Fluorescent probe, fluorescent probe kit and method for detecting sulfate-reducing bacteria

Abstract

The present invention provides a fluorescent probe, a fluorescent probe kit and a method for detecting sulfate-reducing bacteria. The fluorescent probe has the following structural formula. Divalent sulfur anions are used to replace fluorescent molecules in the fluorescent probe, thereby separating metal ions from the fluorescent molecules, and the fluorescent molecules re-exhibit fluorescent properties. Moreover, experiments have shown that when hydrogen sulfide is further used to provide divalent sulfur anions for nucleophilic substitution with the fluorescent probe, the maximum fluorescence intensity of the fluorescent molecule is linearly related to the concentration of hydrogen sulfide, and the concentration of hydrogen sulfide can be characterized. The above-mentioned existing detection methods have proved that the concentration of hydrogen sulfide and the bacterial content of sulfate-reducing bacteria are linearly related, and the above-mentioned fluorescent molecules can be used to make fluorescent probes to detect the bacterial content of sulfate-reducing bacteria. The method for detecting sulfate-reducing bacteria based on the above-mentioned fluorescent probe does not require the expansion and cultivation of sulfate-reducing bacteria, can be used to detect the substance to be detected, and is easy to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of sulfate-reducing bacteria detection, and in particular to a fluorescent probe, a fluorescent probe kit and a sulfate-reducing bacteria detection method. Background Art

[0002] Sulfate-Reducing Bacteria (SRB) is an anaerobic microorganism that is widely found in hypoxic environments such as soil, seawater, underground pipelines, urban pipe networks, human oral cavity, and oil and gas wells. It can use organic matter on the metal surface as a carbon source, and use the hydrogen produced in the bacterial biofilm to reduce sulfate to generate sulfur anions (hydrogen sulfide), which directly causes corrosion failure of metal materials. SRB forms biofilms on the surfaces of various materials through metabolic activities, causing physical and chemical sterilization methods to fail, seriously threatening the service safety of materials. In certain environments, SRB can accumulate on the walls of pipelines, causing local pitting corrosion of the pipelines. Sulfate-reducing bacteria react with organic matter and sulfate to generate hydrogen sulfide, and carbon dioxide will react with iron in the pipeline wall to produce different forms of sulfide iron compounds. Sulfide iron compounds and other forms of sediments in the pipeline form a large amount of black powder, which is harder than the carbon steel of the pipeline, so it will cause serious corrosion damage to the pipeline components.

[0003] As the main harmful bacteria in oilfield pipelines, SRB corrodes metal pipelines, causing pipeline blockage, corrosion perforation, and oil and gas leakage, bringing numerous safety hazards to industrial production. Therefore, it is necessary to quickly detect SRB in pipelines in order to take effective measures to control its hazards. In terms of SRB detection and analysis, traditional detection technologies mainly include the following methods: (1) Liquid culture method: The sample to be tested is diluted in a gradient manner, and the sample to be tested with different dilutions is cultured in a sulfate-reducing bacteria selective culture medium to obtain a sulfate-reducing bacteria culture solution, and the bacterial concentration is determined based on the properties of the culture solution combined with the maximum probable number method (MPN method); (2) Solid culture method: Sulfate-reducing bacteria are screened and separated using a sulfate-reducing bacteria selective solid culture medium, and the bacterial concentration is determined based on the number of colonies. (3) Hemocytometer method: When the cells in the cell suspension to be tested are evenly distributed, the number of cells in a certain volume of suspension can be measured to convert the number of cells in each milliliter of cell suspension. However, the commonly used methods are cumbersome and have a long cycle (at least 7 days), and cannot reflect the problems occurring on site in a timely manner. Therefore, it is hoped that a simple, fast and accurate detection method can be developed to achieve the purpose of rapid detection of SRB in corrosion products such as biofilms, sediments, and rust, and promote its application in the oilfield industry. Summary of the invention

[0004] The main purpose of the present invention is to provide a fluorescent probe, a fluorescent probe kit and a method for detecting sulfate-reducing bacteria, so as to solve the problems of long detection time and complicated method of sulfate-reducing bacteria in the prior art.

