CN113899721A - Fluorescent probe, fluorescent probe kit and method for detecting sulfate reducing bacteria - Google Patents
Fluorescent probe, fluorescent probe kit and method for detecting sulfate reducing bacteria Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The 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. The divalent sulfur negative ions are used for replacing fluorescent molecules in the fluorescent probe, so that the metal ions are separated from the fluorescent molecules, and the fluorescent molecules show the fluorescent characteristic again. Moreover, experiments prove that when hydrogen sulfide is further used for providing divalent sulfur negative ions and the fluorescent probe is used for nucleophilic substitution, the maximum fluorescence intensity of the fluorescent molecule and the concentration of hydrogen sulfide are in a linear relationship, and the concentration of hydrogen sulfide can be further represented. The existing detection method proves that the concentration of the hydrogen sulfide and the bacteria content of the sulfate reducing bacteria are in a linear relation, and further the concentration of the hydrogen sulfide and the bacteria content of the sulfate reducing bacteria can be further detectedThe fluorescent probe is made by the fluorescent molecule to detect the bacteria content of the sulfate reducing bacteria. The method for detecting the sulfate reducing bacteria based on the fluorescent probe does not need to carry out amplification culture on the sulfate reducing bacteria, can detect substances to be detected, and is simple and convenient to operate.
Description
Technical Field
The invention relates to the technical field of sulfate reducing bacteria detection, and particularly relates to a fluorescent probe, a fluorescent probe kit and a method for detecting sulfate reducing bacteria.
Background
Sulfate-Reducing Bacteria (SRB) is an anaerobic microorganism and widely exists in anoxic environments such as soil, seawater, underground pipelines, urban pipe networks, human oral cavities, oil and gas wells and the like. It can utilize organic matter on the surface of metal as carbon source, and utilize hydrogen produced in the bacterial biofilm to reduce sulfate to produce sulfur negative ion (hydrogen sulfide), and can directly result in corrosion failure of metal material. SRB forms biological films on the surfaces of various materials through metabolic activities, so that physical, chemical and other sterilization means are ineffective, and the service safety of the materials is seriously threatened. In certain environments, SRB can collect on the walls of the pipe, making local pitting of the pipe possible. The sulfate reducing bacteria react with organic substances and sulfate to generate hydrogen sulfide and carbon dioxide which react with iron in the wall of the pipeline to generate sulfur-iron compounds in different forms. The ferrous sulfide compounds and other forms of deposits in the pipe form a large amount of black powder which is harder than the carbon steel of the pipe and can pose a serious corrosion hazard to the pipe components.
SRB is used as main harmful bacteria in oil field pipelines, corrodes metal pipelines, causes pipeline blockage, corrosion perforation and oil gas leakage, and brings numerous potential safety hazards to industrial production. Therefore, there is a need for rapid detection of SRB in a pipeline to take effective measures to control its hazards. In the aspect of SRB detection analysis, the conventional detection techniques mainly include the following methods: (1) liquid culture method: carrying out gradient dilution on a sample to be detected, culturing the sample to be detected with different dilutions by using a sulfate reducing bacteria selective culture medium to obtain a sulfate reducing bacteria culture solution, and determining the concentration of bacteria according to the character of the culture solution and the maximum possible number method (MPN method); (2) solid culture method: and (3) screening and separating sulfate reducing bacteria by using a sulfate reducing bacteria selective solid culture medium, and determining the bacterial concentration according to the colony count. (3) Blood cell count method: when the cells in the cell suspension to be detected are uniformly distributed, the number of the cells in each milliliter of the cell suspension can be converted by measuring the number of the cells in a certain volume of the suspension. However, the conventional method is complicated to operate and has a long period (at least 7 days), and the problems occurring on the site cannot be reflected in time, so that a simple, convenient, rapid and accurate detection mode is expected to be developed, so that the purpose of rapidly detecting the SRB in corrosion products such as biomembranes, sediments, rust and the like is achieved, and the method is popularized and applied in the oil field industry.
Disclosure of Invention
The invention mainly aims to provide a fluorescent probe, a fluorescent probe kit and a sulfate reducing bacteria detection method, so as to solve the problems of long detection time and complex method of sulfate reducing bacteria in the prior art.
To achieve the above object, according to one aspect of the present invention, there is provided a fluorescent probe having the following structural formula I:
structural formula I.
