CN113466201B - Pathogenic bacteria rapid detection method based on high-sensitivity fluorescence colorimetric sensing - Google Patents
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Abstract
The invention discloses a pathogenic bacteria rapid detection method based on high-sensitivity fluorescence colorimetric sensing, and belongs to the technical field of food safety detection. The detection method is characterized in that a non-enzymatic hybridization-based up-conversion nano fluorescent probe and a superstructure carbon nitride colorimetric probe are prepared by nano controllable self-assembly, and a high-sensitivity specific pathogenic bacterium detection system is constructed by combining a standard curve method, so that the low-cost, high-sensitivity and specific detection of food-borne pathogenic bacteria in food is realized.
Description
Technical Field
The invention particularly relates to a pathogenic bacterium rapid detection method based on high-sensitivity fluorescence colorimetric sensing, and belongs to the technical field of food safety detection.
Background
According to the world health organization bulletin, food-borne pathogenic bacteria are one of the important causes of food hygiene problems. Common food-borne pathogenic bacteria in food comprise staphylococcus aureus, salmonella, escherichia coli, increases prairie bacillus simplex and the like, the traditional detection methods comprise a microbial detection method, a molecular biology method, an enzyme-linked immunosorbent assay (ELISA) method and the like, instruments and equipment are expensive, the detection cost is high, the steps are complicated, and the real-time rapid detection requirements of the pathogenic bacteria cannot be met. The invention provides a method for quickly detecting pathogenic bacteria based on high-sensitivity fluorescent colorimetric sensing, overcomes the defects of the traditional method, and improves the speed and the accuracy of detecting food-borne pathogenic bacteria in food.
Disclosure of Invention
The invention aims to overcome the technical defects in the prior detection technology, such as: the invention provides a method for quickly detecting pathogenic bacteria based on high-sensitivity fluorescent colorimetric sensing, which is characterized by preparing an up-conversion nano fluorescent probe based on non-enzymatic hybridization and a superstructure carbon nitride colorimetric probe through nano controllable self-assembly, constructing a high-sensitivity specific pathogenic bacteria detection system by combining a standard curve method, and realizing low-cost, high-sensitivity and specific detection of food-borne pathogenic bacteria in food.
Specifically, the technical scheme adopted by the invention is as follows: a pathogenic bacteria rapid detection method based on high-sensitivity fluorescence colorimetric sensing comprises the following steps:
step one, preparation of oleic acid coated erbium-based up-conversion nanoparticles (OA-UCNPs) (Er) ): mixing a certain mass of YCl 3 ·6H 2 O、YbCl 3 ·6H 2 O and ErCl 3 ·6H 2 O is dispersed in a methanol solution, and then the homogeneous mixture is transferred to a three-necked flask, followed by addition of C 18 H 34 O 2 And C 18 H 36 Continuously stirring the mixed solution in a nitrogen environment, then raising the temperature to more than 100 ℃, keeping the temperature for a certain time, cooling to room temperature, and then dropwise adding NH 4 F and NaOH mixed methanol dispersion; then the temperature is increased in a gradient manner until the methanol is volatilized; then continuously heating and stirring the mixture in the nitrogen environment to finally obtain the OA-UCNPs (Er) 。
Step two, preparing carboxyl modified up-conversion nanoparticles (UCNPs-COOH): adding polyacrylic acid mixed with chloroform and toluene into OA-UCNPs by ligand exchange method (Er) Stirring the solution overnight and then centrifuging to obtain UCNPs-COOH;
step three, modifying a surface hairpin structure probe of UCNPs-COOH (UCNPs-H1 and UCNPs-H2): connecting two hairpin probes H1 and H2 modified by amino through an amide condensation reaction, specifically mixing and stirring UCNPs-COOH, NHS and EDC in an ice-water bath, then carrying out centrifugal cleaning, then adding an H1 solution for reaction, and finally centrifuging to obtain UCNPs-H1; the preparation of UCNPs-H2 is carried out as above.
