117] Color reaction; the generated NADH then reacts with the electron acceptor NBT, which is reduced to form bluish purple formazan. This process produces a visible color change.

[0118] Signal enhancement: Graphene quantum dots act as signal enhancers and enhance the light signal of the reaction product through the fluorescence resonance energy transfer (FRET) effect, thereby improving the sensitivity of detection.

[0119] Light-controlled regulation; photosensitizers (azobenzene derivatives) play a regulatory role in the detection process. When conducting the test, the user needs to irradiate the test paper with 365nm ultraviolet light for about 10 seconds, which will cause the inhibitor configuration to change, release the inhibition of the enzyme, and thus start the detection reaction. Photosensitizers (such as 4-((4-hydroxyphenyl)azo)benzoic acid) are in the trans configuration under dark conditions and can inhibit the activity of aldehyde dehydrogenase. When exposed to 365nm ultraviolet light, cis-trans isomerization occurs, turning into a cis configuration, losing the inhibitory effect on the enzyme, and thus starting the detection reaction. This design can accurately control the start time of the reaction and improve the accuracy and reproducibility of the test.

[0120] Self-calibration process: The sample flows through the self-calibration area at the same time, and reacts with the preset 0.5% o-phthalaldehyde standard solution. This process provides an internal reference standard for each test.

[0121] Reading the results: After about 2-3 minutes, the user can read the results in two ways: directly compare the color changes in the reaction area and self-calibration area with the integrated colorimetric card 6 for semi-quantitative analysis, and use a smartphone to scan the color-changing QR code for quantitative analysis through a dedicated application.

[0122] The design of the temperature-responsive hydrogel microspheres makes the detection highly specific. The microspheres swell at room temperature, allowing the target molecules to enter, and shrink after the reaction is completed, effectively blocking interfering substances. The theoretical detection limit can reach 0.01% of o-phthalaldehyde concentration.

[0123] By optimizing the composition and reaction conditions of the enzyme system, the detection range can cover 0.1%-1.0% of o-phthalaldehyde concentration, meeting the needs of most practical applications.

[0124] The surface modification of functionalized gold nanoparticles improves the stability of the system, reduces nonspecific adsorption, and enhances anti-interference ability.

[0125] Graphene quantum dots act as signal enhancers and significantly improve detection sensitivity through the FRET effect, allowing low-concentration samples to be accurately detected.

[0126] The introduction of photosensitizing enzyme inhibitors enables precise control of the detection process, avoids premature reaction of the test strip during storage, and extends the shelf life of the test strip.

[0127] The built-in self-calibration area improves the reliability of the test results, effectively reduces the interference of environmental factors (such as temperature and humidity), and improves the accuracy of the test.

[0128] The integrated colorimetric card 6 allows for rapid semi-quantitative analysis, while the color-changeable QR code supports precise quantitative analysis via smart devices, meeting the needs of different usage scenarios.

[0129] Embodiment 3:

[0130] Figure 1-Figure 5 It is shown that this embodiment describes in detail the preparation method and use method of the o-phthalaldehyde disinfectant concentration detection test paper.

[0131] Step S1, create a microfluidic channel, use CAD software to design the microfluidic channel pattern, input the design pattern into a wax printer, fix the PVDF membrane on the printing platform, heat the paraffin to 65°C to melt it, use precision inkjet technology to print the molten paraffin on the PVDF membrane, and naturally cool it to room temperature to form a hydrophobic boundary.

[0132] Step S2. Preparation of temperature-responsive hydrogel microspheres

[0133] Step M1. Prepare the reaction mixture, oil phase: cyclohexane 50 mL, aqueous phase: deionized water 10 mL, dissolve the following components, N-isopropylacrylamide (NIPAM): 950 mg, N,N'-methylenebisacrylamide: 50 mg, functionalized nanogold particles: 1 mL (concentration 1 mg / mL), aldehyde dehydrogenase: 10 mg, NAD+: 20 mg, NBT: 10 mg, photosensitizer (4-((4-hydroxyphenyl)azo)benzoic acid): 5 mg, surfactant: Span80, 1 mL, mix and disperse by ultrasonication for 5 minutes.