[0005] In order to achieve the above object, according to one aspect of the present invention, a fluorescent probe is provided, which has the following structural formula I:

[0006]

[0007] Structural Formula I.

[0008] According to another aspect of the present invention, a fluorescent probe kit is provided, comprising a fluorescent probe, wherein the fluorescent probe is the fluorescent probe mentioned above.

[0009] Furthermore, the above-mentioned kit also includes a carrier and a color comparison table. The fluorescent probe is loaded on the carrier to form a fluorescent probe test paper. Each color in the color comparison table corresponds to a bacterial count value of sulfate-reducing bacteria or a concentration value of hydrogen sulfide.

[0010] According to another aspect of the present invention, a method for detecting sulfate-reducing bacteria is provided. The method comprises using the above-mentioned fluorescent probe kit to detect corrosion products in the environment where the sulfate-reducing bacteria are located as substances to be detected.

[0011] Furthermore, the fluorescent probe in the above-mentioned fluorescent probe kit is in a free state, and the detection method includes: establishing a first standard curve of the bacterial quantity of sulfate-reducing bacteria and the hydrogen sulfide concentration; establishing a second standard curve of the response value of the fluorescent probe at the maximum fluorescence intensity and fluorescence wavelength and the hydrogen sulfide concentration; in a sealed space, acid-washing the substance to be detected to release hydrogen sulfide, allowing the released hydrogen sulfide to react with the fluorescent probe to obtain a reaction system, and detecting the detection response value of the maximum fluorescence intensity and fluorescence wavelength of the reaction system; determining the hydrogen sulfide concentration in the substance to be detected based on the detection response value and the second standard curve; determining the bacterial quantity of sulfate-reducing bacteria in the environment where the substance to be detected is located based on the hydrogen sulfide concentration and the first standard curve.

[0012] Furthermore, the maximum fluorescence intensity fluorescence wavelength is 460-470 nm.

[0013] Furthermore, the above-mentioned kit includes a fluorescent probe, a carrier and a first color comparison table. The fluorescent probe is loaded on the carrier to form a fluorescent probe test paper. Each color in the first color comparison table corresponds to a bacterial count value of sulfate-reducing bacteria. The detection method includes: setting a space with a radius of 0 to 10 cm centered on the substance to be detected as a test area, and the test area is set in a sealed space; setting the fluorescent probe test paper in the test area; acid-washing the substance to be detected to release hydrogen sulfide, and the fluorescent probe test paper changes color in response to fluorescence after a first predetermined time, and the first predetermined time is preferably 0 to 1 min; comparing the color of the colored fluorescent probe test paper with the first color comparison table to determine the bacterial count value of sulfate-reducing bacteria.

[0014] Furthermore, the above detection method also includes the process of preparing a first color comparison table: establishing a first standard curve of the bacterial count of sulfate-reducing bacteria and the hydrogen sulfide concentration; detecting the color of the fluorescent probe after nucleophilic substitution at different hydrogen sulfide concentrations, and establishing a corresponding relationship between the color and the hydrogen sulfide concentration; establishing a one-to-one correspondence between the bacterial count of sulfate-reducing bacteria and the color based on the first standard curve and the corresponding relationship between the color and the hydrogen sulfide concentration, and then preparing the first color comparison table.

[0015] Furthermore, the above-mentioned kit includes a fluorescent probe, a carrier and a second color comparison table, the fluorescent probe is loaded on the carrier to form a fluorescent probe test paper, each color in the second color comparison table corresponds to a hydrogen sulfide concentration value, and the detection method includes: establishing a first standard curve of the bacterial count of sulfate-reducing bacteria and the hydrogen sulfide concentration; setting a space with a radius of 0 to 10 cm centered on the substance to be detected as a test area, and the test area is set in a sealed space; setting the fluorescent probe test paper in the test area; acid-washing the substance to be detected to release hydrogen sulfide, and the fluorescent probe test paper changes color in response to fluorescence after a second predetermined time, and the second predetermined time is preferably 0 to 1 min; comparing the color of the discolored fluorescent probe test paper with the second color comparison table to determine the concentration value of hydrogen sulfide in the substance to be detected; determining the bacterial count of sulfate-reducing bacteria in the environment where the substance to be detected is located based on the concentration value of hydrogen sulfide in the substance to be detected and the first standard curve.