Step four, constructing a fluorescent colorimetric sensing system: firstly, the specific aptamer of pathogenic bacteria and the corresponding semi-complementary strand thereof are subjected to hybridization reaction to obtain a semi-double-chain structure, and then C is added 3 N 4 UCNPs-H1 and UCNPs-H2, namely the final productBuilding a system;
step five, establishing a food-borne pathogenic detection method: adding pathogenic bacteria solution with different concentrations into the detection system, mixing, and adding TMB and H 2 O 2 Carrying out catalytic color reaction, then carrying out fluorescence signal acquisition, and establishing a quantitative model by using a standard curve method;
further, the fifth step includes that the two-dimensional nano material C is put into 3 N 4 The catalytic colorimetric property, the DNA self-assembly characteristic and the stable luminescence characteristic of the upconversion nanometer fluorescence are combined, and the detection sensitivity can be improved.
Further, the UCNPs-H1 and UCNPs-H2 can be used as fluorescent signal probes and can also be used as probes capable of influencing C 3 N 4 The trigger element of catalytic colorimetric performance improves the colorimetric effect of the detection system and facilitates preliminary naked eye qualitative judgment.
Further, in step one, the YCl 3 ·6H 2 O、YbCl 3 ·6H 2 O and ErCl 3 ·6H 2 The mass proportion range of O is as follows: (2-20): (1-4): 0.1-0.4); c 18 H 34 O 2 And C 18 H 36 The dosage range is as follows: (1-5) and (3-8); NH (NH) 4 The dosage range of F and NaOH is (2-6) to (4-8).
Further, in the second step, chloroform and toluene are added in the volume range of (2.
Furthermore, in the third step, the dosage ratio of UCNPs-COOH, NHS and EDC is (1-2): (1-3): (0.1-2); the amount of H1 and H2 is in the range of 1-500. Mu.L.
Further, in step four, C is added 3 N 4 And the ratio of UCNPs-H1 to UCNPs-H2 is 1.
Further, in step five, TMB and H are added 2 O 2 The ratio of (1-10) to (0.1-2).
Compared with the prior art, the invention has the following beneficial effects:
1. the invention discloses a pathogenic bacterium rapid detection method based on high-sensitivity fluorescence colorimetric sensing, which is characterized in that an up-conversion nano fluorescence probe based on non-enzymatic hybridization and a superstructure carbon nitride colorimetric probe are prepared through nano controllable self-assembly, and a high-sensitivity specificity pathogenic bacterium detection system is constructed by combining a standard curve method, so that the low-cost, high-sensitivity and specificity detection of food-borne pathogenic bacteria in food is realized.
2. The optical sensing detection system constructed by the invention uses the two-dimensional nano material C 3 N 4 The catalytic colorimetric property, the DNA self-assembly characteristic and the stable luminescence characteristic of the upconversion nanometer fluorescence are combined, and the detection sensitivity can be improved.
3. In the optical sensing detection system constructed by the invention, UCNPs-H1 and UCNPs-H2 can be used as fluorescent signal probes and can also be used as probes capable of influencing C 3 N 4 The trigger element of catalytic colorimetric performance improves the colorimetric effect of the detection system and facilitates preliminary naked eye qualitative judgment.
The detection range of the pathogenic bacteria rapid detection method based on the high-sensitivity fluorescence colorimetric sensing to staphylococcus aureus is 27-27 multiplied by 10 6 The cfu/mL is limited by 2cfu/mL, the whole detection process can be completed within 1h, the speed is higher than that of the traditional flat plate counting method, and the potential of online application is realized.
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FIG. 1 OA-UCNPs prepared according to the present invention (Er) And transmission electron micrographs of UCNPs-COOH; a is OA-UCNPs (Er) Transmission electron microscopy images of; b is a transmission electron microscope image of UCNPs-COOH;
FIG. 2 is a schematic verification diagram of the detection system of the present invention;
FIG. 3 is a standard curve for detecting Staphylococcus aureus based on fluorescent probe according to the present invention;
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the following description will clearly describe the embodiments of the present invention in combination with the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following description will explain embodiments of the present invention in further detail with reference to the accompanying drawings.