According to another aspect of the present invention, there is provided a fluorescent probe kit comprising a fluorescent probe as described above.
Furthermore, the kit also comprises a carrier and a color comparison table, wherein the fluorescent probe is loaded on the carrier to form fluorescent probe test paper, and each color in the color comparison table corresponds to the bacterial count value of one sulfate reducing bacterium or the concentration value of one hydrogen sulfide.
According to another aspect of the invention, a method for detecting sulfate-reducing bacteria is provided, which comprises detecting corrosion products of the environment in which the sulfate-reducing bacteria are located as a substance to be detected by using the fluorescent probe kit.
Further, the fluorescent probe in the fluorescent probe kit is in a free state, and the detection method comprises the following steps: establishing a first standard curve of the bacterial load of the sulfate reducing bacteria and the concentration of hydrogen sulfide; establishing a second standard curve of the response value of the fluorescent probe at the maximum fluorescence intensity fluorescence wavelength and the concentration of hydrogen sulfide; in a sealed space, carrying out acid washing on a substance to be detected to enable the substance to be detected to release hydrogen sulfide, enabling the released hydrogen sulfide to react with a fluorescent probe to obtain a reaction system, and detecting a detection response value of the maximum fluorescence intensity fluorescence wavelength of the reaction system; determining the concentration of hydrogen sulfide in the substance to be detected according to the detection response value and the second standard curve; and determining the bacterial load of the sulfate reducing bacteria in the environment where the substance to be detected is located according to the concentration of the hydrogen sulfide and the first standard curve.
Further, the maximum fluorescence intensity fluorescence wavelength is 460 to 470 nm.
Further, the kit comprises a fluorescent probe, a carrier and a first color comparison table, wherein the fluorescent probe is loaded on the carrier to form fluorescent probe test paper, each color in the first color comparison table corresponds to the bacterial count value of one sulfate reducing bacterium, and the detection method comprises the following steps: setting a space with a radius of 0-10 cm and taking a substance to be detected as a center as a test area, wherein the test area is arranged in the sealed space; arranging the fluorescent probe test paper in a test area; carrying out acid washing on a substance to be detected to enable the substance to be detected to release hydrogen sulfide, and changing the color of the fluorescent probe test paper under response fluorescence after first preset time, wherein the first preset time is preferably 0-1 min; and comparing the color of the discolored fluorescent probe test paper with the first color comparison table to determine the bacteria value of the sulfate reducing bacteria.
Further, the detection method further comprises a process of making the first color comparison table: establishing a first standard curve of the bacterial load of the 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 relation between the color and the hydrogen sulfide concentration; and establishing a one-to-one correspondence relationship between the bacterial load and the color of the sulfate reducing bacteria according to the correspondence relationship between the first standard curve and the color and the concentration of the hydrogen sulfide, and further manufacturing a first color comparison table.
Further, the kit comprises a fluorescent probe, a carrier and a second color comparison table, wherein 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, and the detection method comprises the following steps: establishing a first standard curve of the bacterial load of the sulfate reducing bacteria and the concentration of hydrogen sulfide; setting a space with a radius of 0-10 cm and taking a substance to be detected as a center as a test area, wherein the test area is arranged in the sealed space; arranging the fluorescent probe test paper in a test area; pickling the substance to be detected to enable the substance to be detected to release hydrogen sulfide, and changing the color of the fluorescent probe test paper under response fluorescence after a second preset time, wherein the second preset time is preferably 0-1 min; comparing the color of the discolored fluorescent probe test paper with a second color comparison table to determine the concentration value of the hydrogen sulfide in the object to be detected; and determining the bacterial load of the sulfate reducing bacteria in the environment of the substance to be detected according to the concentration value of the hydrogen sulfide in the substance to be detected and the first standard curve.
Further, the detection method further comprises a process of making a second color comparison table: and detecting the color of the fluorescent probe after nucleophilic substitution at different hydrogen sulfide concentrations, establishing a corresponding relation between the color and the hydrogen sulfide concentration, establishing a one-to-one corresponding relation between the hydrogen sulfide concentration and the color, and further manufacturing a second color comparison table.
Further, the wavelength of the response fluorescence is 460-470 nm.