[0016] Furthermore, the above detection method also includes the process of preparing a second color comparison table: detecting the color of the fluorescent probe after nucleophilic substitution at different hydrogen sulfide concentrations, establishing a one-to-one correspondence between color and hydrogen sulfide concentration, and then preparing a second color comparison table.

[0017] Furthermore, the wavelength of the above-mentioned response fluorescence is 460-470 nm.

[0018] By applying the technical solution of the present invention, divalent sulfur anions are used to replace the fluorescent molecules in the fluorescent probe, thereby separating the metal ions from the fluorescent molecules, and the fluorescent molecules re-exhibit fluorescent properties. Moreover, experiments have shown that when further using hydrogen sulfide (or HS - ) provides divalent sulfur anions to perform nucleophilic substitution with fluorescent probes, and the maximum fluorescence intensity of the fluorescent molecule is linearly related to the concentration of hydrogen sulfide, thereby characterizing the concentration of hydrogen sulfide. The above-mentioned existing detection methods have proved that the concentration of hydrogen sulfide is linearly related to the bacterial content of sulfate-reducing bacteria, and thus the above-mentioned fluorescent molecules can be used to prepare fluorescent probes to detect the bacterial content of sulfate-reducing bacteria. The method for detecting sulfate-reducing bacteria based on the above-mentioned fluorescent probe does not require the expansion and cultivation of sulfate-reducing bacteria, can be used to detect the substance to be detected, and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 Shown is a synthesis flow chart of L2Cu according to Example 1 of the present invention;

[0021] Figure 2 The fluorescence spectra of the product 2 and L2Cu according to Example 1 of the present invention are shown;

[0022] Figure 3 Shows that with HS - Fluorescence spectra of L2Cu with increasing concentration;

[0023] Figure 4 A standard curve showing the response value of the fluorescent probe at the maximum fluorescence intensity fluorescence wavelength and the hydrogen sulfide concentration; and

[0024] Figure 5 A standard curve of the bacterial count of sulfate-reducing bacteria and the hydrogen sulfide concentration is shown. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0026] As analyzed in the background technology of this application, the liquid culture method, solid culture method and blood cell counting method in the prior art are time-consuming and complicated to operate. In order to solve this problem, this application provides a fluorescent probe, a fluorescent probe kit and a method for detecting sulfate-reducing bacteria.

[0027] In a typical embodiment of the present application, a fluorescent probe is provided, which has the following structural formula I:

[0028]

[0029] Structural Formula I.

[0030] The above fluorescent probes contain fluorescent molecules with fluorescent properties:

[0031]

[0032] Divalent sulfur anions are used to replace the fluorescent molecules in the fluorescent probe, thereby separating the metal ions from the fluorescent molecules, and the fluorescent molecules re-exhibit fluorescent properties. Moreover, experiments have shown that when further using hydrogen sulfide (or HS - ) provides divalent sulfur anions to perform nucleophilic substitution with fluorescent probes, and the maximum fluorescence intensity of the fluorescent molecule is linearly related to the concentration of hydrogen sulfide, thereby characterizing the concentration of hydrogen sulfide. The above-mentioned existing detection methods have proved that the concentration of hydrogen sulfide is linearly related to the bacterial content of sulfate-reducing bacteria, and thus the above-mentioned fluorescent molecules can be used to prepare fluorescent probes to detect the bacterial content of sulfate-reducing bacteria. The method for detecting sulfate-reducing bacteria based on the above-mentioned fluorescent probe does not require the expansion and cultivation of sulfate-reducing bacteria, can be used to detect the substance to be detected, and is easy to operate.

