A nonadienal specific detection test paper and a preparation method thereof

Abstract

The application provides a nonadienal specific detection test paper and a preparation method thereof. The test paper comprises a non-fluorescent quantitative filter paper base material, a polydimethylsiloxane hydrophobic layer loaded on the surface of the base material, and a reagent layer loaded on the surface of the hydrophobic layer. The reagent layer comprises a chromogenic agent 2,4-dinitrophenylhydrazine and an acid regulator for providing an acidic environment with a pH of 2.0-2.5. The test paper of the application realizes high specificity and high sensitivity detection of nonadienal by isolating water interference through the hydrophobic layer and combining the acid reagent layer with a stabilizer. The detection time is only 1-2 minutes, and the effective period at room temperature is up to 90 days or more, which is significantly better than the prior art and is suitable for instant detection of biological samples such as scalp odor.

Description

Technical Field

[0001] This invention relates to the field of test strip technology, specifically to a nonadienal specific test strip and its preparation method. Background Technology

[0002] Nonadienal (2-Nonenal) is a characteristic aldehyde compound produced by the oxidative degradation of unsaturated fatty acids in human sebum, and is widely recognized as a major source of "age-related odor" and scalp odor. Accurate and rapid detection of nonadienal is of great significance for personal health care and the evaluation of the efficacy of daily chemical products. Currently, the detection of nonadienal mainly relies on large-scale instrumental methods such as gas chromatography-mass spectrometry (GC-MS) or high-performance liquid chromatography (HPLC). While these methods offer high accuracy, they require expensive equipment, specialized operators, and complex sample pretreatment processes, resulting in lengthy processing times and making them unsuitable for rapid on-site detection.

[0003] Based on the principle that 2,4-dinitrophenylhydrazine (2,4-DNPH) specifically condenses with an aldehyde group under acidic conditions to form colored hydrazone compounds, the industry has attempted to develop it into a rapid test strip. However, existing 2,4-DNPH-based test strip products or solution methods have a series of prominent defects in practical applications, making them difficult to commercialize and use daily.

[0004] Specifically, the existing technology has the following main technical defects:

[0005] Firstly, the reagents exhibit extremely poor stability and require stringent storage conditions. Because conventional filter paper substrates are hydrophilic, they readily absorb moisture from the air, causing the loaded 2,4-DNPH reagent to clump and lose activity due to moisture. Therefore, existing test strip products typically require sealed storage in a refrigerator, and their effective shelf life at room temperature after opening is generally less than 48 hours. This is even more inconvenient for solution-based methods, requiring reagent preparation before testing, which is cumbersome and results in significant reagent waste.

[0006] Secondly, the detection reaction is slow, resulting in an excessively long overall time consumption. The condensation reaction of 2,4-DNPH with aldehydes requires a strong acidic catalytic environment. Existing technologies either employ a solution reaction system (requiring a reaction time of 10-15 minutes) or suffer from low reaction efficiency due to the inability of the test strip to effectively maintain a local acidic environment. In addition, the preliminary sampling requires multiple steps such as wiping, transferring, and eluting with cotton swabs, making the total time for a single test as long as 15-20 minutes, which is difficult to meet the needs of immediate and rapid detection.

[0007] Third, it has weak resistance to matrix interference. When testing scalp or skin samples, the samples inevitably contain complex matrices such as sebum and sweat. When existing hydrophilic test strips come into contact with the sample, these matrix components diffuse extensively on the paper fibers along with the liquid, which not only interferes with the uniformity of the colorimetric reaction but may also cause non-specific adsorption or increased colorimetric background, leading to difficulties in interpreting the results or even misinterpretation. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a nonadienal specific detection test strip and its preparation method.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention provides a test strip for the specific detection of nonadienal, the test strip comprising a substrate, a hydrophobic layer, and a reagent layer;

[0011] The substrate is non-fluorescent quantitative filter paper;

[0012] The hydrophobic layer is loaded on the surface of the substrate and is formed of polydimethylsiloxane to make the surface of the substrate hydrophobic.

