CN113588618A - Preparation method and application of up-conversion luminescence flexible biosensor for diethylstilbestrol detection - Google Patents
Preparation method and application of up-conversion luminescence flexible biosensor for diethylstilbestrol detection Download PDFInfo
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Abstract
The invention belongs to the field of food and environment safety detection, and particularly relates to a preparation method and application of an up-conversion luminescence flexible biosensor for diethylstilbestrol detection; the method comprises the following steps: the method comprises the following steps: preparing an oleic acid-coated up-conversion nanomaterial; step two, preparing an amination up-conversion nano material; step three, preparing a flexible up-conversion luminescence sensor; preparing a biological functionalized flexible up-conversion luminescence sensor; the sensor is used for detecting the diethylstilbestrol, and the detection of the diethylstilbestrol content in food and environmental samples is realized through the establishment of a diethylstilbestrol content detection standard curve; according to the invention, the preparation of the up-conversion luminescence flexible biosensor and the detection method of the diethylstilbestrol are provided, so that the diethylstilbestrol in food and environmental samples can be quickly and economically detected, and the up-conversion luminescence flexible biosensor has a wider concentration detection range and a lower detection limit; meanwhile, the sensor has a reproducible function and has good practical prospect.
Description
Technical Field
The invention belongs to the technical field of food and environment detection, and particularly relates to a preparation method and application of an up-conversion luminescence flexible biosensor for diethylstilbestrol detection.
Background
Diethylstilbestrol (DES) is an endocrine disrupter similar to estrogen, primarily used to prevent premature birth, abortion and pregnancy complications, and later used as a veterinary drug to promote animal growth, but poses a potential threat to human health because it interferes with the synthesis and metabolism of normal hormones in humans.
DES has many adverse effects on human health, such as adverse reproductive disorders, long-term changes in sexual behavior, cervical intraepithelial neoplasia, etc., so that detection of DES in food is strictly prohibited in countries such as china and the united states. For example, the Chinese national standard NY 5070-2002 stipulates that the DES cannot be detected in fishery medicine residues in aquatic products, but the potential illegally added DES still threatens the health of consumers. The traditional DES detection method, such as high performance liquid chromatography, enzyme-linked immunosorbent assay and the like, has the difficulties of over-professional operation, high detection cost, complex steps and the like, so that the rapid, convenient and economic detection requirement on the DES content cannot be met.
Disclosure of Invention
The invention aims to provide a preparation and detection method of an up-conversion luminescence flexible biosensor for detecting diethylstilbestrol, which aims to solve the problems in the prior art, so that diethylstilbestrol in food and environment can be quickly and conveniently detected.
In order to achieve the purpose, the invention provides the following scheme:
step one, preparing an oleic acid coated up-conversion nano material: weighing yttrium chloride hexahydrate, ytterbium chloride hexahydrate and thulium chloride hexahydrate in proportion, dispersing the weighed materials in a methanol solvent A, adding oleic acid and 1-octadecene, stirring for the first time under a certain temperature condition, cooling to room temperature after stirring, dropwise adding a mixed solution of sodium hydroxide, ammonium fluoride and the methanol solvent B, heating for a period of time after sealing, stirring for the second time after heating, cooling to room temperature after stirring, carrying out centrifugal separation to obtain an upconversion nano particle precipitate, washing with a mixed solution of cyclohexane and ethanol, and drying to obtain an oleic acid-coated upconversion nano particle;
step two, preparing an amination up-conversion nano material: weighing the oleic acid-coated up-conversion nanoparticles prepared in the step one, and dispersing the oleic acid-coated up-conversion nanoparticles in a hydrochloric acid solution for ultrasonic treatment; after the treatment is finished, adding ethanol, performing ultrasonic dispersion uniformly, and then adding deionized water and ammonia water, and stirring uniformly at a certain temperature; after stirring uniformly, adding tetraethyl orthosilicate for carrying out a first reaction, and then adding 3-aminopropyltriethoxysilane for carrying out a second reaction; after the reaction is finished, performing centrifugal separation, and cleaning the obtained product with distilled water to obtain an amination up-conversion nano material;