[0033] In another typical embodiment of the present application, a fluorescent probe kit is provided, including a fluorescent probe, which is the fluorescent probe described above. The fluorescent probe is in the form of a kit, which is easy to carry and use. When used, a detection method suitable for the field can be formulated according to the principle described above, thereby realizing quick and simple detection of sulfate-reducing bacteria.

[0034] The kit of the present application can be a reagent bottle containing the above-mentioned fluorescent probe, that is, the fluorescent probe exists alone in a free manner. In some embodiments, the above-mentioned kit also includes a carrier and a color comparison table, and the fluorescent probe is loaded on the carrier to form a fluorescent probe test paper. Each color in the color comparison table corresponds to a bacterial amount value of sulfate-reducing bacteria or a concentration value of hydrogen sulfide. In order to facilitate direct comparison with the color comparison table after the fluorescent probe test paper changes color under the action of the substance to be detected, and then quickly determine the bacterial amount value and the concentration value of hydrogen sulfide. If the concentration value of hydrogen sulfide is determined, the corresponding sulfate-reducing bacteria can be directly determined according to the linear relationship between the concentration value of hydrogen sulfide and the bacterial amount value of sulfate-reducing bacteria that has been determined in the art.

[0035] In another typical embodiment of the present application, a method for detecting sulfate-reducing bacteria is provided, the detection method comprising using the above-mentioned fluorescent probe kit to detect corrosion products in the environment where the sulfate-reducing bacteria are located as the substance to be detected. The fluorescent probe is in the form of a kit, which is easy to carry and use. When used, a detection method suitable for the field can be formulated according to the principle described above, thereby realizing quick and simple detection of sulfate-reducing bacteria.

[0036] In some embodiments, when the fluorescent probe in the fluorescent probe kit is in a free state, the above detection method may include: establishing a first standard curve between the bacterial count of sulfate-reducing bacteria and the hydrogen sulfide concentration; establishing a second standard curve between the response value of the fluorescent probe at the maximum fluorescence intensity and fluorescence wavelength and the hydrogen sulfide concentration; in a sealed space, acid-washing the substance to be detected to release hydrogen sulfide, allowing the released hydrogen sulfide to react with the fluorescent probe to obtain a reaction system, and detecting the detection response value of the maximum fluorescence intensity and fluorescence wavelength of the reaction system; determining the hydrogen sulfide concentration in the substance to be detected based on the detection response value and the second standard curve; determining the bacterial count of sulfate-reducing bacteria in the environment in which the substance to be detected is located based on the hydrogen sulfide concentration and the first standard curve.

[0037] In the above detection method, the process of establishing the first standard curve of the bacterial count of sulfate-reducing bacteria and the hydrogen sulfide concentration can refer to the existing technology. When there is no data basis, the above first standard curve is established using liquid culture method, solid culture method or blood cell counting method. Although the detection process is the same as the existing technology and the time to establish the curve is longer, it is not necessary to establish a first standard curve for each detection; the first standard curve can also be established based on an existing database. After the first standard curve is established, it can be reused. Therefore, overall, the detection method of the present application can greatly save detection time; similarly, after the second standard curve is established, it can also be saved and reused. Then, the substance to be detected is acid-washed in a sealed space to release hydrogen sulfide, and the released hydrogen sulfide is reacted with the fluorescent probe to obtain a reaction system; after the reaction is completed, the detection response value of the maximum fluorescence intensity and fluorescence wavelength of the reaction system is detected; the concentration of hydrogen sulfide in the substance to be detected is determined according to the detection response value and the second standard curve; the bacterial amount of sulfate-reducing bacteria in the environment where the substance to be detected is located is determined according to the hydrogen sulfide concentration and the first standard curve. In this process, since both standard curves can be established in advance or according to an existing database, after the detection response value of the maximum fluorescence intensity and fluorescence wavelength of the reaction system is measured, the corresponding bacterial amount can be directly found according to the standard curve, and the detection is simple and quick.