Example 1:
the invention discloses a pathogenic bacteria rapid detection method based on high-sensitivity fluorescence colorimetric sensing, which comprises the following specific steps:
step one, preparation of oleic acid coated erbium-based up-conversion nanoparticles (OA-UCNPs) (Er) ): mixing 0.2366g YCl 3 ·6H 2 O, 0.0775g of YbCl 3 ·6H 2 O and 0.0076g of ErCl 3 ·6H 2 O was dispersed in the methanol solution, and then the homogeneous mixture was transferred to a three-necked flask, followed by addition of 6mL of C 18 H 34 O 2 And 15mL of C 18 H 36 Continuously stirring the mixed solution in a nitrogen environment, then raising the temperature to more than 100 ℃, keeping the temperature for 30min, cooling to room temperature, and then dropwise adding 0.1482g of NH 4 F and 0.1g of NaOH were mixed with the methanol dispersion; then the temperature is increased in a gradient manner until the methanol is volatilized; then continuously heating and stirring the mixture in the nitrogen environment to finally obtain the OA-UCNPs (Er) 。
Step two, preparing carboxyl modified up-conversion nano particles (UCNPs-COOH): polyacrylic acid (200 mg) was mixed with chloroform and toluene (5 mL in total) by ligand exchange method, and added to OA-UCNPs (30 mg) (Er) Stirring the solution overnight and then centrifuging to obtain UCNPs-COOH;
step three, modifying a surface hairpin structure probe of UCNPs-COOH (UCNPs-H1 and UCNPs-H2): connecting two hairpin probes H1 and H2 modified by amino through amide condensation reaction, specifically mixing and stirring 2mg of UCNPs-COOH, 4mg of NHS and 2mg of EDC in ice-water bath, then carrying out centrifugal cleaning, then adding 20 mu L of H1 solution for reaction, and finally carrying out centrifugation to obtain UCNPs-H1; the preparation of UCNPs-H2 is carried out as above.
Step four, constructing a fluorescent colorimetric sensing system: firstly, the specific aptamer of pathogenic bacteria and the corresponding semi-complementary strand are hybridized to obtain a semi-double-chain structure, and then 10 mu L of C is added 3 N 4 50 mu L of UCNPs-H1 and 50 mu L of UCNPs-H2 are the final built system;
step five, establishing a staphylococcus aureus detection method: respectively adding staphylococcus aureus solution with different concentrations into a test system, mixing the system, and then adding 10 mu L of TMB and 10 mu L of H 2 O 2 Carrying out catalytic color development reaction, then carrying out fluorescence signal acquisition, establishing a linear relation standard curve between bacteria concentration and fluorescence intensity, and the result shows that the linear relation between the total number of colonies and the fluorescence intensity is y =958.92x +230.59, R 2 ＝0.9901。
Example 2: the method and the steps of the embodiment 1 of the invention are adopted to measure the content of staphylococcus aureus in milk, and the measurement result is shown in table 1, so that the method has higher conformity with the standard method, and the method can detect the content of pathogenic bacteria in milk with high precision.
TABLE 1 detection of Staphylococcus aureus content in milk by the method of the invention and the Standard method
In conclusion, the invention prepares the upconversion nanometer fluorescent probe based on non-enzymatic hybridization and the superstructure carbon nitride (C) through nanometer controllable self-assembly 3 N 4 ) A colorimetric probe, a high-sensitivity specific pathogen detection system is constructed by combining a standard curve method, a fluorescence up-conversion nano material is synthesized, a hairpin structure probe is modified on the surface of the colorimetric probe to be used as an element for starting carbon nitride catalysis colorimetry, a specific aptamer of the pathogen and a corresponding semi-complementary chain of the specific aptamer undergo a hybridization reaction to obtain a semi-double-chain structure, and then C is added 3 N 4 UCNPs-H1 and UCNPs-H2 are prepared into a pathogenic bacteria detection system in the milk. The constructed detection system for pathogenic bacteria in milk can be used for preliminarily performing qualitative judgment through colorimetric reaction, and can also be used for completing quantitative analysis on various pathogenic bacteria through a fluorescent probe.
Although the embodiments of the present invention have been described in conjunction with the accompanying drawings, those skilled in the art can make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations fall within the scope defined by the appended claims.