[0013] The reagent layer is loaded on the surface of the hydrophobic layer and contains the colorimetric agent 2,4-dinitrophenylhydrazine and an acidity regulator, wherein the acidity regulator is used to provide a reaction environment of pH 2.0-2.5.

[0014] Preferably, as one possible implementation, the thickness of the quantitative filter paper is 0.15-0.20 mm, the pore size is 1.0-1.5 μm, and / or the contact angle of the hydrophobic layer is ≥110°.

[0015] Preferably, as one possible implementation, the loading of the 2,4-dinitrophenylhydrazine is 1.0-1.5 μg / cm²; and / or, the acidity regulator is dilute hydrochloric acid.

[0016] Preferably, as one possible implementation, the reagent layer further comprises a stabilizer, wherein the stabilizer is ascorbic acid.

[0017] Preferably, as one possible implementation, the test strip further includes a detection line (T line) and a control line (C line), which are formed on the surface of the reagent layer by a scribing process; wherein, the detection line (T line) contains 2,4-dinitrophenylhydrazine and an acidity adjuster; the control line (C line) contains a pH indicator or a colorimetric agent that does not react with 2,4-dinitrophenylhydrazine, used to verify whether the test strip is effective and whether the operation is correct.

[0018] This invention provides a method for preparing a nonadienal-specific test strip, comprising the following steps:

[0019] (1) Immerse quantitative filter paper in polydimethylsiloxane solution, remove and dry it to form a hydrophobic layer on the surface of the quantitative filter paper;

[0020] (2) Load the reagent solution containing 2,4-dinitrophenylhydrazine and an acid regulator onto the surface of the hydrophobic layer and dry it to obtain the test paper.

[0021] Preferably, as one possible implementation; in step (1), the polydimethylsiloxane solution is an ethanol solution of polydimethylsiloxane with a concentration of 0.1-1.0%; and / or, the soaking time is 0.5-3 minutes; and / or, the drying temperature is 30-40°C and the time is 20-40 minutes.

[0022] Preferably, as one possible implementation method; in step (2), the reagent solution is loaded onto the surface of the hydrophobic layer by spraying film, the spraying speed is 2-4 cm / s, the pressure is 0.1-0.2 MPa, and the spraying amount is 0.3-0.7 mL / cm²; and / or, the drying includes a first stage of constant temperature drying and a second stage of vacuum drying, the temperature of the first stage is 30-40℃, the time is 0.5-1.5 hours, the temperature of the second stage is 25-35℃, the vacuum degree is -0.08~-0.09 MPa, and the time is 1-2 hours; and / or, the concentration of 2,4-dinitrophenylhydrazine in the reagent solution is 1.0-2.0 g / 100 mL, and the amount of acid regulator added is such that the pH of the reagent solution is 2.0-2.5.

[0023] Preferably, as one possible implementation, the reagent solution further comprises a stabilizer, wherein the stabilizer is ascorbic acid, and its concentration in the reagent solution is 0.02-0.1 g / L.

[0024] This invention provides the application of nonadienal-specific test strips in the preparation of reagents or kits for the detection of nonadienal.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] This invention provides a test strip for the specific detection of nonadienal, the test strip comprising a substrate, a hydrophobic layer, and a reagent layer;

[0027] The substrate is non-fluorescent quantitative filter paper;

[0028] The hydrophobic layer is loaded on the surface of the substrate and is formed of polydimethylsiloxane to make the surface of the substrate hydrophobic.

[0029] The reagent layer is loaded on the surface of the hydrophobic layer and contains the colorimetric agent 2,4-dinitrophenylhydrazine and an acidity regulator, wherein the acidity regulator is used to provide a reaction environment of pH 2.0-2.5.