[0038] In order to improve the sensitivity and accuracy of detection, the above-mentioned maximum fluorescence intensity fluorescence wavelength is preferably 460-470nm, such as 460nm, 461nm, 462nm, 463nm, 464nm, 464.5nm, 465nm, 465.5nm, 466nm, 466.5nm, 467nm, 467.5nm, 468nm, 469nm, and 470nm.

[0039] In other embodiments of the present application, the above-mentioned kit includes a fluorescent probe, a carrier and a first color comparison table. The fluorescent probe is loaded on the carrier to form a fluorescent probe test paper. Each color in the first color comparison table corresponds to a bacterial count value of sulfate-reducing bacteria. At this time, the detection method may include: setting a space with a radius of 0 to 10 cm centered on the substance to be detected as a test area, and the test area is set in a sealed space; setting the fluorescent probe test paper in the test area; acid-washing the substance to be detected to release hydrogen sulfide, and the fluorescent probe test paper changes color in response to fluorescence after a first predetermined time, and the first predetermined time is preferably 0 to 1 min; comparing the color of the colored fluorescent probe test paper with the first color comparison table to determine the bacterial count value of sulfate-reducing bacteria.

[0040] On the basis of the above color comparison table, the bacterial quantity of sulfate-reducing bacteria can be determined by directly comparing the color of the color-changing fluorescent probe test paper with the first color comparison table, which is more convenient and quick.

[0041] In some embodiments, the above detection method also includes the process of preparing a first color comparison table: establishing a first standard curve of the bacterial count of sulfate-reducing bacteria and the hydrogen sulfide concentration; detecting the color of the fluorescent probe after nucleophilic substitution at different hydrogen sulfide concentrations, and establishing a corresponding relationship between the color and the hydrogen sulfide concentration; establishing a one-to-one correspondence between the bacterial count of sulfate-reducing bacteria and the color based on the first standard curve and the corresponding relationship between the color and the hydrogen sulfide concentration, and then preparing the first color comparison table.

[0042] As mentioned above, when hydrogen sulfide is used to provide divalent sulfur anions for nucleophilic substitution with fluorescent probes, the maximum fluorescence intensity of the fluorescent molecule is linearly related to the concentration of hydrogen sulfide, and thus the concentration of hydrogen sulfide can be characterized. The above-mentioned existing detection methods have proved that the concentration of hydrogen sulfide is linearly related to the bacterial content of sulfate-reducing bacteria, and thus the above-mentioned fluorescent molecules can be used to prepare fluorescent probes to detect the bacterial content of sulfate-reducing bacteria. Moreover, the color of the fluorescent probes after nucleophilic substitution at different concentrations gradually changes to dark blue as the concentration of hydrogen sulfide increases. Therefore, those skilled in the art can establish a corresponding relationship between fluorescent colors and sulfate-reducing bacteria, and then prepare a color comparison table.

[0043] In some embodiments of the present application, the above-mentioned kit includes a fluorescent probe, a carrier and a second color comparison table. The fluorescent probe is loaded on the carrier to form a fluorescent probe test paper. Each color in the second color comparison table corresponds to a hydrogen sulfide concentration value. At this time, the detection method may include: establishing a first standard curve of the bacterial count and hydrogen sulfide concentration of sulfate-reducing bacteria; setting a space with a radius of 0 to 10 cm centered on the substance to be detected as a test area, and the test area is set in a sealed space; setting the fluorescent probe test paper in the test area; acid-washing the substance to be detected to release hydrogen sulfide, and the fluorescent probe test paper changes color in response to fluorescence after a second predetermined time, and the second predetermined time is preferably 0 to 1 min; comparing the color of the discolored fluorescent probe test paper with the second color comparison table to determine the concentration value of hydrogen sulfide in the object to be detected; determining the bacterial count of sulfate-reducing bacteria in the environment in which the substance to be detected is located according to the concentration value of hydrogen sulfide in the object to be detected and the first standard curve.