step three, preparing a flexible up-conversion luminescence sensor: cutting a polydimethylsiloxane film according to requirements, soaking the polydimethylsiloxane film in a mixed solution of hydrogen peroxide solution and concentrated sulfuric acid, taking out the polydimethylsiloxane film, washing the polydimethylsiloxane film with distilled water, soaking the polydimethylsiloxane film in a 3-aminopropyltriethoxysilane aqueous solution for heating reaction, and drying the polydimethylsiloxane film with nitrogen after the reaction is finished to obtain an amino-functionalized polydimethylsiloxane film;
activating the upconversion nanoparticles prepared in the step two by using a glutaraldehyde aqueous solution; separating the activated up-conversion nano particles, dispersing the up-conversion nano particles in a phosphate buffer solution, and immersing the amino-functionalized polydimethylsiloxane film into the buffer solution for incubation to prepare the flexible up-conversion luminescence sensor;
step four, preparing a bio-functionalized flexible up-conversion luminescence sensor: immersing the flexible up-conversion luminescence sensor obtained in the third step into a glutaraldehyde aqueous solution for activation; after the reaction is finished, taking out the flexible up-conversion luminescence sensor, washing the flexible up-conversion luminescence sensor by using distilled water, immersing the flexible up-conversion luminescence sensor into a phosphate buffer solution, adding a diethylstilbestrol aptamer complementary sequence solution, and performing primary constant-temperature incubation in a shaking table; after the incubation is finished, adding a diethylstilbestrol aptamer solution marked by 4- ((4- (dimethylamino) phenyl) azo) benzoic acid (Dabcyl), and performing second constant-temperature incubation on a shaking table; and taking out the sensor after the reaction is finished, washing with deionized water, and blow-drying with nitrogen to prepare the bio-functionalized flexible up-conversion luminescence sensor.
The invention also relates to an application of the bio-functionalized flexible up-conversion luminescence sensor in detecting diethylstilbestrol, which comprises the following steps:
(1) the establishment of a diethylstilbestrol content detection standard curve: immersing a biological functionalized flexible up-conversion luminescence sensor into a Tris-HCl buffer solution, and then respectively adding diethylstilbestrol standard solutions with different concentrations, wherein one diethylstilbestrol standard solution corresponds to one sensor, and the concentrations and the sensors are in one-to-one correspondence; obtaining detection solutions with different concentrations, determining the characteristic value of a fluorescence intensity signal of the detection solution after incubation, and performing linear fitting by using the concentration of diethylstilbestrol and the characteristic value of the fluorescence intensity signal to establish a standard curve for detecting the content of diethylstilbestrol;
(2) detection of diethylstilbestrol content in food and environmental samples: pretreating the food and environment samples, extracting to obtain a sample solution containing diethylstilbestrol, immersing a biological functionalized flexible up-conversion luminescence sensor in the sample solution, incubating, measuring the characteristic value of a fluorescence intensity signal of the solution, substituting the characteristic value into the standard curve obtained in the step (1), and calculating the content of the diethylstilbestrol in the food and environment samples.
Further, in the first step, the dosage ratio of the yttrium chloride hexahydrate, the ytterbium chloride hexahydrate, the thulium chloride hexahydrate and the methanol solvent A is 0.2412 g: 0.0775 g: 0.0019 g: 10 mL; the dosage ratio of the methanol solvent A to the oleic acid to the 1-octadecene is 10 mL: 6mL of: 15 mL; the dosage ratio of the methanol solvent A to the sodium hydroxide to the ammonium fluoride to the methanol solvent B is 10 mL: 0.1 g: 0.1482 g: 10 mL.
Further, the certain temperature in the first step is 160 ℃; the first stirring is magnetic stirring for 30 min; the heating temperature after sealing is 50-80 ℃, and the heating time is 40 min; the temperature of the second stirring is 300 ℃, and the stirring time is 1 h.