[0030] The aforementioned test strip for nonadienal specificity testing exhibits ultra-long room temperature stability. This is achieved through a PDMS hydrophobic layer isolating external moisture and combining it with ascorbic acid for antioxidant activity, allowing the test strip to remain effective for over 90 days at room temperature (compared to less than 48 hours with current technology). Simultaneously, its detection is extremely rapid. Integrated directly into the dry reagent layer in an acidic environment, the reaction conditions are met immediately upon sample addition, with color development taking only 1-2 minutes, and a total time of approximately 2-3 minutes – significantly better than the existing 15-20 minutes. It demonstrates high selectivity for nonadienal, exhibits no cross-reactivity with common interfering substances such as formaldehyde and acetaldehyde, and effectively resists interference from matrices such as sebum and sweat. Therefore, it possesses strong specificity and anti-interference capabilities. Attached Figure Description

[0031] Figure 1 This is a flowchart of the preparation method of the nonadienal specific detection test strip of the present invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1

[0034] This invention provides a test strip for the specific detection of nonadienal, the test strip comprising a substrate, a hydrophobic layer, and a reagent layer;

[0035] The substrate is non-fluorescent quantitative filter paper;

[0036] The hydrophobic layer is loaded on the surface of the substrate and is formed of polydimethylsiloxane to make the surface of the substrate hydrophobic.

[0037] The reagent layer is loaded on the surface of the hydrophobic layer and contains the colorimetric agent 2,4-dinitrophenylhydrazine and an acidity regulator, wherein the acidity regulator is used to provide a reaction environment of pH 2.0-2.5.

[0038] This invention, through a three-layer composite structure consisting of "quantitative filter paper substrate - PDMS hydrophobic layer - reagent layer containing DNPH and acidity regulator," integrates hydrophobicity and moisture protection with solid-phase acid supply for the first time. The hydrophobic layer effectively blocks environmental moisture from corroding the reagent layer, solving the problem of test paper easily absorbing moisture and becoming ineffective. At the same time, the acidic environment is fixed on the reagent layer fibers, triggering the reaction upon sample contact, avoiding the cumbersome liquid acid addition steps, and laying the structural foundation for achieving long-term stable storage at room temperature and instantaneous rapid detection.

[0039] Preferably, as one possible implementation, the thickness of the quantitative filter paper is 0.15-0.20 mm, the pore size is 1.0-1.5 μm, and / or the contact angle of the hydrophobic layer is ≥110°.

[0040] In the above technical solution, by limiting the filter paper thickness to 0.15-0.20 mm and the pore size to 1.0-1.5 μm, a uniform chromatographic flow rate of the liquid sample on the test paper is ensured, resulting in clear edges of the colorimetric bands without tailing, thus improving the accuracy of interpretation. The hydrophobic layer with a contact angle ≥110° gives the substrate excellent hydrophobic properties, enabling it to maintain structural integrity for a long time in a strongly acidic environment of pH 2.0-2.5, without fiber hydrolysis or coating peeling, ensuring the durability and stability of the test paper under harsh conditions.

[0041] Preferably, as one possible implementation, the loading of the 2,4-dinitrophenylhydrazine is 1.0-1.5 μg / cm²; and / or, the acidity regulator is dilute hydrochloric acid.

[0042] In the above technical solution, the 2,4-DNPH loading is controlled at 1.0-1.5 μg / cm² to achieve the optimal balance between ensuring colorimetric sensitivity and clarity (the orange-red band is visible to the naked eye) and avoiding excessive background color. Dilute hydrochloric acid is selected as the acidity regulator, as its volatility is lower than other inorganic acids, and most of it can be retained on the fibers during the drying process. This provides a stable and sufficient H⁺ concentration (pH 2.0-2.5) immediately upon use of the test paper, ensuring that the condensation reaction proceeds efficiently and completely.

[0043] Preferably, as one possible implementation, the reagent layer further comprises a stabilizer, wherein the stabilizer is ascorbic acid. As an antioxidant, ascorbic acid preferentially reacts with residual oxygen or oxidizing substances in the system, effectively inhibiting the oxidative degradation of 2,4-DNPH during storage. Experimental data show that the control group without this stabilizer experienced a 50% decrease in sensitivity after 60 days, while the group with the stabilizer showed no significant decrease in sensitivity after 90 days, demonstrating that this technical feature plays a crucial supporting role in achieving an ultra-long shelf life (≥90 days).

