CN115684104A - Shigella rapid detection method based on up-conversion-gold nano fluorescence resonance energy transfer - Google Patents
Shigella rapid detection method based on up-conversion-gold nano fluorescence resonance energy transfer Download PDFInfo
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- CN115684104A CN115684104A CN202211142690.5A CN202211142690A CN115684104A CN 115684104 A CN115684104 A CN 115684104A CN 202211142690 A CN202211142690 A CN 202211142690A CN 115684104 A CN115684104 A CN 115684104A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a method for rapidly detecting shigella in food, and belongs to the field of rapid detection of microorganisms. The detection method comprises the following steps: amino modification and carboxyl of oil-soluble up-conversion fluorescent materialModifying, namely respectively modifying the shigella aptamer and the complementary strand on the surfaces of the gold nanoparticles and the upconversion fluorescent material, and constructing an upconversion-gold nanoparticle fluorescent detection system by utilizing the base complementary pairing effect between the aptamer and the complementary strand; the relation between the concentration of the shigella bacterial liquid and the fluorescence signal intensity is explored by utilizing the fluorescence resonance energy transfer effect between the up-conversion material and the gold nanoparticles. The detection system established by the invention has a wider detection range and a lower detection limit, wherein the linear detection range is 1.2 multiplied by 10 2 CFU/mL‑1.2×10 8 CFU/mL, the detection limit is 30CFU/mL, the sensitivity is higher, and shigella in food can be specifically detected.
Description
Technical Field
The invention belongs to the field of microorganism rapid detection, and particularly relates to a rapid pathogenic bacterium detection technology based on fluorescence resonance energy transfer.
Background
Shigella, also known as dysentery bacterium, belongs to the gram-negative brevibacterium, is widely found in nature, is found in the gastrointestinal tract of both animals and humans, and is the most common pathogenic bacterium of human bacillary dysentery. Bacillary dysentery is the most common intestinal infectious disease, and the most common diseases occur in summer and autumn. The infection source is mainly patients and bacteria carriers, and the oral infection is caused by food, drinking water and the like which pollute the dysentery bacillus. The main clinical manifestations of the disease are general poisoning symptoms, abdominal pain, diarrhea, dysentery heaviness, mucus purulent bloody stool, and bacillary dysentery, which are important intestinal infectious diseases and are the first place in bacterial infection diseases in China.
The common cell culture technology and molecular biology method at present are complicated to operate, time-consuming and labor-consuming, and can not meet the requirement of on-site rapid detection; the reagent of the immunological detection method is difficult to preserve, the enzyme activity is easily influenced by the environment, the accuracy is poor, and the requirement of rapid circulation of food under the current economic globalization condition cannot be met. At present, the method for detecting pathogenic bacteria based on the up-conversion fluorescent material is a new detection method, the up-conversion fluorescent material is utilized to establish a rapid detection method in food circulation, technical support is provided for rapid detection of shigella in food, and the method has very important significance for timely and effectively controlling propagation of shigella, preventing food poisoning and reducing occurrence of food-borne diseases.
Disclosure of Invention
The invention aims to rapidly detect Shigella based on the fluorescence resonance energy transfer effect of an up-conversion material and gold nano-particles, so as to solve the problems of the technology. By constructing an up-conversion-gold nano detection system, the rapid detection of Shigella in food is realized, and the method is superior to the traditional flat plate counting method in detection time.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a shigella rapid detection method based on up-conversion-gold nano fluorescence resonance energy transfer, which comprises the following steps:
Step 3, carboxyl modification of the upconversion fluorescent nano material: and (3) dissolving the water-soluble upconversion fluorescent nano material obtained in the step (2) in a mixed solution of toluene and succinic anhydride, heating in a water bath kettle under the protection of nitrogen in the whole process, washing for many times by using toluene, pure water and acetonitrile after the reaction is finished, and drying in vacuum to obtain the carboxyl modified upconversion fluorescent nano material.
And 4, modifying the complementary strand by using the carboxyl modified up-conversion fluorescent nano material: dispersing the carboxyl modified upconversion fluorescent nano material obtained in the step 3 into a buffer solution, adding glutaraldehyde for oscillation reaction, and washing the reaction product for multiple times by using the buffer solution after the reaction is finished; re-dissolving with buffer solution, adding shigella complementary strand, oscillating, reacting, washing with buffer solution for several times, and re-dissolving with buffer solution.