Further, in the second step, the proportions of the up-conversion nanoparticles, the hydrochloric acid solution, the ethanol, the ammonia water, the tetraethyl orthosilicate and the 3-aminopropyltriethoxysilane are 20mg to 0.5mL to 60mL to 2.5mL to 20uL to 50 uL; the first reaction time is 4-6h, and the second reaction time is 1-2 h; the concentration of the hydrochloric acid solution is 0.1 mol/L; the certain temperature condition is 65 ℃.
Further, in the third step, the size of the polydimethylsiloxane film is 1 × 1 × 0.05 cm; the volume ratio of the hydrogen peroxide solution to the concentrated sulfuric acid mixed solution is 1:3, and the soaking time is 60 s; the volume fraction of the 3-aminopropyltriethoxysilane solution is 5%, the heating reaction temperature is 60-70 ℃, and the reaction time is 3 h; the mass fraction of the hydrogen peroxide solution is 30 percent, and the mass fraction of the concentrated sulfuric acid is 98 percent.
Further, in the third step, the mass fraction of the glutaraldehyde aqueous solution is 25%; the proportion of the up-conversion nanoparticles to the glutaraldehyde aqueous solution is 10mg:1.25mL, and the activation time is 2 h; the pH value of the phosphate buffer solution is 7.4, and the molar concentration is 10 mmol/L; the dosage ratio of the up-conversion nano particles to the phosphate buffer solution is 10mg:5 mL; the incubation time of the up-conversion nano particle solution and the polydimethylsiloxane film is 10-14 h.
Further, in the fourth step, the mass fraction of the glutaraldehyde aqueous solution is 25%, and the activation time is 2 hours; the pH value of the phosphate buffer solution is 7.4, and the molar concentration is 10 mmol/L; the phosphate buffer solution, the diethylstilbestrol aptamer complementary sequence solution and the diethylstilbestrol aptamer solution are mixed according to the proportion of 2 mL: 30 uL: 30 uL; the concentration of the diethylstilbestrol aptamer solution is 10nmol/L, the temperature of the first constant-temperature incubation is 37 ℃, the incubation time is 10-14h, and the rotating speed of a shaking table is 200 r; the concentration of the 4- ((4- (dimethylamino) phenyl) azo) benzoic acid labeled diethylstilbestrol aptamer complementary sequence solution is 10nmol/L, the temperature of the second constant-temperature incubation is 37 ℃, the incubation time is 2h, and the rotating speed of a shaking table is 200 r.
Further, in the step (1), the pH value of the Tris-HCl buffer solution is 8.5, and the molar concentration is 10 mmol/L; the volume ratio of the Tris-HCl buffer solution to the diethylstilbestrol standard solution is 20: 1; the concentration range of the diethylstilbestrol standard solution is 0.05-500 ng/mL; the incubation temperature is 37 ℃, and the incubation time is 20-25 min;
the step of measuring the fluorescence intensity signal characteristic value of the detection solution comprises the following steps: measuring the intensity of a flexible sensor at 477nm under excitation of 980nm excitation lightFluorescence intensity value, recording the fluorescence intensity value F of the flexible sensor without diethylstilbestrol0Adding diethylstilbestrol standard solution to react, and then obtaining the fluorescence intensity value F of the flexible sensor; (F-F)0)/F0The fluorescence intensity signal characteristic value of the detection solution is obtained.
Further, in the step (2), the sample pretreatment method comprises: homogenizing solid food, adding into acetonitrile-acetone mixed solution, oscillating, blowing with nitrogen, centrifuging, extracting with acetonitrile, collecting supernatant, blowing with nitrogen, and re-dissolving with methanol solution;
the volume ratio of acetonitrile to acetone in the acetonitrile-acetone mixed solution is 4: 1; the dosage ratio of the solid food to the acetonitrile-acetone mixed solution is 1 g: 3 mL; the mass fraction of the methanol solution is 40%. The relationship between the use amount of the methanol solution during redissolution and the use amount of solid food is 1-2 ml: 2g of the total weight of the mixture; the incubation temperature is 37 ℃, and the incubation time is 20-25 min.