[0134] Step M2. Polymerization reaction: Add the mixture in M1 into a three-necked flask containing the oil phase, introduce nitrogen for 15 minutes to deoxygenate, stir in a 70°C water bath at 500 rpm, quickly add 0.1 mL of 0.1 mol / L ammonium persulfate (APS) solution to start polymerization, and react for 4 hours.

[0135] Step M3. Post-treatment: After the reaction is completed, the mixture is cooled to room temperature, centrifuged at 6000 rpm for 10 minutes, the precipitate is collected, washed alternately with deionized water and ethanol for 3 times, and finally dispersed in 10 mL of PBS buffer (pH 7.4).

[0136] Step S3. Applying hydrogel microspheres: Use a micropipette to evenly apply 100 μL of the hydrogel microsphere suspension on the reaction area and dry naturally at room temperature for 2 hours.

[0137] Step S4. Apply a signal enhancer, prepare a graphene quantum dot solution with a concentration of 1 mg / mL, use a sprayer to evenly spray 5 μL / cm² of the quantum dot solution on the hydrogel microsphere layer, and dry it at room temperature for 30 minutes.

[0138] Step S5. Add standard solution, prepare 0.5% o-phthalaldehyde standard solution, use a micropipette to drop 2 μL of the standard solution into the self-calibration area, and dry in a 37°C oven for 1 hour.

[0139] Step S6. Set up the colorimetric card and indicator element, use anti-fading ink, print the integrated colorimetric card 6 on the PET film by screen printing technology, use photosensitive color-changing ink, print the QR code next to the result display area by micro-jet printing technology, and cure it under 365nm ultraviolet light for 10 seconds.

[0140] Step S7. Assemble the layers, place the bottom layer 1 (PET film, thickness 0.1 mm) on the clean workbench, accurately align the treated PVDF membrane with the bottom layer 1 and paste it, and paste the water-absorbing layer 5 modified cellulose, thickness 0.5 mm, on the sample input area.

[0141] Step S8. Cover the protective layer, cut the PE protective film to a size slightly larger than the test paper, cover the PE film on the surface of the test paper, press lightly to ensure the fit, and design an easy-tear pull tab at one end of the protective layer.

[0142] Step S9. Freeze drying treatment: Place the assembled test paper on the freeze drying machine tray, set the parameters: temperature -50°C, pressure 0.05mbar, freeze drying, time 12 hours, after completion, seal the test paper and store it away from light.

[0143] Use process;

[0144] Step B1. Sample addition: tear off the protective film on the surface of the test paper, use a pipette to absorb 50 μL of the o-phthalaldehyde sample to be tested, and drop the sample into the input area of ​​the test paper.

[0145] Step B2. Light activation: use a UV flashlight with a wavelength of 365 nm to irradiate the test paper at a distance of 5 cm for 10 seconds.

[0146] Step B3. Wait for the reaction. Place the test paper at room temperature (about 25°C) for 3 minutes and wait for the reaction to complete.

[0147] Step B4. Visual comparison: observe the color changes of the reaction area and the self-calibration area, compare the colors with the integrated colorimetric card 6, and estimate the approximate concentration range of o-phthalaldehyde.

[0148] Step B5. Smart Scan, open the dedicated mobile app, use the mobile phone camera to aim at the color-changing QR code, keep the phone stable, and wait for the app to automatically scan and recognize it.

[0149] Step B6. Data analysis and management. The application automatically analyzes the color changes of the QR code, calculates the precise concentration of o-phthalaldehyde according to the preset algorithm, and displays the concentration value and judgment result on the application interface. The user can choose to save the test results, including date, time, concentration value and other information. The application provides a data export function, which can share or transmit the results in CSV or PDF format.