By applying the technical scheme of the invention, divalent sulfur negative ions are used for replacing fluorescent molecules in the fluorescent probe, so that metal ions are separated from the fluorescent molecules, and the fluorescent molecules show the fluorescent characteristic again. Furthermore, it has been shown through tests that further use of hydrogen sulfide (or HS) is possible-) When the divalent sulfur negative ions and the fluorescent probe are provided for nucleophilic substitution, the maximum fluorescence intensity of the fluorescent molecule and the concentration of hydrogen sulfide are in a linear relationship, and the concentration of hydrogen sulfide can be further represented. The existing detection method proves that the concentration of the hydrogen sulfide and the bacteria content of the sulfate reducing bacteria are in a linear relation, and then the fluorescent molecules can be used for manufacturing fluorescent probes to detect the bacteria content of the sulfate reducing bacteria. The method for detecting the sulfate reducing bacteria based on the fluorescent probe does not need to carry out amplification culture on the sulfate reducing bacteria, can detect substances to be detected, and is simple and convenient to operate.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows L according to embodiment 1 of the present invention2A synthetic scheme of Cu;
FIG. 2 shows the products 2 and L of example 1 according to the invention2A fluorescence spectrum of Cu;
FIG. 3 shows following HS-Increase in concentration L2A fluorescence spectrum of Cu;
FIG. 4 shows a standard curve of the response of a fluorescent probe at the fluorescence wavelength of maximum fluorescence intensity versus the concentration of hydrogen sulfide; and
FIG. 5 shows a standard curve of the bacterial load of sulfate-reducing bacteria and the concentration of hydrogen sulfide.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
As analyzed in the background of the application, the liquid culture method, the solid culture method and the blood counting method in the prior art all take longer time and are complex to operate, and in order to solve the problem, the application provides a fluorescent probe, a fluorescent probe kit and a detection method of sulfate reducing bacteria.
In one exemplary embodiment of the present application, a fluorescent probe is provided, the fluorescent probe having the following structural formula I:
structural formula I.
The fluorescent probe comprises a fluorescent molecule with fluorescent characteristics:
the divalent sulfur negative ions are used for replacing fluorescent molecules in the fluorescent probe, so that the metal ions are separated from the fluorescent molecules, and the fluorescent molecules show the fluorescent characteristic again. Furthermore, it has been shown through tests that further use of hydrogen sulfide (or HS) is possible-) When the divalent sulfur negative ions and the fluorescent probe are provided for nucleophilic substitution, the maximum fluorescence intensity of the fluorescent molecule and the concentration of hydrogen sulfide are in a linear relationship, and the concentration of hydrogen sulfide can be further represented. The existing detection method proves that the concentration of the hydrogen sulfide and the bacteria content of the sulfate reducing bacteria are in a linear relation, and then the fluorescent molecules can be used for manufacturing fluorescent probes to detect the bacteria content of the sulfate reducing bacteria. The method for detecting the sulfate reducing bacteria based on the fluorescent probe does not need to carry out amplification culture on the sulfate reducing bacteria, can detect substances to be detected, and is simple and convenient to operate.
In another exemplary embodiment of the present application, a fluorescent probe kit is provided, which includes a fluorescent probe as described above. The fluorescent probe is used as a kit form, so that the kit is convenient to carry and use, and when the kit is used, a detection method suitable for a field can be formulated according to the described principle, so that the sulfate reducing bacteria can be quickly and simply detected.
The kit of the present application may be a reagent bottle containing the above-mentioned fluorescent probe, that is, the fluorescent probe exists alone in a free form. In some embodiments, the kit further includes a carrier and a color comparison table, 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 one bacterial count value of the sulfate-reducing bacteria or one concentration value of the hydrogen sulfide. So that the fluorescent probe test paper is directly contrasted with the color contrast table after the fluorescent probe test paper changes color under the action of the substance to be detected, and then the bacterial quantity value and the concentration value of the hydrogen sulfide are rapidly determined, if the concentration value of the hydrogen sulfide is determined, the corresponding bacterial quantity of the sulfate reducing bacteria can be directly determined according to the linear relation between the concentration value of the hydrogen sulfide and the bacterial quantity value of the sulfate reducing bacteria which is determined in the field.
In another exemplary embodiment of the present application, a method for detecting sulfate-reducing bacteria is provided, which includes detecting corrosion products of an environment in which sulfate-reducing bacteria are located as a substance to be detected by using the above-mentioned fluorescent probe kit. The fluorescent probe is used as a kit form, so that the kit is convenient to carry and use, and when the kit is used, a detection method suitable for a field can be formulated according to the described principle, so that the sulfate reducing bacteria can be quickly and simply detected.
