A colorimetric sensing test paper for detecting listeria monocytogenes by metabolic biomarkers, a preparation method and application thereof

Abstract

The present application relates to a colorimetric sensing test paper for detecting Listeria monocytogenes by metabolic biomarkers, a preparation method and applications. The colorimetric sensing test paper is composed of metal hydroxide, guanidine compound, metal molybdate, agar powder and test paper, and has a specific chemical reaction with the metabolic biomarkers of Listeria monocytogenes, resulting in color change. The advantages of the colorimetric sensing test paper are that it can be observed by naked eye, is simple to operate and does not require expensive equipment, and can realize high selectivity, high sensitivity and rapid detection of metabolic biomarkers of Listeria monocytogenes. The colorimetric sensing test paper for detecting Listeria monocytogenes by metabolic biomarkers has potential application prospects in the field of food pathogenic bacteria detection.

Description

TECHNICAL FIELD

[0001] The present application relates to a colorimetric sensing test paper for detecting Listeria monocytogenes by metabolic biomarkers, a preparation method and application, and provides a preparation method of a colorimetric sensing test paper for detecting Listeria monocytogenes by metabolic biomarkers, belonging to the technical field of sensing. BACKGROUND

[0002] Listeria monocytogenes, simply referred to as Listeria monocytogenes, can cause various complications such as bacteremia, meningitis, and miscarriage in pregnant women. Listeria monocytogenes has strong tolerance to low temperature environment and can parasitize in meat, vegetables and seafood, known as "refrigerator killer". Therefore, reliable detection of Listeria monocytogenes is an important means to ensure food safety and maintain people's health. However, the traditional methods for detecting Listeria monocytogenes include biochemical tests, nucleic acid analysis and immune tests, which have the disadvantages of long time consumption, complex operation procedures and expensive instruments. Therefore, it is of important application value to develop an easy-to-operate and efficient method for detecting Listeria monocytogenes.

[0003] Listeria monocytogenes produces volatile organic compounds (VOCs) in the metabolic process, which can be used as a biomarker gas for detecting Listeria monocytogenes. In recent years, metal oxide semiconductor-based resistance-type gas sensors have become a novel means for detecting microbial metabolic VOCs due to their non-invasive, rapid, simple operation and high sensitivity. For example, in January 2022, the Swiss journal "Sensors and Actuators B: Chemical" reported a gas sensor based on cobalt-doped zinc oxide particles for detecting the biomarker 3-hydroxy-2-butanone of Listeria monocytogenes (Sensors and Actuators B: Chemical 2022, 358, 131482). Although such research has made progress, the selectivity of this gas sensor is poor, which limits its industrial application.

[0004] Colorimetric sensors have attracted widespread attention due to their easy preparation, simple operation, and naked-eye observation, and show potential in selective detection of target substances. For example, in November 2023, a formaldehyde colorimetric sensor based on wood-based materials achieved high sensitivity and selectivity in detecting formaldehyde (ACS Sustainable Chemistry & Engineering 2023, 11, 17206), and was applied to detect formaldehyde present in indoor environments. Therefore, developing a colorimetric sensing test paper with high selectivity to achieve rapid and sensitive detection of Listeria monocytogenes is expected to solve the above problems, but there are still technical challenges. SUMMARY

[0005] This invention aims to solve existing technical problems by developing a colorimetric sensor strip for detecting Listeria monocytogenes using metabolic biomarkers, its preparation method, and its application. The colorimetric sensor strip can undergo a specific chemical reaction with Listeria monocytogenes metabolic biomarkers, producing a color change. Its advantages include being visually observable, simple to operate, and requiring no expensive equipment, enabling high selectivity, high sensitivity, and rapid detection of Listeria monocytogenes metabolic biomarkers.

[0006] The preparation steps of the colorimetric sensor test strip for detecting Listeria monocytogenes using metabolic biomarkers are as follows:

[0007] (1) Preparation of sensing reagent: First, add a mixture of 5-15 mol (M) metal hydroxide and 0.15-1 M guanidine compound to ultrapure water and sonicate at room temperature for 0.5-5 hours (h) to obtain solution A; then, add 0.1-0.5 M metal molybdate and 0.05-0.5 M agar powder to solution A and stir at room temperature for 0.5-2 h to obtain mixture B; finally, incubate mixture B at 50-90 degrees Celsius ( o C) The sensing reagent is obtained by stirring in a water bath for 0.2-2 h;

[0008] (2) Preparation of colorimetric sensor paper: Measure 5-50 μL of the above-mentioned sensing reagent with a pipette, drop it onto a test paper with a diameter of 2-15 mm, and let it stand at room temperature for 2-15 minutes to obtain the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers.