[0044] Preferably, as one possible implementation, the test strip further includes a detection line (T line) and a control line (C line), which are formed on the surface of the reagent layer by a scribing process; wherein, the detection line (T line) contains 2,4-dinitrophenylhydrazine and an acidity adjuster; the control line (C line) contains a pH indicator or a colorimetric agent that does not react with 2,4-dinitrophenylhydrazine, used to verify whether the test strip is effective and whether the operation is correct.

[0045] In the above technical solution, the C-line (control line) and T-line (detection line) formed by the membrane stretching process construct the test strip into a standardized lateral chromatography immunoassay / chemical analysis format. The presence of the C-line provides an internal reference, which can effectively determine whether the test strip is ineffective or whether the operation is standardized (if the C-line does not develop color, the result is invalid), greatly improving the reliability of the test results.

[0046] Example 2

[0047] See Figure 1 This invention provides a method for preparing a nonadienal specific test strip, comprising the following steps:

[0048] (1) Immerse quantitative filter paper in polydimethylsiloxane solution, remove and dry it to form a hydrophobic layer on the surface of the quantitative filter paper;

[0049] (2) Load the reagent solution containing 2,4-dinitrophenylhydrazine and an acid regulator onto the surface of the hydrophobic layer and dry it to obtain the test paper.

[0050] After the reagent layer is prepared, a scribing instrument is used to scribble the test paper at different locations to form T-lines and C-lines.

[0051] T-line streaking solution: Same composition as the reagent solution in step (2) (containing 1.0-2.0 g / 100 mL 2,4-DNPH, diluted hydrochloric acid adjusted to pH 2.0-2.5, and ascorbic acid). C-line streaking solution: Ethanol solution containing 0.1-0.5% (w / v) methyl orange (or bromophenol blue, Congo red, or other pH indicators), dried after streaking. Streaking parameters: speed 2-4 cm / s, membrane width 1-2 mm, spacing between two lines 5-8 mm.

[0052] The above preparation method is carried out according to the preparation approach of "first hydrophobic treatment, then reagent loading". This process ensures a clear separation and tight bond between the hydrophobic layer as the bottom barrier and the reagent layer as the functional reaction layer, avoiding the reagent from being embedded inside the hydrophobic layer and losing its reactivity.

[0053] Preferably, as one possible implementation; in step (1), the polydimethylsiloxane solution is an ethanol solution of polydimethylsiloxane with a concentration of 0.1-1.0%; and / or, the soaking time is 0.5-3 minutes; and / or, the drying temperature is 30-40°C and the time is 20-40 minutes.

[0054] In the above technical solution, controlling the PDMS solution concentration at 0.1-1.0% and soaking for 0.5-3 minutes can form a uniform, dense, and appropriately thick nanoscale hydrophobic coating on the surface of the filter paper fibers. This ensures good hydrophobicity without clogging the fiber pores and affecting the chromatography rate. The mild drying conditions of 30-40℃ prevent high temperatures from damaging the filter paper fiber structure, thus ensuring the strength of the substrate.

[0055] Preferably, as one possible implementation method; in step (2), the reagent solution is loaded onto the surface of the hydrophobic layer by spraying film, the spraying speed is 2-4 cm / s, the pressure is 0.1-0.2 MPa, and the spraying amount is 0.3-0.7 mL / cm²; and / or, the drying includes a first stage of constant temperature drying and a second stage of vacuum drying, the temperature of the first stage is 30-40℃, the time is 0.5-1.5 hours, the temperature of the second stage is 25-35℃, the vacuum degree is -0.08~-0.09 MPa, and the time is 1-2 hours; and / or, the concentration of 2,4-dinitrophenylhydrazine in the reagent solution is 1.0-2.0 g / 100 mL, and the amount of acid regulator added is such that the pH of the reagent solution is 2.0-2.5.

[0056] In the above technical solution, a spraying machine is used to load the reagent solution at a specific speed, pressure, and spray volume to achieve precise and uniform spreading of the reagent solution on the hydrophobic layer surface, resulting in a very small coefficient of variation (CV%≤2%). The unique two-stage process of "constant temperature drying + vacuum drying" rapidly removes a large amount of solvent in the first stage, and gently removes residual solvent in the second stage under negative pressure and low temperature, effectively preventing the "coffee ring effect" (i.e., solute migration to the edge), and ensuring that DNPH and acid regulators are uniformly fixed inside the fiber, guaranteeing consistency within and between batches.

