Colorimetric sensing test paper for detecting listeria monocytogenes through metabolic biomarkers as well as preparation method and application of colorimetric sensing test paper

Abstract

The invention relates to colorimetric sensing test paper for detecting listeria monocytogenes through metabolic biomarkers as well as a preparation method and application of the colorimetric sensing test paper. The colorimetric sensing test paper is composed of metal hydroxide, a guanidyl compound, metal molybdate, agar powder and test paper, and the colorimetric sensing test paper and a metabolic biomarker of listeria monocytogenes are subjected to a specific chemical reaction to generate color change. The colorimetric sensing test paper has the advantages that the colorimetric sensing test paper can be observed by naked eyes, the operation is simple, expensive equipment is not needed, and high-selection, high-sensitivity and rapid detection of listeria monocytogenes metabolism biomarkers can be realized. The colorimetric sensing test paper for detecting the listeria monocytogenes through the metabolic biomarker has a potential application prospect in the field of food pathogenic bacterium detection.

Description

Technical Field

[0001] The invention relates to a colorimetric sensor test paper for detecting Listeria monocytogenes through metabolic biomarkers, a preparation method and application thereof, provides a preparation method for the colorimetric sensor test paper for detecting Listeria monocytogenes through metabolic biomarkers, and belongs to the field of sensor technology. Background Art

[0002] Listeria monocytogenes, abbreviated as Listeria monocytogenes, can cause a variety of complications such as bacteremia, meningitis and miscarriage in pregnant women. Listeria monocytogenes has a strong tolerance to low temperature environments and can parasitize in meat, vegetables and seafood. It is called the "refrigerator killer". Therefore, reliable detection of Listeria monocytogenes is an important means to ensure food safety and maintain people's health. However, the existing traditional methods for detecting Listeria monocytogenes include biochemical tests, nucleic acid analysis and immunoassays, which have the disadvantages of being time-consuming, complicated operating procedures and expensive instruments. In view of this, it is of great application value to develop an easy-to-operate and efficient method to detect Listeria monocytogenes.

[0003] Listeria monocytogenes produces volatile organic compounds (VOCs) during its metabolism, which can be used as biomarker gases for detecting Listeria monocytogenes. In recent years, metal oxide semiconductor-based resistive gas sensors have become a novel means of detecting microbial metabolic VOCs due to their advantages of being non-invasive, rapid, simple to operate, and highly sensitive. 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 the detection of 3-hydroxy-2-butanone, a biomarker for Listeria monocytogenes (Sensors and Actuators B: Chemical 2022, 358, 131482). Although such research has made progress, the poor selectivity of this gas sensor limits its industrial application.

[0004] Colorimetric sensors have attracted widespread attention due to their advantages such as easy preparation, simple operation, and observability with the naked eye. They have shown potential in the selective detection of target substances. For example, in November 2023, the research on formaldehyde colorimetric sensors developed based on wood substrates achieved high sensitivity and high selectivity for formaldehyde detection, and was used to detect formaldehyde in indoor environments (ACS Sustainable Chemistry & Engineering 2023, 11, 17206). Therefore, the development of a highly selective colorimetric sensing paper to achieve rapid and sensitive detection of Listeria monocytogenes is expected to solve the above problems, but there are still technical challenges. Summary of the invention

[0005] The present invention will solve the existing technical problems, and aims to invent a colorimetric sensor test paper, preparation method and application for detecting Listeria monocytogenes through metabolic biomarkers. The colorimetric sensor test paper can undergo a specific chemical reaction with the metabolic biomarkers of Listeria monocytogenes to produce a color change, which has the advantages of being observable with the naked eye, simple to operate and not requiring expensive equipment, and can achieve high selectivity, high sensitivity and rapid detection of metabolic biomarkers of Listeria monocytogenes.

[0006] The steps for preparing the colorimetric sensor test paper for detecting Listeria monocytogenes by metabolic biomarkers are as follows: (1) Preparation of sensing reagents: First, a mixture of 5-15 mol (M) metal hydroxide and 0.15-1 M guanidine compound was added to ultrapure water and ultrasonically dispersed at room temperature for 0.5-5 hours (h) to obtain solution A; then, 0.1-0.5 M metal molybdate and 0.05-0.5 M agar powder were added to the above solution A and stirred at room temperature for 0.5-2 h to obtain mixture B; finally, the above mixture B was heated at 50-90 degrees Celsius ( o C) stirring in a water bath for 0.2 - 2 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor test paper: Use a pipette to measure 5-50 microliters (μL) of the above-mentioned sensor reagent, and drop it onto a test paper with a diameter of 2-15 millimeters (mm). After standing at room temperature for 2-15 minutes (min), the colorimetric sensor test paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

[0007] Preferably, in step (1), the metal hydroxide is any one of 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), or a mixture of two or more thereof.