[0150] The following are the main chemical reactions and chemical formulas involved in the o-phthalaldehyde disinfectant concentration test paper:

[0151] Reaction 1: Oxidation of o-phthalaldehyde (catalyzed by aldehyde dehydrogenase):

[0152]

[0153] in:

[0154] represents o-phthalaldehyde

[0155] Phthalic acid

[0156] Nicotinamide adenine dinucleotide (oxidized form)

[0157] NADH is nicotinamide adenine dinucleotide (reduced form)

[0158] Reaction 2: Reduction reaction of NADH and NBT (tetranitrotetrazolyl blue):

[0159] NADH+NBT (oxidized form, yellow) → +NBT (reduced form, blue-purple)

[0160] Here, the reduction of NBT forms bluish-purple formazan.

[0161] Reaction 3: Photoisomerization reaction of photosensitizing enzyme inhibitors (taking azobenzene derivatives as an example):

[0162] Azobenzene (trans, inhibitory activity) + →Azobenzene (cis, no inhibitory activity)

[0163] in Represents the energy of a photon.

[0164] Reaction 4: Polymerization reaction of hydrogel microspheres:

[0165]

[0166]

[0167] in:

[0168] N-isopropylacrylamide (NIPAM)

[0169] N,N'-methylenebisacrylamide (cross-linking agent)

[0170] Reaction 5: Surface modification reaction of gold nanoparticles:

[0171]

[0172] Wherein HS-R represents a thiol compound, and R is an organic group.

[0173] These chemical reactions together constitute the working principle of the o-phthalaldehyde disinfectant concentration test strip, achieving high sensitivity and specificity detection. Reactions 1 and 2 are the core detection reactions, reaction 3 controls the start of the detection, and reactions 4 and 5 occur during the preparation process and are used to construct the smart response materials required for detection.

[0174] This embodiment describes in detail the whole process of the o-phthalaldehyde disinfectant concentration test paper from preparation to use. By accurately controlling each step, the high quality and stability of the test paper are guaranteed. At the same time, the simplified use process and intelligent result analysis make this detection method suitable for professionals and also convenient for ordinary users to use. This test paper has a wide range of application prospects in the fields of medical health, food safety, environmental monitoring, etc.

[0175] In view of current practical needs, the above-mentioned implementation mode adopted in this application is not limited to the scope of protection. Various changes made within the knowledge scope of technical personnel in this field without departing from the concept of this application still fall within the scope of protection of the present invention.