[0044] On the basis of the above second color comparison table, the concentration of hydrogen sulfide can be determined by directly comparing the color of the color-changing fluorescent probe test paper with the color comparison table, and the bacterial count of sulfate-reducing bacteria can be obtained according to the first standard curve, which is convenient and quick.

[0045] In some embodiments, the above detection method also includes the process of making a second color comparison table: detecting the color of the fluorescent probe after nucleophilic substitution with different hydrogen sulfide concentrations, establishing a corresponding relationship between color and hydrogen sulfide concentration, establishing a one-to-one correspondence between hydrogen sulfide concentration and color, and then making a second color comparison table. As mentioned above, when hydrogen sulfide is used to provide divalent sulfur anions for nucleophilic substitution with fluorescent probes, the maximum fluorescence intensity of the fluorescent molecule is linearly related to the hydrogen sulfide concentration, and then the concentration of hydrogen sulfide can be characterized. The color of the fluorescent probe after nucleophilic substitution with different concentrations is different. As the concentration of hydrogen sulfide increases, the color of the probe gradually changes to dark blue. Therefore, those skilled in the art can establish a corresponding relationship between the fluorescent color and the hydrogen sulfide concentration value, and then make a color comparison table.

[0046] The above-mentioned response fluorescence selection of the fluorescent probe of the present application can produce fluorescence within the wavelength range with maximum fluorescence intensity. For example, the wavelength of the above-mentioned response fluorescence is preferably 460-470nm, such as 460nm, 461nm, 462nm, 463nm, 464nm, 464.5nm, 465nm, 465.5nm, 466nm, 466.5nm, 467nm, 467.5nm, 468nm, 469nm, and 470nm.

[0047] The beneficial effects of the present application will be further illustrated below in combination with embodiments and comparative examples.

[0048] Example 1

[0049] 3 mmol of 1,8-naphthalene dicarboxylic anhydride (NA) was dissolved in 30 mL of DMF and heated under reflux until NA was completely dissolved. 4 mmol of hydrazine hydrate was added and the temperature was raised to 110°C and refluxed for about 1 to 2 h. The mixture after the reaction was poured into water and filtered, and the filter cake was washed with distilled water. The product 1 was obtained by vacuum drying with a yield of 92%.

[0050] 1.2 mmol of product 1 was weighed and dissolved in 20 mL of DMF. 3 mL of ethanol solution containing salicylaldehyde (including 2 mL of salicylaldehyde and 1 mL of ethanol) was added dropwise. The mixture was refluxed at 110° C. for 1 to 1.5 h. The reaction mixture was poured into water and filtered. After vacuum drying, a solid product 2 was obtained with a yield of 88%.

[0051] Weigh 1 mmol of product 2 and dissolve it in 15 mL of DMF. Add 4 mL of ethanol solution containing 0.5 mol of CuCl2 and 0.5 mL of triethylamine dropwise. Reflux at 70°C for 1 to 1.5 h. Pour the reaction mixture into water and filter it. After drying, a solid product, namely the desired complex L2Cu, is obtained with a yield of 88%.

[0052] The synthesis flow chart of L2Cu is as follows Figure 1 shown.

[0053] L2Cu is used as a H2S fluorescent probe. NA fluorescent molecules have fluorescent properties, and when combined with CuCl2, energy transfer occurs, which leads to fluorescence quenching, weakening or eliminating its fluorescent properties. After adding H2S, due to the strong nucleophilicity of H2S, it can undergo a nucleophilic substitution reaction with L2Cu, and the color development reaction of copper ions makes it have strong fluorescent characteristics. The above process synthesizes a fluorescence-enhanced H2S fluorescent probe. The fluorescence emission spectra of product 2 and L2Cu are shown in Figure 2 From the process of forming the complex in Example 1 above, it can be seen that as the amount of CuCl2 added increases, the fluorescence intensity weakens, and when the molar ratio of product 2 to copper chloride is 2:1, the fluorescence effect is quenched, indicating that the above structural formula of L2Cu is correct.