Regeneration of flexible up-conversion luminescence biosensor: incubating the sensor reacted with the diethylstilbestrol solution to be detected and a Dabcyl (dye) -labeled diethylstilbestrol aptamer complementary sequence again in a shaking table at constant temperature; and taking out the sensor after the reaction is finished, washing with deionized water, and drying with nitrogen to realize regeneration of the flexible up-conversion luminescence biosensor.
The methanol solvent A and the methanol solvent B mentioned in the steps are both methanol, and different letters are only used for distinguishing in terms of names.
The invention has the following beneficial effects:
1. the invention discloses a preparation method of an up-conversion luminescence flexible biosensor, which is characterized by modifying up-conversion nano particles on the surface of polydimethylsiloxane through a glutaraldehyde crosslinking method, then combining an aptamer complementary sequence on the surface of the sensor through the glutaraldehyde crosslinking method, and finally self-assembling a quencher-labeled aptamer sequence on the surface of the sensor through a base complementary pairing principle.
2. The invention discloses a diethylstilbestrol detection method, which is used for detecting DES (data encryption standard) by a flexible biosensor based on up-conversion luminescence, and particularly uses Dabcyl dye at the tail end of an aptamer sequence and up-conversion nanoparticles as a fluorescence acceptor and a fluorescence donor for fluorescence resonance energy transfer respectively.
3. The specificity detection system constructed by the invention, particularly the up-conversion nanoparticle-Dabcyl fluorescence resonance energy transfer system which is optimally designed and connected with the aptamer, has strong fluorescence responsiveness to DES, can effectively eliminate the interference of background fluorescence and other molecules, has high specificity to the detection of DES, can realize high-sensitivity detection on DES content, overcomes the defects of the traditional method, and is of great importance to guarantee the safety of food and environment.
4. The DES concentration and fluorescence intensity signal characteristic value linear concentration range established by the invention is 0.05-500ng/mL, the DES concentration and fluorescence intensity signal characteristic value linear concentration range has a wider linear detection range, the detection limit LOD is 0.032ng/mL, the high-sensitivity detection of the AFB1 content in food can be met, the DES concentration and fluorescence intensity signal characteristic value linear concentration range has good universality, and the detection precision and sensitivity are higher than those of the traditional method.
5. The upconversion luminescence flexible biosensor constructed by the invention has a reproducible function, can be effectively reproduced for 7 times by taking 80% detection precision as a threshold value, and has good portability and economy, so that the detection method has good practical prospect.
Drawings
FIG. 1 is a schematic diagram of the preparation, regeneration and DES detection of the upconversion luminescence flexible biosensor according to the present invention.
Fig. 2 is a transmission electron micrograph of the upconversion nanoparticles prepared in example 1.
FIG. 3 is a scanning electron micrograph of the upconversion luminescent flexible biosensor prepared in example 1.
FIG. 4 is a standard curve established for the detection of DES at different concentrations in example 1; wherein, A is a fluorescence signal diagram of a sensor for detecting DES with different concentrations; b is a standard curve established by DES concentration logarithm and a characteristic value of a fluorescence intensity signal of the sensor at 477 nm.
FIG. 5 is a graph of performance evaluation of an upconversion luminescent flexible biosensor; wherein, A is a regeneration performance evaluation chart of the up-conversion luminescence flexible biosensor in the embodiment 1; b is the characteristic value of the fluorescence intensity signal of different interferents of comparative examples 1-8.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The "parts" in the present invention are all parts by mass unless otherwise specified. The diethylstilbestrol aptamer complementary sequence solution and the diethylstilbestrol aptamer solution used in the invention are purchased from biological engineering (Shanghai) GmbH.