[0009] Preferably, in step (1), the metal hydroxide is composed of any one, two or more of the following: sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), barium hydroxide (Ba(OH)2), lithium hydroxide (LiOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), strontium hydroxide (Sr(OH)2), and thallium hydroxide (TlOH).

[0010] Preferably, in step (1), the guanidine compound is composed of any one, two or more of the following: guanidinebutamine, guanidinepropionic acid, guanidineacetic acid, guanidine phosphate, thioguanidine, guanidine hydrochloride, guanidinebenzoic acid, arginine and creatine.

[0011] Preferably, in step (1), the metal molybdate is composed of any one, two or more of sodium molybdate (Na2MoO4), potassium molybdate (K2MoO4) and ammonium molybdate ((NH4)2MoO4).

[0012] Preferably, in step (2), the test paper is any one of rapid qualitative filter paper, medium-speed qualitative filter paper, slow-speed qualitative filter paper, rapid quantitative filter paper, medium-speed quantitative filter paper, and slow-speed quantitative filter paper.

[0013] When the colorimetric sensor strip comes into contact with metabolic biomarkers of Listeria monocytogenes, the color change becomes more significant as the concentration of the metabolic biomarkers increases, providing a quantitative basis for assessing the quantity of Listeria monocytogenes.

[0014] The following is an example of a colorimetric sensor strip for detecting Listeria monocytogenes using the metabolic biomarker 3-hydroxy-2-butanone:

[0015] (1) Colorimetric sensing test: After fixing the above colorimetric sensing test paper in the test chamber, the required concentration of 3-hydroxy-2-butanone can be introduced to evaluate the colorimetric sensing performance.

[0016] (2) Colorimetric sensing application: The colorimetric sensing test strip for detecting Listeria monocytogenes using the metabolic biomarker 3-hydroxy-2-butanone in this invention has the advantages of being visually observable, easy to operate and not requiring expensive equipment. It can achieve high selectivity, high sensitivity and rapid detection of the metabolic biomarker 3-hydroxy-2-butanone, and has potential application prospects in the field of detection of food pathogen Listeria monocytogenes. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the application of the colorimetric sensor test paper described above.

[0018] Figure 2 The above colorimetric sensor paper shows the color change performance of different concentrations of 3-hydroxy-2-butanone, with a response time of 20 minutes for all samples.

[0019] Figure 3 The graphs show the color change performance of the above colorimetric sensor paper for 3-hydroxy-2-butanone, ethanol, acetone, toluene, and formaldehyde, with a gas concentration of 10 ppm for each gas. Detailed Implementation

[0020] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention are also within the scope of protection of this invention. All raw materials and reagents used in this invention are commercially available.

[0021] Example 1

[0022] (1) Preparation of sensing reagent: First, a mixture of 5 M NaOH and 0.3 M guanidine was added to ultrapure water and ultrasonically dispersed at room temperature for 1 h to obtain solution A; then, 0.2 M Na2MoO4 and 0.2 M agar powder were added to solution A and stirred at room temperature for 1 h to obtain mixture B; finally, mixture B was heated at 80 °C. o The sensing reagent was obtained by stirring in a water bath at C for 1 h.

[0023] (2) Preparation of colorimetric sensor paper: 5 μL of the above-mentioned sensing reagent was measured with a pipette and dropped onto medium-speed qualitative filter paper with a diameter of 8 mm. After standing at room temperature for 5 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0024] Example 2

[0025] (1) Preparation of sensing reagent: First, a mixture of 10 M KOH and 1 M arginine was added to ultrapure water and ultrasonically dispersed at room temperature for 5 h to obtain solution A; then, 0.1 M Na2MoO4 and 0.4 M agar powder were added to solution A and stirred at room temperature for 1 h to obtain mixture B; finally, mixture B was subjected to a 50°C immersion test. o The sensing reagent was obtained by stirring in a water bath at C for 2 h.

[0026] (2) Preparation of colorimetric sensor paper: 50 μL of the above-mentioned sensing reagent was measured with a pipette and dropped onto a slow quantitative filter paper with a diameter of 15 mm. After standing at room temperature for 15 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0027] Example 3

[0028] (1) Preparation of sensing reagent: First, a mixture of 7 M Ca(OH)2 and 1 M creatine was added to ultrapure water and ultrasonically dispersed at room temperature for 2 h to obtain solution A; then, 0.1 M K2MoO4 and 0.05 M agar powder were added to solution A and stirred at room temperature for 1 h to obtain mixture B; finally, mixture B was heated at 90°C. o The sensing reagent was obtained by stirring in a water bath at C for 0.2 h.