Step 5, synthesizing a gold nano material: dispersing chloroauric acid in ultrapure water, heating to boil, keeping the boiling state, adding trisodium citrate dispersed in ultrapure water, keeping the boiling state after reboiling, turning off a heat source after the reaction is finished, and cooling the solution for later use.
Step 7, verifying the specificity of the detection system: different standard strains are selected to repeat the detection process of the invention, and the intensity of the output fluorescence signal is recorded.
Preferably, the mass ratio of yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate in step 1 is as follows: 0.3492 g: 0.2676 g: 0.1860 g: 0.0180g, ethanol: oleic acid: octadecene =1 mL: 3 mL: 7mL; the first heating temperature is 150-170 ℃, and the heating time is 30min; the mass ratio of the ammonium fluoride to the sodium hydroxide is as follows: 0.1472 g: 0.1g; the two-stage water bath conditions are as follows: stirring in water bath at 50 deg.C for 40min, and water bath at 70 deg.C for 30min; the temperature of the second heating is 290-310 ℃, and the heating time is 1h.
Preferably, the mass of alendronate in step 2 is 50mg, and the upconverter material chloroform ethanol =200 mg: 10 mL: 4mL.
Preferably, in step 3, the upconverter material toluene succinic anhydride =40 mg: 15 ml: 5ml. The heating temperature is 80 ℃, and the heating time is 12h.
Preferably, in step 4, the upconverting material MES buffer =5 mg: 2.5ml; EDC, NHS, pure water =160mg, 80ml; the PBS buffer =2.5ml; the complementary strand =500 μ L (1 μmol/ml).
Preferably, chloroauric acid to ultrapure water =16.98 ml: 50ml in step 5; the ratio of trisodium citrate to ultrapure water is =0.05 g: 4.95ml, and the boiling state of the two times is kept for 10min.
Preferably, the concentration of the aptamer in the step 6 is 1 mu mol/ml, and the concentration of the up-conversion material is 4mg/ml; the up-conversion material, gold nano-particles and Shigella =140 muL, 140 muL and 20 muL.
The invention discloses the following technical effects:
(1) The invention discloses a fluorescent signal detection method of shigella in food, which is characterized in that a steady-state specific shigella detection system is constructed based on the principle of fluorescence resonance energy transfer of up-conversion fluorescent nanoparticles and gold nanoparticles, and the detection schematic diagram is shown in figure 1;
(2) The specificity detection system constructed by the invention shows high selectivity to Shigella, can eliminate the interference of other bacteria, can realize the specificity detection of the Shigella content in food, overcomes the defects of the traditional detection method, and has important significance for the guarantee of food safety.
(3) The linear range concentration of the shigella and the fluorescence signal intensity established by the invention is 1.2 multiplied by 10 2 -1.2×10 8 CFU/mL, has a wider linear detection range, has a detection limit LOD of 30CFU/mL, can well meet the requirement of high-sensitivity detection of Shigella in food, and has better universality.