In some embodiments, when the fluorescent probe in the fluorescent probe kit is in a free state, the detection method may include: establishing a first standard curve of the bacterial load of the sulfate reducing bacteria and the concentration of hydrogen sulfide; establishing a second standard curve of the response value of the fluorescent probe at the maximum fluorescence intensity fluorescence wavelength and the concentration of hydrogen sulfide; in a sealed space, carrying out acid washing on a substance to be detected to enable the substance to be detected to release hydrogen sulfide, enabling the released hydrogen sulfide to react with a fluorescent probe to obtain a reaction system, and detecting a detection response value of the maximum fluorescence intensity fluorescence wavelength of the reaction system; determining the concentration of hydrogen sulfide in the substance to be detected according to the detection response value and the second standard curve; and determining the bacterial load of the sulfate reducing bacteria in the environment where the substance to be detected is located according to the concentration of the hydrogen sulfide and the first standard curve.
In the detection method, the process of establishing the first standard curve of the bacterial load of the sulfate reducing bacteria and the concentration of the hydrogen sulfide can refer to the prior art, and the first standard curve is established by using a liquid culture method, a solid culture method or a blood counting method when no data base exists; the first standard curve can be established according to the existing database and can be reused after being established, so that the detection method can greatly save the detection time in the whole view; similarly, the second standard curve can be stored for repeated use after being established. Then, carrying out acid washing on the substance to be detected in the sealed space to enable the substance to be detected to release hydrogen sulfide, and enabling the released hydrogen sulfide to react with the fluorescent probe to obtain a reaction system; detecting the detection response value of the maximum fluorescence intensity fluorescence wavelength of the reaction system after the reaction is finished; determining the concentration of hydrogen sulfide in the substance to be detected according to the detection response value and the second standard curve; according to the concentration of hydrogen sulfide and the first standard curve, the bacterial load of sulfate reducing bacteria in the environment where the substance to be detected is located is determined, in the process, because the two standard curves can be established in advance or established according to the existing database, after the detection response value of the maximum fluorescence intensity fluorescence wavelength of the reaction system is measured, the corresponding bacterial load can be directly searched according to the standard curve, and the detection is simple, convenient and quick.
In order to improve the sensitivity and accuracy of detection, the fluorescence wavelength of the maximum fluorescence intensity is preferably 460-470 nm, such as 460nm, 461nm, 462nm, 463nm, 464nm, 464.5nm, 465nm, 465.5nm, 466nm, 466.5nm, 467nm, 467.5nm, 468nm, 469nm and 470 nm.
In other embodiments of the present application, the kit includes a fluorescent probe, a carrier, and a first color comparison table, where the fluorescent probe is loaded on the carrier to form a fluorescent probe test paper, and each color in the first color comparison table corresponds to a bacterial count value of sulfate-reducing bacteria, and in this case, the detection method may include: setting a space with a radius of 0-10 cm and taking a substance to be detected as a center as a test area, wherein the test area is arranged in the sealed space; arranging the fluorescent probe test paper in a test area; carrying out acid washing on a substance to be detected to enable the substance to be detected to release hydrogen sulfide, and changing the color of the fluorescent probe test paper under response fluorescence after first preset time, wherein the first preset time is preferably 0-1 min; and comparing the color of the discolored fluorescent probe test paper with the first color comparison table to determine the bacteria value of the sulfate reducing bacteria.
On the basis of the color comparison table, the color of the color-changing fluorescent probe test paper is directly compared with the first color comparison table, so that the bacterial load of the sulfate reducing bacteria can be determined, and the method is more convenient and quicker.
In some embodiments, the above detection method further comprises a process of making a first color comparison table: establishing a first standard curve of the bacterial load of the 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 relation between the color and the hydrogen sulfide concentration; and establishing a one-to-one correspondence relationship between the bacterial load and the color of the sulfate reducing bacteria according to the correspondence relationship between the first standard curve and the color and the concentration of the hydrogen sulfide, and further manufacturing a first color comparison table.