FIG. 1 is a schematic diagram of the preparation, regeneration and DES detection of an upconversion luminescence flexible biosensor according to the present invention; the specific steps are detailed in the examples;
example 1:
step one, preparing an oleic acid coated up-conversion nano material: 0.2412g of yttrium chloride hexahydrate, 0.0775g of ytterbium chloride hexahydrate and 0.0019g of thulium chloride hexahydrate are accurately weighed and ultrasonically dispersed in 10mL of methanol solvent, 6mL of oleic acid and 15mL of 1-octadecene are added, then the mixture is magnetically stirred and reacted for 30min at 160 ℃ under the protection of nitrogen, and after the mixture is cooled to room temperature, a mixed solution containing 0.1g of sodium hydroxide and 0.1482g of ammonium fluoride dissolved in 10mL of methanol is dropwise added. Sealing the flask, heating at 80 ℃ for 40min to volatilize methanol, magnetically stirring at 300 ℃ for 1h, cooling to room temperature, performing centrifugal separation to obtain an upconversion nanoparticle precipitate, cleaning with a mixed solution of cyclohexane and ethanol in a volume ratio of 1:1 for three times, and drying to obtain pure oleic acid-coated upconversion nanoparticles;
FIG. 2 is a transmission electron micrograph of the prepared upconverting nanoparticles;
step two, preparing an amination up-conversion nano material: weighing 20mg of oleic acid-coated up-conversion nanoparticles, dispersing in 1mL of hydrochloric acid solution with the concentration of 0.1mol/L, and carrying out ultrasonic treatment to remove surface oleic acid groups; after the treatment is finished, dispersing the up-conversion nanoparticles in 60mL of ethanol and performing ultrasonic dispersion, then adding 10mL of deionized water and 2.5mL of ammonia water and uniformly stirring at 65 ℃; adding 20uL tetraethyl orthosilicate for reaction for 4h, and then adding 50uL 3-aminopropyltriethoxysilane for reaction for 2 h; after the reaction is finished, performing centrifugal separation, and collecting to obtain an amination up-conversion nano material;
step three, preparing a flexible up-conversion luminescence sensor: cutting a polydimethylsiloxane film (1 multiplied by 1cm), soaking for 60s by using 2mL of mixed solution (1:3, V/V) of hydrogen peroxide and concentrated sulfuric acid, and then soaking in 2mL of 3-aminopropyltriethoxysilane aqueous solution (5 percent, V/V) for heating reaction at the temperature of 70 ℃ for 3 h; after the reaction is finished, drying the mixture by using nitrogen to obtain an amino-functionalized polydimethylsiloxane film;
activating the 10mg of upconversion nanoparticles prepared in the step two by using 1.25mL of glutaraldehyde aqueous solution for 2 h; dispersing the activated up-conversion nanoparticles in 2mL of phosphate buffer solution, and incubating with an amino-functionalized polydimethylsiloxane film for 12h to prepare a flexible up-conversion luminescence sensor;
FIG. 3 is a scanning electron microscope image of the prepared upconversion luminescence flexible biosensor;
step four, flexible up-conversion luminescence sensor biological functionalization: immersing the flexible up-conversion luminescence sensor into 1.25mL of glutaraldehyde aqueous solution to activate surface amino for 2 h; after the reaction is finished, cleaning the flexible up-conversion luminescence sensor by using deionized water, immersing the flexible up-conversion luminescence sensor into 2mL of phosphate buffer solution again, adding 10nmol/L of diethylstilbestrol aptamer complementary sequence solution, and incubating for 12h at the constant temperature of 37 ℃ of a shaking table at the rotating speed of 200 r; after the incubation is finished, adding 10nmol/L Dabcyl labeled diethylstilbestrol aptamer solution, and incubating for 2h at the constant temperature of 37 ℃ by using a shaking table at the rotating speed of 200 r; after the reaction is finished, taking out the sensor, washing with deionized water, and drying with nitrogen to prepare the bio-functionalized flexible up-conversion luminescence sensor;
detecting the content of diethylstilbestrol in prawns:
(1) the establishment of a diethylstilbestrol content detection standard curve: immersing the bio-functionalized flexible up-conversion luminescence sensor obtained in the fourth step into 2mL of Tris-HCl buffer solution (pH 8.5), then respectively adding diethylstilbestrol standard solutions with different concentrations to obtain detection solutions with different concentrations, after incubation, measuring fluorescence intensity signal characteristic values of the detection solutions (figure 4A), performing linear fitting according to the diethylstilbestrol concentration and the fluorescence intensity signal characteristic values, and establishing a standard curve (figure 4B) for detecting the diethylstilbestrol content; the specific positions are as follows: preparing DES solutions with different concentrations of 0.05, 0.1, 1, 5, 50, 100, 500ng/mL and the like, respectively adding 100uL into a specific detection system, and collecting characteristic values of fluorescence signals after incubating for 25min at 37 ℃;
wherein, the step of measuring the fluorescence intensity signal characteristic value of the detection solution comprises the following steps: measuring the fluorescence intensity value of the flexible sensor at 477nm under the excitation of 980nm exciting light, and recording the fluorescence intensity value F of the flexible sensor without diethylstilbestrol0Adding diethylstilbestrol standard solution to react, and then obtaining the fluorescence intensity value F of the flexible sensor; (F-F)0)/F0The fluorescence intensity signal characteristic value of the detection solution is obtained.
FIG. 4 is a standard curve established for the detection of DES at different concentrations in example 1;
as can be seen from FIG. 4, the fluorescence signal at 477nm increased with increasing DES concentration; obtaining a standard curve (F-F) for DES content detection by linear fitting0)/F0＝1.1582log(CDES) +1.9561 (FIG. 4B), correlation coefficient R20.9897, the limit of detection LOD is 0.032ng/mL, and the linear range is 0.05-500 ng/mL.
(2) Detecting the content of diethylstilbestrol in prawns: weighing 2g of prawn sample, homogenizing, adding 6mL of acetonitrile-acetone mixed solution, shaking for 2min, and centrifuging at 15 ℃ for 10min at 4000 rmp; taking 3mL of supernatant, and drying the supernatant at 60 ℃ by using nitrogen; adding 0.5mL of chloroform, shaking for 20s, adding 2mL of sodium hydroxide solution (2mol/L), shaking for 30s, and centrifuging for 5min at 4000 rmp; taking 1mL of supernatant, adding 200uL of phosphoric acid solution (6mol/L), and shaking for 5 s; adding 3mL of acetonitrile for extraction, shaking for 2min, centrifuging at 4000rmp at room temperature for 10min, taking 1.5mL of upper organic phase, and drying by using nitrogen at 60 ℃; dissolving with 1mL of methanol solution (40%, V/V), shaking and mixing uniformly for 30 s; and (3) obtaining a prawn detection solution, finally taking 50uL for detection, after incubating with the flexible up-conversion luminescence biosensor for 25min, determining a characteristic value of a fluorescence signal of the sensor, substituting the characteristic value into the standard curve obtained in the step (1), and calculating the content of diethylstilbestrol in the prawn to be 8.65 ng/mL.
Regeneration of flexible up-conversion luminescence biosensor: the sensor reacted with the solution to be detected of the diethylstilbestrol is incubated with the sequence complementary to the diethylstilbestrol aptamer marked by Dabcyl (dye) again in a shaking table at constant temperature according to the fourth step; after the reaction is finished, taking out the sensor, washing with deionized water, and drying with nitrogen to realize regeneration of the flexible up-conversion luminescence biosensor; the same sensor was used for DES repeated detection and regeneration at 5ng/ml, and fig. 5A is a graph evaluating the regeneration performance of the upconversion luminescence flexible biosensor. As can be seen from fig. 5A, the sensor can be reproduced within 3 times to ensure the detection accuracy to be more than 90%, and the sensor can be reproduced within 7 times to ensure the detection accuracy to be more than 80%.