(4) The invention realizes the high specificity and sensitivity detection of the shigella in the food by constructing the specific shigella detection system, and has wider detection linear range and lower detection limit, thereby having good practical prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of Shigella detection based on fluorescence resonance energy transfer according to the present invention;
FIG. 2 is a transmission electron micrograph of water-soluble up-converting nanomaterial prepared according to example 1;
FIG. 3 is a spectrum of fluorescence signals of a specific detection system at different Shigella concentrations in example 1;
FIG. 4 is a standard curve of fluorescence detection at different Shigella concentrations in example 1;
FIG. 5 is a graph showing fluorescence intensity signals of different food-borne pathogens in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the following description clearly describes the embodiments of the present invention in combination with the technical solutions, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the 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 rapid detection method of shigella based on fluorescence resonance energy transfer, which comprises the following specific steps:
0.3492g of yttrium chloride hexahydrate, 0.2676g of gadolinium chloride hexahydrate, 0.1860g of ytterbium chloride hexahydrate and 0.0180g of erbium chloride hexahydrate are weighed out and dissolved in 6mL of methanol for 20min by ultrasonic. Adding 1mL of the above solution, 1mL of ethanol, 3mL of oleic acid, and 7mL of octadecenoic acid into a three-neck flask, and introducing water and air under sealed condition for 2min. The temperature is raised to 160 ℃, nitrogen is introduced in the whole process, the mixture is stirred for 30min at a constant speed, and then the mixture is cooled to the room temperature. 0.1472g of NH 4 F and 0.1g of NaOH were dissolved in 10mL of a methanol solution, and the solution was slowly dropped into a three-necked flask using a needle while keeping stirring. After all the liquid is dripped, the three-neck flask is sealed by a preservative film, transferred into a water bath kettle and stirred at a constant temperature of 50 DEG CAfter 40min, the temperature of the water bath kettle is raised to 70 ℃, and formaldehyde is volatilized for 30min. Building the device again, heating to 300 ℃, introducing nitrogen in the whole process, stirring at a constant speed for 1h, and cooling to room temperature. Finally transferring the solution into a 50mL centrifuge tube, adding 2.5mL pure water and 10mL ethanol, and centrifuging at 6000rpm for 3min; adding 10mL of cyclohexane for vortex dispersion, and centrifuging at 5000rpm for 2min; and adding 10mL of ethanol, centrifuging at 6000rpm for 3min, and placing the centrifugal tube in a vacuum drying oven at 60 ℃ to obtain the up-conversion fluorescent nano material of the oil phase.
Step 3, carboxyl-modified upconversion fluorescent nano material: the up-conversion fluorescent material modified by the amino group is dissolved in 15mL of toluene, ultrasonic treatment is carried out for 10min, and then 5mL0.16M succinic anhydride is added. Under nitrogen, heat to 80 ℃ in a water bath and keep at this temperature with constant stirring for 12h, then cool to room temperature. The cooled solution was placed in a 50mL centrifuge tube, centrifuged at 11000rpm for 15min, washed three times with toluene and deionized water and twice with acetonitrile. Vacuum drying at 60 ℃ for 4h to obtain the carboxyl modified up-conversion nano material (COOH-UCNPs).
And 4, modifying the complementary strand by using the carboxyl modified up-conversion fluorescent nano material: first, 5mg of ucnps-COOH was dispersed in 2.5 mm mes buffer (50mm, ph = 6) by sonication for 30 minutes. Then, 80mL of the mixed solution (2 mg/mL EDC and 2mg/mL NHS) was added to the UCNPs-COOH solution and reacted for 2h with stirring. Then, UCNPs-COOH was washed three times with PBS buffer solution (pH = 7.4) and re-dispersed in 2.5mL of PBS buffer solution (pH = 6.4). Then, 500. Mu.L of the complementary strand was added to the UCNPs-COOH solution, and reacted for 2h with stirring at room temperature. Finally, the complementary strand-modified UCNPs were washed with PBS buffer solution.
Step 5, synthesizing a gold nano material: 0.01698g of chloroauric acid is weighed and dispersed in 50mL of ultrapure water, then the chloroauric acid solution is placed in a three-neck flask, a condenser tube is installed and a glass plug is plugged, and the three-neck flask is stirred and heated to boiling state and kept at the boiling state for 10min. Thereafter, 5mL trisodium citrate (1%) was added rapidly, and after reheating to boiling, the boiling was maintained for 10min, during which vigorous stirring was maintained. After the reaction is finished, the heat source is closed, the reaction product is cooled to room temperature, and the reaction product is stored at 4 ℃.
Optimizing a reaction system:
first, the upconversion solution modified by the complementary strand is adjusted to appropriate fluorescence intensity through dilution, then 1 muL of aptamer is diluted according to 1/10, 1/12.5, 1/25, 1/50, 1/100 and 1/200 respectively, 10 muL of diluted aptamer solution is added into 130 muL of gold nanometer solution for reaction for 15min, then 140 muL of pure water and 20 muL of upconversion solution are added for reaction for enough time, and the fluorescence intensity is measured after the reaction is finished. The optimal aptamer concentration is 1/100.