As mentioned above, when hydrogen sulfide is used to provide sulfide anions and the fluorescent probe is used for nucleophilic substitution, the maximum fluorescence intensity of the fluorescent molecule and the concentration of hydrogen sulfide are in a linear relationship, and the concentration of hydrogen sulfide can be further characterized. The existing detection method proves that the concentration of the hydrogen sulfide and the bacteria content of the sulfate reducing bacteria are in a linear relation, and then the fluorescent molecules can be used for manufacturing fluorescent probes to detect the bacteria content of the sulfate reducing bacteria. And the color of the fluorescent probe after nucleophilic substitution with different concentrations gradually changes to dark blue along with the increase of the concentration of the hydrogen sulfide, so that a person skilled in the art can establish the corresponding relationship between the fluorescent color and the sulfate reducing bacteria to further manufacture a color comparison table.
In some embodiments of the present application, the kit includes a fluorescent probe, a carrier, and a second color comparison table, where the fluorescent probe is loaded on the carrier to form a fluorescent probe test strip, and each color in the second color comparison table corresponds to a concentration value of hydrogen sulfide, and in this case, the detection method may include: establishing a first standard curve of the bacterial load of the sulfate reducing bacteria and the concentration of hydrogen sulfide; setting a space with a radius of 0-10 cm and taking a substance to be detected as a center as a test area, wherein the test area is arranged in the sealed space; arranging the fluorescent probe test paper in a test area; pickling the substance to be detected to enable the substance to be detected to release hydrogen sulfide, and changing the color of the fluorescent probe test paper under response fluorescence after a second preset time, wherein the second preset time is preferably 0-1 min; comparing the color of the discolored fluorescent probe test paper with a second color comparison table to determine the concentration value of the hydrogen sulfide in the object to be detected; and determining the bacterial load of the sulfate reducing bacteria in the environment of the substance to be detected according to the concentration value of the hydrogen sulfide in the substance to be detected and the first standard curve.
On the basis of the second color comparison table, the color of the color-changing fluorescent probe test paper is directly compared with the color comparison table, so that the concentration of the hydrogen sulfide can be determined, the bacterial load of the sulfate reducing bacteria can be obtained according to the first standard curve, and the method is convenient and quick.
In some embodiments, the above detection method further comprises a process of making a second color comparison table: and detecting the color of the fluorescent probe after nucleophilic substitution at different hydrogen sulfide concentrations, establishing a corresponding relation between the color and the hydrogen sulfide concentration, establishing a one-to-one corresponding relation between the hydrogen sulfide concentration and the color, and further manufacturing a second color comparison table. As mentioned above, when hydrogen sulfide is used to provide sulfide anions and the fluorescent probe is used for nucleophilic substitution, the maximum fluorescence intensity of the fluorescent molecule and the concentration of hydrogen sulfide are in a linear relationship, and the concentration of hydrogen sulfide can be further characterized. The color of the fluorescent probe after nucleophilic substitution with different concentrations is different, and the color of the probe gradually changes to dark blue along with the increase of the concentration of hydrogen sulfide, so that a person skilled in the art can establish the corresponding relationship between the fluorescent color and the concentration value of hydrogen sulfide, and further make a color comparison table.
The fluorescent probe of the present application is selected to generate fluorescence in a wavelength range having the maximum fluorescence intensity, and preferably, the wavelength of the response fluorescence is 460 to 470nm, such as 460nm, 461nm, 462nm, 463nm, 464nm, 464.5nm, 465nm, 465.5nm, 466nm, 466.5nm, 467nm, 467.5nm, 468nm, 469nm, 470 nm.
The advantageous effects of the present application will be further described below with reference to examples and comparative examples.
Example 1
Dissolving 3mmol of 1, 8-Naphthalic Anhydride (NA) in 30mL of DMF, heating and refluxing until the NA is completely dissolved, adding 4mmol of hydrazine hydrate, heating to 110 ℃, refluxing for about 1-2 h, pouring the reacted mixture into water, performing suction filtration, washing a filter cake with distilled water, and performing vacuum drying to obtain a product 1 with the yield of 92%.
Weighing 1.2mmol of product 1, dissolving in 20mL of DMF, dropwise adding 3mL of ethanol solution (2 mL of salicylaldehyde and 1mL of ethanol) dissolved with salicylaldehyde, refluxing at 110 ℃ for 1-1.5 h, pouring the reacted mixture into water, filtering, and vacuum drying to obtain a solid product 2 with the yield of 88%.