[0008] Preferably, in step (1), the guanidine compound is any one of agmatine, guanidine propionic acid, guanidine acetic acid, guanidine phosphate, thioguanidine, guanidine hydrochloride, guanidine benzoic acid, arginine and creatine, or a mixture of two or more thereof.

[0009] Preferably, in step (1), the metal molybdate is any one of sodium molybdate (Na2MoO4), potassium molybdate (K2MoO4) and ammonium molybdate ((NH4)2MoO4), or a mixture of two or more thereof.

[0010] Preferably, in step (2), the test paper is any one of rapid qualitative filter paper, medium-speed qualitative filter paper, slow qualitative filter paper, rapid quantitative filter paper, medium-speed quantitative filter paper and slow quantitative filter paper.

[0011] After the colorimetric sensor test paper comes into contact with the metabolic biomarker of Listeria monocytogenes, as the concentration of the metabolic biomarker increases, the color changes more significantly, providing a quantitative basis for evaluating the number of Listeria monocytogenes.

[0012] Taking the colorimetric sensor paper for detecting Listeria monocytogenes by the metabolic biomarker 3-hydroxy-2-butanone as an example, the following is explained: (1) Colorimetric sensing test: After the colorimetric sensing test paper is fixed in the test chamber, the required concentration of 3-hydroxy-2-butanone can be introduced to evaluate the colorimetric sensing performance; (2) Colorimetric sensing application: The colorimetric sensing test paper of the present invention for detecting Listeria monocytogenes by using the metabolic biomarker 3-hydroxy-2-butanone has the advantages of being observable with the naked eye, simple 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 pathogenic bacteria Listeria monocytogenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the application of the above colorimetric sensor paper; Figure 2 The color change performance diagram of the above colorimetric sensor paper to different concentrations of 3-hydroxy-2-butanone, the response time is 20 minutes; Figure 3 This is the color change performance diagram of the above colorimetric sensor paper to 3-hydroxy-2-butanone, ethanol, acetone, toluene, and formaldehyde, and the gas concentration is 10 ppm. DETAILED DESCRIPTION

[0014] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited 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 present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available.

[0015] Example 1 (1) Preparation of sensing reagents: First, a mixture of 5 M NaOH and 0.3 M agmatine 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 the above solution A and stirred at room temperature for 1 h to obtain mixture B. Finally, the above mixture B was heated at 80 °C for 24 h. o C. and stirring in a water bath for 1 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 5 μL of the above-mentioned sensor reagent, and drop it onto a medium-speed qualitative filter paper with a diameter of 8 mm. After standing at room temperature for 5 minutes, the colorimetric sensor paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

[0016] Example 2 (1) Preparation of sensing reagents: 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 the above solution A and stirred at room temperature for 1 h to obtain mixture B. Finally, the above mixture B was stirred at 50 °C for 2 h. o C. and stirred in a water bath for 2 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 50 μL of the above-mentioned sensor reagent, and drop it onto a slow quantitative filter paper with a diameter of 15 mm. After standing at room temperature for 15 minutes, the colorimetric sensor paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

[0017] Example 3 (1) Preparation of sensing reagents: 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 the above solution A and stirred at room temperature for 1 h to obtain mixture B. Finally, the above mixture B was heated at 90 °C for 24 h. o C. and stirred in a water bath for 0.2 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 10 μL of the above-mentioned sensor reagent, and drop it 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 through metabolic biomarkers is obtained.

[0018] Example 4 (1) Preparation of sensing reagents: First, a mixture of 15 M Ba(OH)2 and 0.5 M guanidine propionic 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 the above solution A and stirred at room temperature for 0.5 h to obtain mixture B. Finally, the above mixture B was heated at 60 o C. and stirring in a water bath for 0.5 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 5 μL of the above-mentioned sensor reagent, and drop it 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 through metabolic biomarkers is obtained.

[0019] Example 5 (1) Preparation of sensing reagents: First, a mixture of 2 M KOH, 2 M LiOH, 0.15 M creatine, and 0.2 M guanidine acetic 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 MK2MoO4, and 0.1 M agar powder were added to the above solution A and stirred at room temperature for 0.5 h to obtain mixture B. Finally, the above mixture B was heated at 90 °C for 24 h. o C. and stirred in a water bath for 2 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 12 μL of the above-mentioned sensor reagent, and drop it onto a medium-speed qualitative filter paper with a diameter of 6 mm. After standing at room temperature for 6 minutes, the colorimetric sensor paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

[0020] Example 6 (1) Preparation of sensing reagents: First, a mixture of 3 M NaOH, 3 M RbOH, 0.5 M arginine, and 0.5 M guanidine propionic 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 the above solution A and stirred at room temperature for 0.8 h to obtain mixture B. Finally, the above mixture B was heated at 50 °C for 2 h and then heated to 40 °C for 3 h. o C. and stirred in a water bath for 1.5 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 7 μL of the above-mentioned sensor reagent, and drop it onto a medium-speed quantitative filter paper with a diameter of 11 mm. After standing at room temperature for 14 minutes, the colorimetric sensor paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