And secondly, connecting the optimized aptamer with the gold nanoparticles according to the result obtained in the first step, adding 140 mu L of pure water and 20 mu L of up-conversion solution with the concentration of 1, 2, 3, 4 and 5mg/mL respectively after the reaction is finished, reacting for a long time, and measuring the fluorescence intensity after the reaction is finished. The optimal upconversion concentration was obtained at 4mg/mL.
And thirdly, reacting the gold nano modified by the optimized aptamer and the upconversion modified by the complementary strand, changing the reaction time of the gold nano modified by the optimized aptamer and the upconversion modified by the complementary strand to 3, 5, 7, 9, 11, 13 and 15min respectively, and measuring the fluorescence intensity after the reaction is finished. The optimal upconversion and gold nanoparticle reaction time is 15min.
The fourth step is to140 μ L Shigella (10 high concentration) 6 CFU/mL) is added into 140 mu L of aptamer modified gold nano solution, reaction is carried out for 5min, 10min, 15min, 20min, 25min and 30min respectively, then 20 mu L of complementary strand modified up-conversion solution is added into the solution which reacts for different time, reaction is carried out for 15min, and fluorescence intensity is measured after the reaction is finished. The optimal reaction time of the gold nano-particles and the bacteria is 25min.
Step 7, constructing standard curves for detecting shigella at different concentrations: firstly, 130 mu L of gold nano-particles and 10 mu L of sulfhydryl modified aptamer react for 10min at normal temperature, and after the reaction is finished, 140 mu L of gold nano-particles with the concentration of 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 Respectively adding 10CFU/mL shigella into 140 mu L aptamer modified gold nano solution, reacting for 25min, then adding 20 mu L complementary strand modified upconversion solution into the solution, reacting for 15min, measuring fluorescence, taking the fluorescence signal value as Y, establishing a standard curve with the concentration of shigella as X, and obtaining a shigella standard curve Y =191.62X +2298.9, R 2 =0.9695, LOD =3S according to the formula 0 /K(S 0 K is the slope of the standard curve for repeated determination of the relative standard deviation of ten blank experiments) to obtain a detection limit of 30CFU/mL.
Example 2:
the invention discloses a rapid detection method of shigella based on fluorescence resonance energy transfer, which comprises the following specific steps:
0.3492g of yttrium chloride hexahydrate, 0.2676g of gadolinium chloride hexahydrate, 0.1860g of ytterbium chloride hexahydrate and 0.0180g of erbium chloride hexahydrate are weighed out and dissolved in 6mL of methanol for 20min by ultrasonic. Adding 1ml of above solution, 1ml of ethanol, 3ml of oleic acid, and 7ml of octadecenoic acid into a three-neck flask, and introducing water and gas for 2min under sealed condition. The temperature is increased to 150 ℃, nitrogen is introduced into the whole process, the mixture is stirred at a constant speed for 30min, and then the mixture is cooled to the room temperature. 0.1472g of NH 4 F and 0.1g of NaOH were dissolved in 10ml of a methanol solution, and the solution was slowly dropped into a three-necked flask using a needle while keeping stirring. After all the water drops are finished, sealing the three-neck flask with a preservative film, transferring the three-neck flask into a water bath kettle, stirring the three-neck flask at a constant temperature of 50 ℃ for 40min, then heating the water bath kettle to 70 ℃, and volatilizing formaldehyde for 30min. Building the device again, heating to 290 ℃, introducing nitrogen in the whole process, stirring at a constant speed for 1h, and cooling to room temperature. Finally transferring the solution into a 50mL centrifuge tube, adding 2.5mL of pure water and 10mL of ethanol, and centrifuging at 6000rpm for 3min; adding 10mL of cyclohexane for vortex dispersion, and centrifuging at 5000rpm for 2min; adding 10mL of ethanol, centrifuging at 6000rpm for 3min, placing the centrifugal tube at 60 ℃, and drying in vacuum to obtain the up-conversion fluorescent nano material of the oil phase.
Step 3, carboxyl modification of the upconversion fluorescent nano material: ADA-UCNPs were dissolved in 15mL toluene, sonicated for 10min, and then 5mL0.16M succinic anhydride was added. Under the protection of nitrogen, the mixture is heated to 80 ℃ in a water bath kettle and kept at the temperature for stirring for 12 hours, and then cooled to room temperature. The cooled solution was placed in a 50ml centrifuge tube, centrifuged at 11000rpm for 15min, washed three times with toluene and deionized water and twice with acetonitrile. Vacuum drying at 60 deg.C for 4h. And obtaining the carboxyl modified upconversion nanometer material (COOH-UCNPs).