[0054] Comparative Example 1

[0055] Weigh 1 mmol of product 2 and dissolve it in 15 mL of DMF. Add 4 mL of ethanol solution containing 0.4 mol of ZnCl2 and 0.5 mL of triethylamine dropwise. Reflux at 70°C for 1 to 1.5 h. Pour the reaction mixture into water and filter it. After drying, a solid product, namely the desired complex L2Zn, is obtained with a yield of 64%.

[0056] NA fluorescent molecules have fluorescent properties, and they also transfer energy when combined with ZnCl2, which leads to fluorescence quenching, weakening or disappearance of fluorescence properties. After adding H2S, due to its strong nucleophilicity, H2S can undergo nucleophilic substitution reaction with L2Zn. However, since zinc ions are colorless and the fluorescence is not very obvious, the detection results are not sensitive when it is used as a fluorescent probe for the detection of sulfate-reducing bacteria.

[0057] Testing process:

[0058] Detection of Hydrogen Sulfide by Fluorescent Probe

[0059] Prepare a 5 mol / L L2Cu solution, add a 5 mol / L NaHS aqueous solution to an equal volume of L2Cu solution, shake well until the reaction is complete, remove the upper layer of liquid and measure the fluorescence excitation and fluorescence emission spectra of the solution at 300-800 nm. The results of the fluorescence detection of hydrogen sulfide test are shown in Figure 3 , it can be seen that after adding NaHS to L2Cu, the maximum emission wavelength is at 466nm, and the intensity is enhanced by two times. With the addition of NaHS, the fluorescence energy changes significantly, indicating that the fluorescent probe can effectively detect H2S. Figure 3 With HS - As the concentration increases, a shoulder peak can be seen in the fluorescence spectrum. The maximum fluorescence intensity of the solution fluorescence spectrum at 466nm is similar to that of HS - The concentration is linearly related, the regression equation is Y = 0.0541X-0.0490, and the standard curve is shown in Figure 4 Comparing the color change of the fluorescent probe after adding H2S, the color of the probe gradually changes from colorless to dark blue.

[0060] Test on quantitative relationship between bacterial count and hydrogen sulfide

[0061] The culture solution inoculated with SRB was placed in a constant temperature incubator for different periods of time, and the hydrogen sulfide content in the bottle under different bacterial counts was tested by the traditional liquid culture method, and a standard curve was drawn, such as Figure 5 As shown, it can be seen that SRB produces hydrogen sulfide under different bacterial dosage conditions.

[0062] The amount of bacteria in the corrosion product was tested using the fluorescent probe test paper of Example 1:

[0063] The fluorescent probe is loaded on the filter paper to make it into a pH test paper type, which is convenient to carry and quick to detect. Figure 4 and Figure 5 The standard curve was used to determine the amount of SRB corresponding to different colors and a color comparison table was established.

[0064] The corrosion products in the pipeline steel of Shengli Oilfield were taken into a sealed box, and the pickling spray was applied to the corrosion products (the composition of the pickling spray was: 0.5g of Sb2O3 and 50g of SnCl2 were added to every 25mL of concentrated hydrochloric acid). The test paper loaded with fluorescent probe was placed close to the corrosion products for measurement (the two were about 5cm apart). After about 20 seconds, the bacterial count value of 10 could be directly read by comparing the color depth. 6 Pieces / mL.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting sulfate-reducing bacteria, characterized in that: The detection method comprises using a fluorescent probe kit to detect corrosion products in the environment of sulfate-reducing bacteria as substances to be detected; The fluorescent probe kit comprises a fluorescent probe, a carrier and a color comparison table, wherein the fluorescent probe is loaded on the carrier to form a fluorescent probe test paper, and each color in the color comparison table corresponds to a bacterial count value of sulfate-reducing bacteria or a concentration value of hydrogen sulfide; The fluorescent probe has the following structural formula I: Structural Formula I; The fluorescent probe in the fluorescent probe kit is in a free state, and the detection method comprises: Establishing the first standard curve of the bacterial count of sulfate-reducing bacteria and the concentration of hydrogen sulfide; Establishing a second standard curve between the response value of the fluorescent probe at the maximum fluorescence intensity fluorescence wavelength and the hydrogen sulfide concentration; In a sealed space, the substance to be detected is acid-washed to release hydrogen sulfide, the released hydrogen sulfide reacts with a fluorescent probe to obtain a reaction system, and the detection response value of the maximum fluorescence intensity and fluorescence wavelength of the reaction system is detected; Determine the concentration of hydrogen sulfide in the substance to be detected according to the detection response value and the second standard curve; Determine the bacterial count of sulfate-reducing bacteria in the environment where the substance to be detected is located according to the hydrogen sulfide concentration and the first standard curve; Wherein, the pickling is carried out using an acid pickling spray, and the composition of the acid pickling spray is: 0.5g of Sb2O3 and 50g of SnCl2 are added to every 25mL of concentrated hydrochloric acid.