Claims

1. A test paper for detecting the concentration of o-phthalaldehyde disinfectant, Features: comprising a base layer (1) made of a hydrophobic material; A microfluidic layer (2) disposed on the bottom layer (1), the microfluidic layer (2) being divided into a plurality of functional areas by hydrophobic boundaries; the plurality of functional areas comprising a sample input area, a reaction area, a self-calibration area and a result display area; An intelligent response detection layer (3) disposed on the microfluidic layer (2), the intelligent response detection layer (3) comprising temperature-responsive hydrogel microspheres and a signal enhancer; the intelligent response detection layer (3) is disposed above the reaction area of ​​the microfluidic layer (2); A self-calibration layer (4) is arranged on the microfluidic layer (2); the self-calibration layer (4) is arranged above the self-calibration area of ​​the microfluidic layer (2) The sample input area is located at one end of the test paper, and the sample input area is provided with a water absorbing layer (5); A result display area located at the other end of the test paper, wherein the result display area is provided with an integrated colorimetric card (6); A color-variable indicating element disposed above the intelligent response detection layer (3), the self-calibration layer (4) and the integrated colorimetric card (6); The bottom layer (1) is made of at least one material selected from the group consisting of polyethylene terephthalate (PET), polypropylene (PP) and polyethylene (PE); The microfluidic layer (2) is made of at least one hydrophilic porous membrane material selected from the group consisting of polyvinylidene fluoride (PVDF) membrane, nitrocellulose membrane, and cellulose acetate membrane; The hydrophobic boundary is formed of at least one material selected from the group consisting of wax, polydimethylsiloxane (PDMS), or a photocurable resin; The temperature-responsive hydrogel microspheres are prepared by copolymerization of N-isopropylacrylamide (NIPAM) and at least one crosslinking agent selected from N,N'-methylenebisacrylamide or N,N'-ethylenebisacrylamide; The temperature-responsive hydrogel microspheres contain functionalized nano-gold particles with a particle size of 5-100 nm, the surface of which is modified with a thiol compound; an enzyme system comprising aldehyde dehydrogenase, a coenzyme and an electron acceptor; and a photosensitizer. The aldehyde dehydrogenase is selected from aldehyde dehydrogenases of bacterial or fungal origin; The coenzyme is nicotinamide adenine dinucleotide (NAD+) or its reduced form (NADH); The electron acceptor is selected from at least one of tetrazolium blue (NBT), dichloroindophenol (DCIP) or methylene blue; The photosensitizer is selected from at least one of azobenzene derivatives, spiropyran derivatives or diarylethene derivatives; The signal enhancer is selected from at least one of graphene quantum dots, carbon quantum dots or semiconductor quantum dots; When the signal enhancer is graphene quantum dots, its particle size is 1-20nm and the emission wavelength is 400-700nm; The self-calibration layer (4) contains a preset o-phthalaldehyde solution of known concentration, the concentration of which ranges from 0.1% to 1.0%; The water-absorbing layer (5) is made of at least one of modified cellulose, glass fiber or polyester fiber; The integrated colorimetric card (6) comprises a plurality of color regions, each color region corresponding to a predetermined o-phthalaldehyde concentration range; The color-variable indicator element is a two-dimensional code or a bar code printed with ink that changes with the concentration of o-phthalaldehyde; It also includes a peelable transparent protective layer covering the surface of the test paper, and the protective layer is made of at least one material selected from polyethylene, polypropylene or polyethylene terephthalate.

2. A method for preparing a test paper for detecting concentration of o-phthalaldehyde disinfectant according to claim 1, Features: The steps include: S1. Creating microfluidic channels using hydrophobic material printing technology on hydrophilic porous membranes; S2, preparing temperature-responsive hydrogel microspheres comprising functionalized gold nanoparticles, an enzyme system and a photosensitized enzyme inhibitor; S3, applying the hydrogel microsphere suspension to the reaction zone; S4, applying a signal enhancer on the hydrogel microsphere layer; S5 adds a known concentration of o-phthalaldehyde standard solution to the self-calibration area; S6, providing an integrated colorimetric card (6) and a color-variable indicator element; S7, assembling the bottom layer (1), the treated hydrophilic porous membrane and the water absorbing layer (5); S8, covering with protective layer; S9, treating the coated test paper using freeze drying technology, wherein the freeze drying is performed at -40°C to -60°C, 0.01-0.1 mbar, for 6-24 hours; The method for preparing the hydrogel microspheres in step S2 comprises: M1, in the presence of a water-oil surfactant, dispersing N-isopropylacrylamide monomer, a cross-linking agent, functionalized nano-gold particles, an enzyme system and a photosensitizer in an oil phase; M2. Under nitrogen protection, add initiator to carry out polymerization reaction; M3. Centrifuge to obtain hydrogel microspheres, and wash with deionized water.

3. A method for using the o-phthalaldehyde disinfectant concentration detection test paper according to claim 1, Features: The steps include: B1. Add the o-phthalaldehyde sample to be tested to the sample input area; B2. Irradiate the test paper with light of wavelength 350-400nm for 5-15 seconds to activate the detection reaction; B3. Wait for 30 seconds to 5 minutes at 15-30°C to allow the reaction to complete; B4. Observe the color changes of the reaction area and the self-calibration area, and compare the colors with the integrated colorimetric card (6); B5. Use smart devices to scan the color-changing indicator element to obtain quantitative results B6. Use a dedicated application on a smart device to analyze the color change of the color-changing indicator element, calculate the precise concentration of o-phthalaldehyde, and record, store and transmit the test results.

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