And 4, modifying the complementary strand by using the carboxyl modified up-conversion fluorescent nano material: first, 5mg of UCNPs-COOH was dispersed in 2.5ml of MES buffer (50mm, ph = 6) for 30 minutes by sonication. Then, 80mL of the mixed solution (2 mg/mL EDC and 2mg/mL NHS) was added to the UCNPs-COOH solution and reacted for 2 hours with stirring. Then, UCNPs-COOH was washed three times with PBS buffer solution (pH = 7.4) and re-dispersed in 2.5ml of PBS buffer solution (pH = 6.4). Then, 500. Mu.L of the complementary strand was added to the UCNPs-COOH solution, and reacted for 2 hours at room temperature with stirring. Finally, the complementary strand-modified UCNPs were washed with PBS buffer solution.
Step 5, synthesizing a gold nano material: 0.01698g of chloroauric acid is weighed and dispersed in 50ml of ultrapure water, then the chloroauric acid solution is placed in a three-neck flask, a condenser tube is added and a glass plug is plugged, and the three-neck flask is stirred and heated to boiling state and kept at the boiling state for 10min. 5ml trisodium citrate (1%) is then added rapidly, and after reheating to boiling, the boiling state is maintained for 10min, during which vigorous stirring is maintained. After the reaction is finished, the heat source is closed, the reaction product is cooled to room temperature, and the reaction product is stored at 4 ℃.
Optimizing a reaction system:
in the first step, the fluorescence intensity of the complementary strand modified up-conversion solution is adjusted to a proper fluorescence intensity by dilution, then 1 uL of aptamer is diluted according to 1/10, 1/12.5, 1/25, 1/50, 1/100 and 1/200 respectively, then 10 uL of diluted aptamer solution is added into 130 uL of gold nanometer solution for reaction for 15min, then 140 uL of pure water and 20 uL of up-conversion solution are added, the reaction is carried out for a sufficient time, and the fluorescence intensity is measured after the reaction is finished. The optimal aptamer concentration is 1/100.
And secondly, connecting the optimized aptamer with the gold nanoparticles according to the result obtained in the first step, adding 140 mu L of pure water and 20 mu L of up-conversion solution with the concentration of 1, 2, 3, 4 and 5mg/ml respectively after the reaction is finished, reacting for a long time, and measuring the fluorescence intensity after the reaction is finished. The optimum upconversion concentration was found to be 4mg/ml.
And thirdly, reacting the gold nano modified by the optimized aptamer with the upconversion modified by the complementary strand, changing the reaction time of the gold nano modified by the optimized aptamer and the upconversion modified by the complementary strand, controlling the reaction time to be 3, 5, 7, 9, 11, 13 and 15 minutes respectively, and measuring the fluorescence intensity after the reaction is finished. The optimal reaction time of up-conversion and gold nano-particles is 15min.
Fourthly, 140 mu L of Shigella (high concentration 10) 6 CFU/mL) is added into 140 mu L of aptamer modified gold nano solution, reaction is carried out for 5min, 10min, 15min, 20min, 25min and 30min respectively, then 20 mu L of complementary strand modified up-conversion solution is added into the solution which reacts for different time, the optimized time of the third step is reacted, and the fluorescence intensity is measured after the reaction is finished. The optimal reaction time of the gold nano-particles and the bacteria is 25min.
Step 7, constructing standard curves for detecting shigella at different concentrations: firstly, 125 mu L of gold nano-particles and 5 mu L of thiol-modified aptamer react for 10min at normal temperature, and after the reaction is finished, the concentration of 140 mu L is respectively 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 And 10CFU/mL shigella is respectively added into 140 muL of aptamer modified gold nano solution, reaction is carried out for 25min, then 20 muL of complementary strand modified up-conversion solution is respectively added into the solution, fluorescence is detected after reaction is carried out for 15min, the fluorescence signal value is taken as Y, a standard curve with the concentration of shigella as X is established, a shigella standard curve Y =191.6X +2299 is obtained, R is subjected to 2 =0.9544, according to the formula LOD =3S 0 /K(S 0 K is the slope of the standard curve for repeated determination of the relative standard deviation of ten blank experiments) to obtain a detection limit of 30CFU/mL.