[0029] (2) Preparation of colorimetric sensor paper: 10 μL of the above-mentioned sensing reagent was measured with a pipette and dropped onto a slow qualitative filter paper with a diameter of 9 mm. After standing at room temperature for 8 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0030] Example 4

[0031] (1) Preparation of sensing reagent: First, a mixture of 15 M Ba(OH)2 and 0.5 M guanidinopropionic acid was added to ultrapure water and ultrasonically dispersed at room temperature for 3 h to obtain solution A; then, 0.5 M (NH4)2MoO4 and 0.2 M agar powder were added to solution A and stirred at room temperature for 0.5 h to obtain mixture B; finally, mixture B was subjected to a 60°C test. o The sensing reagent was obtained by stirring in a water bath at C for 0.5 h.

[0032] (2) Preparation of colorimetric sensor paper: 5 μL of the above-mentioned sensing reagent was measured with a pipette and dropped onto a rapid quantitative filter paper with a diameter of 2 mm. After standing at room temperature for 2 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0033] Example 5

[0034] (1) Preparation of sensing reagents: First, a mixture of 2 M KOH, 2 M LiOH, 0.15 M creatine and 0.2 M guanidinoacetic acid was added to ultrapure water and ultrasonically dispersed at room temperature for 3 h to obtain solution A; then, 0.2 M (NH4)2MoO4, 0.2 M K2MoO4 and 0.1 M agar powder were added to solution A and stirred at room temperature for 0.5 h to obtain mixture B; finally, mixture B was heated at 90°C. o The sensing reagent was obtained by stirring in a water bath at C for 2 h.

[0035] (2) Preparation of colorimetric sensor paper: 12 μL of the above-mentioned sensing reagent was measured with a pipette and dropped onto medium-speed qualitative filter paper with a diameter of 6 mm. After standing at room temperature for 6 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0036] Example 6

[0037] (1) Preparation of sensing reagent: First, a mixture of 3 M NaOH, 3 M RbOH, 0.5 M arginine and 0.5 M guanidinopropionic acid was added to ultrapure water and ultrasonically dispersed at room temperature for 2 h to obtain solution A; then, 0.3 M Na2MoO4, 0.1 M K2MoO4 and 0.2 M agar powder were added to solution A and stirred at room temperature for 0.8 h to obtain mixture B; finally, mixture B was heated at 50 °C. o The sensing reagent was obtained by stirring in a water bath at temperature C for 1.5 h.

[0038] (2) Preparation of colorimetric sensor paper: 7 μL of the above-mentioned sensing reagent was measured with a pipette and dropped onto medium-speed quantitative filter paper with a diameter of 11 mm. After standing at room temperature for 14 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0039] Example 7

[0040] (1) Preparation of sensing reagents: First, a mixture of 3 M NaOH, 2 M CsOH, 3 M Sr(OH)2, 0.5 M creatine and 0.5 M guanidinoacetic acid was added to ultrapure water and ultrasonically dispersed at room temperature for 3 h to obtain solution A; then, 0.2 M Na2MoO4, 0.2 M (NH4)2MoO4 and 0.5 M agar powder were added to solution A and stirred at room temperature for 2 h to obtain mixture B; finally, mixture B was heated at 90°C. o The sensing reagent was obtained by stirring in a water bath at C for 2 h.

[0041] (2) Preparation of colorimetric sensor paper: 13 μL of the above-mentioned sensing reagent was measured by pipette and dropped onto a slow quantitative filter paper with a diameter of 11 mm. After standing at room temperature for 9 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0042] Example 8

[0043] (1) Preparation of sensing reagent: First, a mixture of 4 M NaOH, 3 M LiOH, 5 M Ti(OH)2, 0.3 M arginine and 0.5 M guanidinopropionic acid was added to ultrapure water and ultrasonically dispersed at room temperature for 4 h to obtain solution A; then, 0.1 M K2MoO4, 0.1 M (NH4)2MoO4 and 0.3 M agar powder were added to solution A and stirred at room temperature for 1 h to obtain mixture B; finally, mixture B was subjected to a 60°C test. o The sensing reagent was obtained by stirring in a water bath at temperature C for 1.5 h.