Claims (4)
1. A pathogenic bacteria rapid detection method based on high-sensitivity fluorescence colorimetric sensing is characterized in that: the method comprises the following steps:
step one, preparing OA-UCNPs by oleic acid coating erbium-based up-conversion nanoparticles (Er) : mixing a certain mass of YCl 3 ·6H 2 O、YbCl 3 ·6H 2 O and ErCl 3 ·6H 2 O is dispersed in methanol solution, then the homogeneous mixture is transferred to a three-necked flask, followed by the addition of C 18 H 34 O 2 And C 18 H 36 Continuously stirring the mixed solution in a nitrogen environment, then raising the temperature to more than 100 ℃, keeping the temperature for a certain time, cooling to room temperature, and then dropwise adding NH 4 F and NaOH mixed methanol dispersion; then the temperature is increased in a gradient manner until the methanol is volatilized; then continuously heating and stirring the mixture in the nitrogen environment to finally obtain the OA-UCNPs (Er) ；
In step one, YCl 3 ·6H 2 O、YbCl 3 ·6H 2 O and ErCl 3 ·6H 2 The mass proportion range of O is as follows: (2-20): 1-4): 0.1-0.4); c 18 H 34 O 2 And C 18 H 36 The dosage range is as follows: (1-5) and (3-8); NH 4 The dosage ranges of F and NaOH are (2-6) to (4-8);
step two, preparing UCNPs-COOH by using the carboxyl modified upconversion nanoparticles: adding polyacrylic acid mixed with chloroform and toluene into OA-UCNPs by ligand exchange method (Er) Stirring the solution overnight and then centrifuging to obtain UCNPs-COOH;
in the second step, chloroform and toluene are added in the volume range of 2;
step three, modifying UCNPs-H1 and UCNPs-H2 by a surface hairpin structure probe of UCNPs-COOH: connecting two hairpin probes H1 and H2 modified by amino through amide condensation reaction, specifically mixing and stirring UCNPs-COOH, NHS and EDC in ice-water bath, then carrying out centrifugal cleaning, then adding H1 solution for reaction, and finally centrifuging to obtain UCNPs-H1; the preparation steps of UCNPs-H2 are the same as above;
in the third step, the dosage ratio of UCNPs-COOH, NHS and EDC is (1-2): (1-3): 0.1-2); the dosage range of H1 and H2 is 1-500 mu L;
step four, constructing a fluorescent colorimetric sensing system: firstly, the specific aptamer of pathogenic bacteria and the corresponding semi-complementary strand are hybridized to obtain a semi-double-chain structure, and then C is added 3 N 4 UCNPs-H1 and UCNPs-H2 are finally built systems;
in step four, C is added 3 N 4 The ratio range of UCNPs-H1 to UCNPs-H2 is 1;
step five, establishing a food-borne pathogenic disease detection method: adding pathogenic bacteria solution with different concentrations into the detection system, mixing, and adding TMB and H 2 O 2 Carrying out catalytic color reaction, then carrying out fluorescence signal acquisition, and establishing a quantitative model by using a standard curve method.
2. The method for rapidly detecting pathogenic bacteria based on high-sensitivity fluorescence colorimetric sensing according to claim 1, characterized in that the fifth step further comprises the step of adding a two-dimensional nanomaterial C 3 N 4 The catalytic colorimetric performance, the DNA self-assembly characteristic and the stable luminescence characteristic of the upconversion nanometer fluorescence are combined, and the detection sensitivity is improved.
3. The method for rapidly detecting pathogenic bacteria based on high-sensitivity fluorescent colorimetric sensing according to claim 1, wherein the UCNPs-H1 and UCNPs-H2 can be used as fluorescent signal probes and can also be used as probes capable of influencing C 3 N 4 A trigger element for catalyzing colorimetric performance.
4. The method for rapidly detecting pathogenic bacteria based on high-sensitivity fluorescent colorimetric sensing according to claim 1, characterized in that in the fifth step, TMB and TMB are addedH 2 O 2 The ratio of (1-10) to (0.1-2).
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