Example 3:
the invention discloses a rapid detection method of shigella based on fluorescence resonance energy transfer, which comprises the following specific steps:
0.3492g of yttrium chloride hexahydrate, 0.2676g of gadolinium chloride hexahydrate, 0.1860g of ytterbium chloride hexahydrate and 0.0180g of erbium chloride hexahydrate are weighed and dissolved in 6mL of methanol by ultrasound for 20min. Adding 1ml of the above solution, 1ml of ethanol, 3ml of oleic acid, and 7ml of octadecenoic acid into a three-neck flask, and introducing water and air under sealed condition for 2min. The temperature is increased to 170 ℃, nitrogen is introduced into the whole process, the mixture is stirred at a constant speed for 30min, and then the mixture is cooled to room temperature. 0.1472g of NH 4 F and 0.1g of NaOH were dissolved in 10ml of a methanol solution, and the solution was slowly dropped into a three-necked flask using a needle while keeping stirring. After all the water drops are finished, sealing the three-neck flask with a preservative film, transferring the three-neck flask into a water bath kettle, stirring the three-neck flask at a constant temperature of 50 ℃ for 40min, then heating the water bath kettle to 70 ℃, and volatilizing formaldehyde for 30min. Building the device again, heating to 310 ℃, introducing nitrogen in the whole process, stirring at a constant speed for 1h, and cooling to room temperature. Finally transferring the solution into a 50mL centrifuge tube, adding 2.5mL of pure water and 10mL of ethanol, and centrifuging at 6000rpm for 3min; adding 10mL of cyclohexane for vortex dispersion, and centrifuging at 5000rpm for 2min; adding 10mL of ethanol, centrifuging at 6000rpm for 3min, placing the centrifugal tube at 60 ℃, and drying in vacuum to obtain the up-conversion fluorescent nano material of the oil phase.
Step 3, carboxyl modification of the upconversion fluorescent nano material: ADA-UCNPs were dissolved in 15mL toluene, sonicated for 10min, and then 5mL0.16M succinic anhydride was added. Under the protection of nitrogen, the mixture is heated to 80 ℃ in a water bath kettle and kept at the temperature for stirring for 12 hours, and then cooled to room temperature. The cooled solution was placed in a 50ml centrifuge tube, centrifuged at 11000rpm for 15min, washed three times with toluene and deionized water and twice with acetonitrile. Vacuum drying at 60 deg.C for 4h. Obtaining the carboxyl modified up-conversion nano material (COOH-UCNPs).
And 4, modifying the complementary strand by using the carboxyl modified up-conversion fluorescent nano material: first, 5mg of ucnps-COOH was dispersed in 2.5ml of MES buffer (50mm, ph = 6) for 30 minutes by sonication. Then, 80mL of the mixed solution (2 mg/mL EDC and 2mg/mL NHS) was added to the UCNPs-COOH solution and reacted for 2 hours with stirring. Then, UCNPs-COOH was washed three times with PBS buffer solution (pH = 7.4) and redispersed in 2.5ml of PBS buffer solution (pH = 6.4). Then, 500. Mu.L of the complementary strand was added to the UCNPs-COOH solution, and reacted for 2 hours at room temperature with stirring. Finally, the complementary strand modified UCNPs were washed with PBS buffer solution.
Step 5, synthesizing a gold nano material: 0.01698g of chloroauric acid is weighed and dispersed in 50ml of ultrapure water, then the chloroauric acid solution is placed in a three-neck flask, a condenser tube is added and a glass plug is plugged, and the three-neck flask is stirred and heated to boiling state and kept at the boiling state for 10min. 5ml trisodium citrate (1%) is then added rapidly, and after reheating to boiling, the boiling state is maintained for 10min, during which vigorous stirring is maintained. After the reaction is finished, the heat source is closed, the reaction product is cooled to room temperature, and the reaction product is stored at 4 ℃.