[0044] (2) Preparation of colorimetric sensor paper: 10 μL of the above-mentioned sensing reagent was measured with a pipette and dropped onto a rapid qualitative filter paper with a diameter of 4 mm. After standing at room temperature for 5 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0045] Example 9

[0046] (1) Preparation of sensing reagents: First, a mixture of 2 M Ba(OH)2, 2 M CsOH, 3 M RbOH, 0.2 M guanidine hydrochloride and 0.5 M thioguanidine was added to ultrapure water and ultrasonically dispersed at room temperature for 5 h to obtain solution A; then, 0.1 M Na2MoO4, 0.2 M (NH4)2MoO4 and 0.06 M agar powder were added to solution A and stirred at room temperature for 1 h to obtain mixture B; finally, mixture B was subjected to a 50°C test. o The sensing reagent was obtained by stirring in a water bath at C for 1 h.

[0047] (2) Preparation of colorimetric sensor paper: 25 μL of the above-mentioned sensing reagent was measured with a pipette and dropped onto medium-speed quantitative filter paper with a diameter of 7 mm. After standing at room temperature for 3 min, the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers was obtained.

[0048] Example 10

[0049] (1) Preparation of sensing reagent: First, a mixture of 2 M TlOH, 3 M KOH, 1 M LiOH, 0.15 M guanidinobenzoic acid and 0.5 M guanidine phosphate was added to ultrapure water and ultrasonically dispersed for 3 h at room temperature to obtain solution A; then, 0.2 M K2MoO4, 0.2 M K2MoO4 and 0.45 M agar powder were added to solution A and stirred at room temperature for 2 h to obtain mixture B; finally, mixture B was heated at 80 °C. o The sensing reagent was obtained by stirring in a water bath at C for 2 h.

[0050] (2) Preparation of colorimetric sensor paper: Measure 20 μL of the above-mentioned sensing reagent with a pipette, drop it onto a slow qualitative filter paper with a diameter of 5 mm, and let it stand at room temperature for 10 min to obtain the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers.

Claims

1. A colorimetric sensor test strip for detecting Listeria monocytogenes by metabolic biomarkers, characterized in that, The colorimetric sensor test paper changes color after exposure to the metabolic biomarker of Listeria monocytogenes, which is 3-hydroxy-2-butanone. The colorimetric sensor test paper is prepared as follows: (1) Prepare the sensing reagent: first, add a mixture of 5-15 moles of metal hydroxide and 0.15-1 moles of guanidino compound to ultrapure water, and ultrasonically disperse at room temperature for 0.5-5 hours to obtain solution A; then, add 0.1-0.5 moles of metal molybdate and 0.05-0.5 moles of agar powder to the above solution A, and stir at room temperature for 0.5-2 hours to obtain mixture B; finally, stir the above mixture B in a water bath at 50-90 degrees Celsius for 0.2-2 hours to obtain the sensing reagent; (2) Prepare the colorimetric sensor test paper: use a pipette to take 5-50 microliters of the above sensing reagent, and drop it onto a test paper with a diameter of 2-15 millimeters, and let it stand at room temperature for 2-15 minutes to obtain the colorimetric sensor test paper for detecting Listeria monocytogenes through metabolic biomarkers.

2. The method for preparing a colorimetric sensing test paper for detecting Listeria monocytogenes through metabolic biomarkers according to claim 1, characterized in that: In step (1), the metal hydroxide is any one of sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, lithium hydroxide, rubidium hydroxide, cesium hydroxide, strontium hydroxide, thallium hydroxide, or a mixture of two or more thereof.

3. The method for preparing a colorimetric sensing test paper for detecting Listeria monocytogenes by metabolic biomarkers according to claim 1, characterized in that: In step (1), the guanidino compound is any one of agmatine, guanidinopropionic acid, guanidinoacetic acid, guanidine phosphate, thioguanidine, guanidine hydrochloride, guanidinobenzoic acid, arginine, and creatine, or a mixture of two or more thereof.

4. The method for preparing a colorimetric sensing test paper for detecting Listeria monocytogenes by metabolic biomarkers according to claim 1, characterized in that: In step (1), the metal molybdate is any one of sodium molybdate, potassium molybdate, and ammonium molybdate, or a mixture of two or more thereof.

5. The method for preparing a colorimetric sensing test paper for detecting Listeria monocytogenes by metabolic biomarkers according to claim 1, characterized in that: In step (2), the test paper is any one of rapid qualitative filter paper, medium-speed qualitative filter paper, slow-speed qualitative filter paper, rapid quantitative filter paper, medium-speed quantitative filter paper, and slow-speed quantitative filter paper.

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