[0057] Preferably, as one possible implementation, the reagent solution further comprises a stabilizer, wherein the stabilizer is ascorbic acid, and its concentration in the reagent solution is 0.02-0.1 g / L. Limiting the ascorbic acid concentration to 0.02-0.1 g / L (preferably 0.05 g / L) represents the optimal concentration window found between effective antioxidant properties and non-interference with the colorimetric reaction. If the concentration is too low, the protective effect is insufficient; if the concentration is too high, the weak acidity and reducing properties of ascorbic acid itself may competitively consume DNPH or affect the stability of the condensation product. This range has been experimentally verified to simultaneously ensure long shelf life and high colorimetric sensitivity.

[0058] This invention provides a method for preparing the above-mentioned nonadienal specific test strip, comprising the following steps: (1) immersing quantitative filter paper in a polydimethylsiloxane solution, removing and drying it to form a hydrophobic layer on the surface of the quantitative filter paper; (2) loading a reagent solution containing 2,4-dinitrophenylhydrazine and an acidity regulator onto the surface of the hydrophobic layer, drying it to obtain the test strip. In a preferred embodiment, the concentration of the polydimethylsiloxane solution in step (1) is 0.1-1.0%, the immersion time is 0.5-3 minutes, the drying temperature is 30-40°C, and the drying time is 20-40 minutes. In the preferred embodiment, in step (2), a film-spraying method is used for loading, with a spraying speed of 2-4 cm / s, a pressure of 0.1-0.2 MPa, and a spraying amount of 0.3-0.7 mL / cm²; the drying method is a first-stage constant temperature drying (30-40℃, 0.5-1.5 hours) and a second-stage vacuum drying (25-35℃, vacuum degree -0.08~-0.09 MPa, 1-2 hours);

[0059] The following are the comparative experimental results between the scheme of this Example 2 and the prior art (DNPH solution method / ordinary test paper):

[0060] 1. Comparison of operation time and steps:

[0061] Sampling preparation Reagents need to be prepared on-site or moisture-sensitive test strips need to be taken out of the refrigerator. Take the test strips directly from room temperature. Sampling method After collection with cotton swabs, the sample needs to be washed into the solution, or the test strip may be easily interfered with by sebum if it comes into direct contact with the sample. Apply the test strip directly to the scalp and press for 30 seconds, or apply a drop of swab solution. reaction time The solution method requires standing for 10-15 minutes; ordinary test strips are prone to uneven reaction due to moisture absorption. Observe the results after letting it stand for 1-2 minutes. Total time Approximately 15-20 minutes Approximately 2-3 minutes stability Reagents must be prepared and used immediately, or test strips must be kept for less than 48 hours. Test strips can be stored at room temperature for ≥90 days.

[0062] Stability data:

[0063] Experimental conditions: room temperature (25±2℃), humidity (50±5%RH), aluminum foil encapsulation (containing desiccant / deoxidizer).

[0064] Data performance:

[0065] Day 0: 100% pass rate.

[0066] 30 days: 100% pass rate, no moisture absorption, no clumping.

[0067] 60 days: 100% pass rate, no significant decrease in colorimetric sensitivity.

[0068] 90 days: 100% pass rate, clear T-line color development, and stable C-line.

[0069] Comparative data: The control group without hydrophobic coating began to absorb moisture after 15 days, and the pass rate dropped to 0% after 30 days; the control group without stabilizer showed a 50% decrease in sensitivity after 60 days.

[0070] Coefficient of variation (CV%): CV% ≤ 2% for intra-batch and inter-batch test results over a 90-day storage period.

[0071] 2. Specific experimental data

[0072] List of interfering substances and test results:

[0073] Interference substances tested: formaldehyde, acetaldehyde, propionaldehyde, and butenaldehyde (all at a concentration of 10 ppm).

[0074] Test results:

[0075] 2-Nonenal (10ppm): T line shows color (orange-red), C line shows color, positive.