Weighing 1mmol of product 2, dissolving in 15mL of DMF, and dropwise adding 0.5mol of CuCl2And 0.5mL of triethylamine in 4mL of ethanol solution, refluxing for 1-1.5 h at 70 ℃, pouring the reacted mixture into water, filtering, and drying to obtain a solid product, namely the required complex L2Cu, yield 88%.
L2The scheme for the synthesis of Cu is shown in FIG. 1.
L2Cu as H2S fluorescent probe, NA fluorescent molecule has fluorescent characteristic and reacts with CuCl2The binding will undergo a transfer of energy, resulting in quenching of the fluorescence, weakening or eliminating its fluorescent properties. And H is added2After S, due to H2S has a strong nucleophilicity with L2The Cu undergoes nucleophilic substitution reaction and the color development reaction of copper ions, so that the Cu has strong fluorescence characteristics, and the fluorescence-enhanced H is synthesized by the process2And (3) an S fluorescent probe. Products 2 and L2The fluorescence emission spectrum of Cu is shown in FIG. 2. The formation of the complex by example 1 above can be seenOut with CuCl2The dropwise addition amount is increased, the fluorescence intensity is weakened, and when the molar ratio of the product 2 to the copper chloride is 2:1, the fluorescence effect is quenched, which shows that L2Correctness of the above structural formula of Cu.
Comparative example 1
Weighing 1mmol of product 2, dissolving in 15mL of DMF, and dropwise adding 0.4mol of ZnCl2And 0.5mL of triethylamine in 4mL of ethanol solution, refluxing for 1-1.5 h at 70 ℃, pouring the reacted mixture into water, filtering, and drying to obtain a solid product, namely the required complex L2Zn was obtained in a yield of 64%.
The NA fluorescent molecule has the fluorescent characteristic of ZnCl2The binding also effects a transfer of energy, resulting in quenching of the fluorescence and a weakening or disappearance of the fluorescent properties. Addition of H2After S, due to H2S has a strong nucleophilicity with L2The nucleophilic substitution reaction of Zn occurs. However, the zinc ions are colorless and the fluorescence is not very obvious, so when the zinc ions are used as a fluorescent probe to detect the sulfate reducing bacteria, the detection result is not sensitive.
And (3) detection process:
detection of hydrogen sulfide by fluorescent probes
Preparing L with the concentration of 5mol/L2Adding NaHS aqueous solution with concentration of 5mol/L into Cu solution until L with equal volume concentration2Shaking up in the Cu solution, removing the upper layer liquid after the reaction is sufficient, and measuring the fluorescence excitation and fluorescence emission spectrum of the solution at the position of 300-800 nm. The results of the fluorescence detection of hydrogen sulfide are shown in FIG. 3, and it can be seen that L is2After NaHS is added into Cu, the maximum emission wavelength is at 466nm, and the intensity is enhanced by two times. With the addition of NaHS, the fluorescence energy is obviously changed, which shows that the fluorescent probe can effectively detect H2And S. Following HS in FIG. 3-As the concentration increased, a shoulder was observed in the fluorescence spectrum. Maximum fluorescence intensity and HS of fluorescence spectrum of solution at 466nm-The concentration is linear, the regression equation is 0.0541X-0.0490, and the standard curve is shown in figure 4. Adding H into contrast fluorescent probe2The color changed after S, the color of the probe gradually changed from colorless to dark blue.
Quantitative relation test of bacterial load and hydrogen sulfide
The culture solution inoculated with the SRB is placed in a constant temperature incubator to be cultured for different time, the hydrogen sulfide content in the bottle under the condition of different bacterial load is tested by the traditional liquid culture method, and a standard curve is made, as shown in figure 5, so that the hydrogen sulfide generated by the SRB under the condition of different bacterial load can be known.
The amount of bacteria in the corrosion product was tested using the fluorescent probe paper of example 1:
loading the fluorescent probe on filter paper, making into pH-like test paper type, carrying conveniently, rapidly detecting, determining bacterial amount of SRB corresponding to different colors according to the standard curves of FIG. 4 and FIG. 5, and establishing color comparison table.
Taking the corrosion product in the pipeline steel of the Shenglii oil field into a sealed box, and applying acid cleaning spray on the corrosion product (the acid cleaning spray component is that 0.5g Sb is added into 25mL concentrated hydrochloric acid per one2O3And 50g of SnCl2) Measuring the corrosion product with the test paper loaded with the fluorescent probe close to the corrosion product (the distance between the two is about 5cm), and directly reading the bacterial quantity value of 10 by color depth contrast within about 20 seconds6one/mL.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A fluorescent probe, characterized in that it has the following structural formula I:
2. a fluorescent probe kit comprising a fluorescent probe, wherein the fluorescent probe is the fluorescent probe of claim 1.