2. The detection method according to claim 1, characterized in that: The maximum fluorescence intensity fluorescence wavelength is 460~470nm.

3. The detection method according to claim 1, characterized in that: The kit includes a fluorescent probe, a carrier and a first color comparison table. The fluorescent probe is loaded on the carrier to form a fluorescent probe test paper. Each color in the first color comparison table corresponds to a bacterial count value of sulfate-reducing bacteria. The detection method includes: A space with a radius of 0 to 10 cm and centered on the substance to be detected is set as a test area, and the test area is set in a sealed space; placing the fluorescent probe test paper in the test area; The substance to be detected is acid-washed to release hydrogen sulfide, and the fluorescent probe test paper changes color in response to fluorescence after a first predetermined time, and the first predetermined time is 0 to 1 minute; The color of the color-changing fluorescent probe test paper is compared with the first color comparison table to determine the bacterial count value of the sulfate-reducing bacteria.

4. The detection method according to claim 3, characterized in that: The detection method also includes the process of making the first color comparison table: Establishing the first standard curve of the bacterial count of sulfate-reducing bacteria and the concentration of hydrogen sulfide; Detecting the color of the fluorescent probe after nucleophilic substitution at different hydrogen sulfide concentrations, and establishing a corresponding relationship between the color and the hydrogen sulfide concentration; A one-to-one correspondence between the bacterial count of the sulfate-reducing bacteria and the color is established according to the first standard curve and the correspondence between the color and the hydrogen sulfide concentration, and then the first color comparison table is prepared.

5. The detection method according to claim 1, characterized in that: The kit includes a fluorescent probe, a carrier and a second color comparison table. The fluorescent probe is loaded on the carrier to form a fluorescent probe test paper. Each color in the second color comparison table corresponds to a concentration value of hydrogen sulfide. The detection method includes: Establishing the first standard curve of the bacterial count of sulfate-reducing bacteria and the concentration of hydrogen sulfide; A space with a radius of 0 to 10 cm and centered on the substance to be detected is set as a test area, and the test area is set in a sealed space; placing the fluorescent probe test paper in the test area; The substance to be detected is acid-washed to release hydrogen sulfide, and the fluorescent probe test paper changes color in response to fluorescence after a second predetermined time, and the second predetermined time is 0 to 1 minute; Comparing the color of the color-changing fluorescent probe test paper with the second color comparison table to determine the concentration value of hydrogen sulfide in the substance to be detected; According to the concentration value of hydrogen sulfide in the substance to be detected and the first standard curve, the bacterial quantity of sulfate-reducing bacteria in the environment where the substance to be detected is located is determined, The detection method also includes the process of making the second color comparison table: The color of the fluorescent probe after nucleophilic substitution at different hydrogen sulfide concentrations is detected, and a corresponding relationship between the color and the hydrogen sulfide concentration is established, and a one-to-one corresponding relationship between the hydrogen sulfide concentration and the color is established, thereby preparing a second color comparison table.

6. The detection method according to any one of claims 3 to 5, characterized in that: The wavelength of the response fluorescence is 460-470 nm.

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