Optimizing a reaction system:
in the first step, the fluorescence intensity of the complementary strand modified up-conversion solution is adjusted to a proper fluorescence intensity by dilution, then 1 uL of aptamer is diluted according to 1/10, 1/12.5, 1/25, 1/50, 1/100 and 1/200 respectively, then 10 uL of diluted aptamer solution is added into 130 uL of gold nanometer solution for reaction for 15min, then 140 uL of pure water and 20 uL of up-conversion solution are added, the reaction is carried out for a sufficient time, and the fluorescence intensity is measured after the reaction is finished. The optimal aptamer concentration is 1/100.
And secondly, connecting the optimized aptamer with the gold nanoparticles according to the result obtained in the first step, adding 140 mu L of pure water and 20 mu L of up-conversion solution with the concentration of 1, 2, 3, 4 and 5mg/ml respectively after the reaction is finished, reacting for a long time, and measuring the fluorescence intensity after the reaction is finished. The optimum upconversion concentration was found to be 3mg/ml.
And thirdly, reacting the gold nano modified by the optimized aptamer with the upconversion modified by the complementary strand, changing the reaction time of the gold nano modified by the optimized aptamer and the upconversion modified by the complementary strand, controlling the reaction time to be 3, 5, 7, 9, 11, 13 and 15 minutes respectively, and measuring the fluorescence intensity after the reaction is finished. The optimal upconversion and gold nanoparticle reaction time is 15min.
Fourthly, 140 mu L of shigella (with high concentration of 10 percent) 6 CFU/mL) is added into 140 mu L of aptamer modified gold nano solution to react for 5min, 10min, 15min, 20min, 25min and 30min respectively, then 20 mu L of complementary strand modified upconversion solution is added into the solution reacting at different time, the optimized time in the third step is reacted, and the fluorescence intensity is measured after the reaction is finished. The optimal reaction time of the gold nano-particles and the bacteria is 25min.
Step 7, constructing standard curves for detecting shigella at different concentrations: firstly, 130 mu L of gold nano-particles and 10 mu L of sulfhydryl-modified aptamer react for 10min at normal temperature, and after the reaction is finished, the concentration of 140 mu L is respectively 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 Respectively adding 10CFU/mL shigella into 140 mu L aptamer modified gold nano solution, reacting for 25min, respectively adding 20 mu L complementary strand modified upconversion solution into the solution, reacting for 15min, measuring fluorescence, taking the fluorescence signal value as Y, establishing a standard curve with the concentration of shigella as X, and obtaining a shigella standard curve Y =190.43X +2303.2, R 2 =0.9646, according to the formula LOD =3S 0 /K(S 0 K is the slope of the standard curve for repeated determination of the relative standard deviation of ten blank experiments) to calculate a detection limit of 30CFU/mL.
Claims (7)
1. A shigella rapid detection method based on up-conversion-gold nano fluorescence resonance energy transfer is characterized by comprising the following steps:
step 1, preparing oil-soluble up-conversion fluorescent nanoparticles: mixing four materials of yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate with ethanol, oleic acid and 1-octadecene, placing the mixture in a three-neck flask, heating the mixture under the protection of nitrogen in the whole process, and then cooling the mixture to room temperature; dispersing ammonium fluoride and sodium hydroxide in methanol solution, slowly adding into a cooling system, and heating in water bath in two stages. And under the protection of nitrogen, heating the reaction system again, cooling to room temperature after the reaction is finished, performing centrifugal cleaning on ethanol and cyclohexane for many times, and performing vacuum drying to obtain the up-conversion nano material of the oil phase.
Step 2, preparing alendronate modified upconversion fluorescent nanoparticles: and (2) dissolving the oil phase up-conversion fluorescent nano material obtained in the step (1) in a mixed solution of chloroform, ethanol and alendronic acid, performing ultrasonic treatment, placing the mixture in a centrifugal tube, and adjusting the pH value by using pH test paper to stir and mix the whole system in an acidic environment. After the reaction is finished, the material is washed by pure water and ethanol for multiple times and finally dispersed in the pure water, so that the water-soluble up-conversion fluorescent material is obtained.