3. The fluorescent probe kit of claim 2, further comprising 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 one bacterial count value of sulfate-reducing bacteria or one concentration value of hydrogen sulfide.
4. A method for detecting sulfate-reducing bacteria, which is characterized by comprising the step of detecting corrosion products of the environment where the sulfate-reducing bacteria are located as substances to be detected by using the fluorescent probe kit of claim 2.
5. The detection method according to claim 4, wherein the fluorescent probe in the fluorescent probe kit is in a free state, and the detection method comprises:
establishing a first standard curve of the bacterial load of the sulfate reducing bacteria and the concentration of hydrogen sulfide;
establishing a second standard curve of the response value of the fluorescent probe at the maximum fluorescence intensity fluorescence wavelength and the concentration of hydrogen sulfide;
in a sealed space, carrying out acid washing on the substance to be detected to enable the substance to be detected to release hydrogen sulfide, enabling the released hydrogen sulfide to react with a fluorescent probe to obtain a reaction system, and detecting a detection response value of the maximum fluorescence intensity fluorescence wavelength of the reaction system;
determining the concentration of the hydrogen sulfide in the substance to be detected according to the detection response value and the second standard curve;
and determining the bacterial load of the 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.
6. The detection method according to claim 5, wherein the maximum fluorescence intensity fluorescence wavelength is 460 to 470 nm.
7. The detection method according to claim 4, wherein the kit comprises 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 strip, each color in the first color comparison table corresponds to the bacterial count value of one sulfate-reducing bacterium, and the detection method comprises the following steps:
setting a space with a radius of 0-10 cm and taking the substance to be detected as a center as a test area, wherein the test area is arranged in the sealed space;
disposing the fluorescent probe strip in the test region;
carrying out acid washing on the substance to be detected to enable the substance to be detected to release hydrogen sulfide, and changing the color of the fluorescent probe test paper under response fluorescence after first preset time, wherein the first preset time is preferably 0-1 min;
and comparing the color of the discolored fluorescent probe test paper with the first color comparison table to determine the bacterial count value of the sulfate reducing bacteria.
8. The inspection method according to claim 7, further comprising a process of making the first color look-up table:
establishing a first standard curve of the bacterial load of the 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 relation between the color and the hydrogen sulfide concentration;
and establishing a one-to-one correspondence relationship between the bacterial load and the color of the sulfate reducing bacteria according to the first standard curve and the correspondence relationship between the color and the concentration of the hydrogen sulfide, and further manufacturing the first color comparison table.
9. The detection method according to claim 4, wherein the kit comprises a fluorescent probe, a carrier and a second color comparison table, the fluorescent probe is loaded on the carrier to form a fluorescent probe strip, each color in the second color comparison table corresponds to a concentration value of hydrogen sulfide, and the detection method comprises:
establishing a first standard curve of the bacterial load of the sulfate reducing bacteria and the concentration of hydrogen sulfide;
setting a space with a radius of 0-10 cm and taking the substance to be detected as a center as a test area, wherein the test area is arranged in the sealed space;
disposing the fluorescent probe strip in the test region;
carrying out acid washing on the substance to be detected to enable the substance to be detected to release hydrogen sulfide, and changing the color of the fluorescent probe test paper under response fluorescence after a second preset time, wherein the second preset time is preferably 0-1 min;
comparing the color of the color-changing fluorescent probe test paper with the second color comparison table to determine the concentration value of the hydrogen sulfide in the object to be detected;
determining the bacterial load of the sulfate reducing bacteria in the environment of the substance to be detected according to the concentration value of the hydrogen sulfide in the substance to be detected and the first standard curve,
preferably, the detection method further comprises a process of making the second color look-up table:
detecting the color of the fluorescent probe after nucleophilic substitution at different hydrogen sulfide concentrations, establishing the corresponding relation between the color and the hydrogen sulfide concentration, establishing the one-to-one corresponding relation between the hydrogen sulfide concentration and the color, and further manufacturing a second color comparison table.
10. The detection method according to any one of claims 7 to 9, wherein the wavelength of the response fluorescence is 460 to 470 nm.
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