[0021] Example 7 (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 guanidine acetic 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 the above solution A and stirred at room temperature for 2 h to obtain mixture B. Finally, the above mixture B was heated at 90 °C for 24 h. o C. and stirred in a water bath for 2 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 13 μL of the above-mentioned sensor reagent, and drop it onto a slow quantitative filter paper with a diameter of 11 mm. After standing at room temperature for 9 minutes, the colorimetric sensor paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

[0022] Example 8 (1) Preparation of sensing reagents: First, a mixture of 4 M NaOH, 3 M LiOH, 5 M Ti(OH)2, 0.3 M arginine, and 0.5 M guanidine propionic 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 the above solution A and stirred at room temperature for 1 h to obtain mixture B. Finally, the above mixture B was heated at 60 °C for 24 h and then heated to 40 °C for 24 h. o C. and stirred in a water bath for 1.5 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 10 μL of the above-mentioned sensor reagent, and drop it onto a rapid qualitative filter paper with a diameter of 4 mm. After standing at room temperature for 5 minutes, the colorimetric sensor paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

[0023] Example 9 (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 the above solution A and stirred at room temperature for 1 h to obtain mixture B. Finally, the above mixture B was heated at 50 °C for 24 h and then heated to 40 °C for 30 min. o C. and stirring in a water bath for 1 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 25 μL of the above-mentioned sensor reagent, and drop it onto a medium-speed quantitative filter paper with a diameter of 7 mm. After standing at room temperature for 3 minutes, the colorimetric sensor paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

[0024] Example 10 (1) Preparation of sensing reagents: First, a mixture of 2 M TlOH, 3 M KOH, 1 M LiOH, 0.15 M guanidine benzoic acid, and 0.5 M guanidine phosphate was added to ultrapure water and ultrasonically dispersed at room temperature for 3 h to obtain solution A. Then, 0.2 M K2MoO4, 0.2 M K2MoO4, and 0.45 M agar powder were added to the above solution A and stirred at room temperature for 2 h to obtain mixture B. Finally, the above mixture B was heated at 80 °C for 24 h and stirred for 3 h. o C. and stirred in a water bath for 2 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor paper: Use a pipette to measure 20 μL of the above-mentioned sensor reagent, and drop it onto a slow qualitative filter paper with a diameter of 5 mm. After standing at room temperature for 10 min, the colorimetric sensor paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

Claims

1. A colorimetric sensor test paper for detecting Listeria monocytogenes by metabolic biomarkers, characterized in that: The colorimetric sensor test paper changes color after being exposed to the metabolic biomarker of Listeria monocytogenes, and the preparation steps are as follows: (1) Preparation of sensing reagents: First, a mixture of 5-15 mol (M) metal hydroxide and 0.15-1 M guanidine compound was added to ultrapure water and ultrasonically dispersed at room temperature for 0.5-5 hours (h) to obtain solution A; then, 0.1-0.5 M metal molybdate and 0.05-0.5 M agar powder were added to the above solution A and stirred at room temperature for 0.5-2 h to obtain mixture B; finally, the above mixture B was heated at 50-90 degrees Celsius ( o C) stirring in a water bath for 0.2 - 2 h to obtain the sensing reagent; (2) Preparation of colorimetric sensor test paper: Use a pipette to measure 5-50 μL of the above-mentioned sensor reagent, and drop it onto a test paper with a diameter of 2-15 mm. After standing at room temperature for 2-15 minutes, the colorimetric sensor test paper for detecting Listeria monocytogenes through metabolic biomarkers is obtained.

2. The method for preparing a colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers according to claim 1, characterized in that: In step (1), the metal hydroxide is any one of 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), or a mixture of two or more thereof.

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

4. The method for preparing a colorimetric sensor 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 (Na2MoO4), potassium molybdate (K2MoO4) and ammonium molybdate ((NH4)2MoO4), or a mixture of two or more thereof.

5. The method for preparing a colorimetric sensor 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 qualitative filter paper, rapid quantitative filter paper, medium-speed quantitative filter paper and slow quantitative filter paper.

6. The color change of the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers according to claim 1, characterized in that: After the colorimetric sensor test paper comes into contact with the metabolic biomarker of Listeria monocytogenes, the color changes more significantly as the concentration of the metabolic biomarker increases.

7. The use of the colorimetric sensor paper for detecting Listeria monocytogenes by metabolic biomarkers according to claim 1, characterized in that: It can achieve highly selective, highly sensitive and rapid detection of Listeria monocytogenes metabolic biomarkers, and has potential application prospects in the field of food pathogenic bacteria detection.

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