Step 3, preparing carboxyl modified upconversion fluorescent nanoparticles: and (3) dissolving the water-soluble upconversion fluorescent nano material obtained in the step (2) in toluene, performing ultrasonic treatment, then adding succinic anhydride, heating in a water bath kettle under the protection of nitrogen in the whole process, raising the temperature, keeping for a period of time, after the reaction is finished, washing for many times by using toluene, pure water and acetonitrile, and performing vacuum drying to obtain the carboxyl modified upconversion fluorescent nano material.
And 4, modifying the complementary strand by using the carboxyl modified up-conversion fluorescent nano material: and (3) dispersing the carboxyl modified upconversion fluorescent nanoparticles prepared in the step (3) in a morpholine ethanesulfonic acid buffer solution (MES) for ultrasonic treatment. Adding a mixed solution of carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the solution to perform stirring reaction, washing the solution for multiple times by Phosphate Buffer Solution (PBS) after the reaction is finished, and re-dispersing the solution in the PBS solution. And adding the aptamer complementary strand of the Shigella into the solution, stirring for reaction, and finally washing for multiple times by using PBS (phosphate buffer solution) to obtain the upconversion fluorescent nano material modified by the complementary strand.
Step 5, synthesizing a gold nano material: heating the chloroauric acid solution to boiling, adding the trisodium citrate solution, reacting for a period of time after reboiling, turning off the heat source after the reaction is finished, and cooling the solution for later use.
Step 6, constructing an up-conversion-gold nanometer detection system: connecting the gold nanoparticles with the Shigella aptamer, adding an upconversion fluorescent nanomaterial for modifying a Shigella complementary strand, finally adding Shigella to measure the intensity of a fluorescent signal, and constructing a standard curve by taking the concentration of the Shigella as a horizontal coordinate and the intensity of the fluorescent signal as a vertical coordinate.
Step 7, verifying the specificity of the detection system: different kinds of standard strains are selected to repeat the detection process of the invention, and the intensity of the output fluorescence signal is recorded.
2. The shigella rapid detection method based on up-conversion-gold nano fluorescence resonance energy transfer as claimed in claim 1, wherein in step 1, the mass ratio of yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate is: 0.3492 g: 0.2676 g: 0.1860 g: 0.0180g, ethanol oleic acid octadecene =1 mL: 3 mL: 7mL; the first heating temperature is as follows: heating at 150-170 deg.C for 30min; the mass ratio of ammonium fluoride to sodium hydroxide is as follows: 0.1472 g: 0.1g, 10mL of methanol; the two-stage water bath conditions were: stirring in water bath at 50 deg.C for 40min, and water bath at 70 deg.C for 30min; the temperature of the second heating is 290-310 ℃, and the heating time is 1h.
3. The shigella rapid detection method based on upconversion-gold nanofluorescence resonance energy transfer as claimed in claim 1, wherein in step 2, the mass of alendronic acid is 50mg, and the mass of upconversion material is chloroform to ethanol =200mg to 10mL to 4mL; the acidic condition is pH =2-5, and the stirring time is 30min.
4. The shigella rapid detection method based on upconversion-gold nanofluorescence resonance energy transfer as claimed in claim 1, wherein in step 3, the upconversion material: toluene: succinic anhydride =40 mg: 15 mL: 5mL; the heating temperature is 80 ℃, and the heating time is 12h.
5. The method for rapidly detecting Shigella based on upconversion-gold nanofluorescence resonance energy transfer as claimed in claim 1, wherein in step 4, the upconversion material/MES buffer solution =5 mg: 2.5mL, the EDC/NHS/pure water =160 mg: 80mL, the PBS buffer solution is 2.5mL, and the complementary strand is 500 μ L (1 μmol/mL).
6. The shigella rapid detection method based on upconversion-gold nanofluorescence resonance energy transfer as claimed in claim 1, wherein in step 5, chloroauric acid to ultrapure water =16.98 mL: 50mL, trisodium citrate to ultrapure water =0.05 g: 4.95mL, and boiling states of two times are kept for 10min.
7. The shigella rapid detection method based on up-conversion-gold nanofluorescent resonance energy transfer as claimed in claim 1, wherein in step 6, the concentration of the aptamer is 1 μmol/mL, and the concentration of the up-conversion material is 4mg/mL; upconverter material gold nanoparticles shigella =140 μ L20 μ L.
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