[0076] Formaldehyde, acetaldehyde, propionaldehyde, and butenaldehyde: No color development on the T line, only color development on the C line, negative.

[0077] Blank control (physiological saline): No color development on the T line, only color development on the C line, negative.

[0078] Conclusion: No cross-reaction was observed. At the tested concentration, no significant color reaction was observed against common interfering substances such as formaldehyde, acetaldehyde, propionaldehyde, and butenal. It should be noted that the B / G ratio concept here mainly applies to fluorescence or specific instrument readings. This qualitative test strip is based on the visually visible color bands; therefore, it is recorded as colorimetric / non-colorimetric.

[0079] Example 3

[0080] This invention provides the application of nonadienal-specific test strips in the preparation of reagents or kits for the detection of nonadienal.

[0081] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A test strip for the specific detection of nonadienal, characterized in that, The test strip comprises a substrate, a hydrophobic layer, and a reagent layer; The substrate is non-fluorescent quantitative filter paper; The hydrophobic layer is loaded on the surface of the substrate and is formed of polydimethylsiloxane to make the surface of the substrate hydrophobic. The reagent layer is loaded on the surface of the hydrophobic layer and contains the colorimetric agent 2,4-dinitrophenylhydrazine and an acidity regulator, wherein the acidity regulator is used to provide a reaction environment of pH 2.0-2.

5.

2. The test strip according to claim 1, characterized in that, The quantitative filter paper has a thickness of 0.15-0.20 mm and a pore size of 1.0-1.5 μm; and / or, the contact angle of the hydrophobic layer is ≥110°.

3. The test strip according to claim 1 or 2, characterized in that, The loading of the 2,4-dinitrophenylhydrazine is 1.0-1.5 μg / cm²; and / or the acidity regulator is dilute hydrochloric acid.

4. The test strip according to any one of claims 1-3, characterized in that, The reagent layer also contains a stabilizer, which is ascorbic acid.

5. The test strip according to any one of claims 1-4, characterized in that, The test strip also includes a detection line (T line) and a control line (C line), which are formed on the surface of the reagent layer by a scribing process; wherein, the detection line (T line) contains 2,4-dinitrophenylhydrazine and an acidity adjuster; the control line (C line) contains a pH indicator or a colorimetric agent that does not react with 2,4-dinitrophenylhydrazine, used to verify whether the test strip is effective.

6. A method for preparing a nonadienal-specific test strip as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Immerse quantitative filter paper in polydimethylsiloxane solution, remove and dry it to form a hydrophobic layer on the surface of the quantitative filter paper; (2) Load the reagent solution containing 2,4-dinitrophenylhydrazine and an acid regulator onto the surface of the hydrophobic layer and dry it to obtain the test paper.

7. The preparation method according to claim 6, characterized in that, In step (1), the polydimethylsiloxane solution is an ethanol solution of polydimethylsiloxane with a concentration of 0.1-1.0%; and / or, the soaking time is 0.5-3 minutes; and / or, the drying temperature is 30-40°C and the time is 20-40 minutes.

8. The preparation method according to claim 6 or 7, characterized in that, In step (2), the reagent solution is loaded onto the surface of the hydrophobic layer by spraying film, with a spraying speed of 2-4 cm / s, a pressure of 0.1-0.2 MPa, and a spraying amount of 0.3-0.7 mL / cm²; and / or, the drying includes a first stage of constant temperature drying and a second stage of vacuum drying, with the temperature of the first stage being 30-40℃ and the time being 0.5-1.5 hours, and the temperature of the second stage being 25-35℃, the vacuum degree being -0.08~-0.09 MPa, and the time being 1-2 hours; and / or, the concentration of 2,4-dinitrophenylhydrazine in the reagent solution is 1.0-2.0 g / 100 mL, and the amount of acid adjuster added is such that the pH of the reagent solution is 2.0-2.

5.

9. The preparation method according to any one of claims 6-8, characterized in that, The reagent solution also contains a stabilizer, which is ascorbic acid, and its concentration in the reagent solution is 0.02-0.1 g / L.

10. The use of the test strip according to any one of claims 1-5 in the preparation of reagents or kits for detecting nonadienal.

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