Example 2:
step one, preparing an oleic acid coated up-conversion nano material: 0.2412g of yttrium chloride hexahydrate, 0.0775g of ytterbium chloride hexahydrate and 0.0019g of thulium chloride hexahydrate are accurately weighed and ultrasonically dispersed in 10mL of methanol solvent, 6mL of oleic acid and 15mL of 1-octadecene are added, then the mixture is magnetically stirred and reacted for 30min at 160 ℃ under the protection of nitrogen, and after the mixture is cooled to room temperature, a mixed solution containing 0.1g of sodium hydroxide and 0.1482g of ammonium fluoride dissolved in 10mL of methanol is dropwise added. Sealing the flask, heating at 50 ℃ for 40min to volatilize methanol, magnetically stirring at 300 ℃ for 1h, cooling to room temperature, performing centrifugal separation to obtain an upconversion nanoparticle precipitate, cleaning with a mixed solution of cyclohexane and ethanol in a volume ratio of 1:1 for three times, and drying to obtain pure oleic acid-coated upconversion nanoparticles;
step two, preparing an amination up-conversion nano material: weighing 20mg of oleic acid-coated up-conversion nanoparticles, dispersing in 1mL of hydrochloric acid solution with the concentration of 0.1mol/L, and carrying out ultrasonic treatment to remove surface oleic acid groups; after the treatment is finished, dispersing the up-conversion nanoparticles in 60mL of ethanol and performing ultrasonic dispersion, then adding 10mL of deionized water and 2.5mL of ammonia water and uniformly stirring at 65 ℃; adding 20uL tetraethyl orthosilicate for reaction for 6h, and then adding 50uL 3-aminopropyltriethoxysilane for reaction for 1 h; after the reaction is finished, performing centrifugal separation, and collecting to obtain an amination up-conversion nano material;
step three, preparing a flexible up-conversion luminescence sensor: cutting a polydimethylsiloxane film (1 multiplied by 1cm), soaking for 60s by using 2mL of mixed solution (1:3, V/V) of hydrogen peroxide and concentrated sulfuric acid, then soaking in 2mL of 3-aminopropyltriethoxysilane aqueous solution (5 percent, V/V) to react with 60 ℃ by heating, and drying by blowing nitrogen after the reaction is finished to obtain the amino functionalized polydimethylsiloxane film; activating the 10mg of upconversion nanoparticles prepared in the step two by using 1.25mL of glutaraldehyde aqueous solution for 2 h; dispersing the activated up-conversion nanoparticles in 2mL of phosphate buffer solution, and incubating with an amino-functionalized polydimethylsiloxane film for 14h to prepare a flexible up-conversion luminescence sensor;
step four, flexible up-conversion luminescence sensor biological functionalization: immersing the flexible up-conversion luminescence sensor into 1.25mL of glutaraldehyde aqueous solution to activate surface amino for 2 h; after the reaction is finished, washing the flexible up-conversion luminescence sensor by using deionized water, immersing the flexible up-conversion luminescence sensor into 2mL of phosphate buffer solution again, adding 10nmol/L of diethylstilbestrol aptamer complementary sequence solution, and incubating for 14h at the constant temperature of 37 ℃ of a shaking table at the rotating speed of 200 r; after the incubation is finished, adding 10nmol/L Dabcyl labeled diethylstilbestrol aptamer solution, and incubating for 2h at the constant temperature of 37 ℃ by a shaking table at the rotating speed of 200 r; after the reaction is finished, taking out the sensor, washing with deionized water, and drying with nitrogen to prepare the bio-functionalized flexible up-conversion luminescence sensor;
detecting the content of diethylstilbestrol in fish meat:
(1) the establishment of a diethylstilbestrol content detection standard curve: immersing the bio-functionalized flexible up-conversion luminescence sensor obtained in the fourth step into 2mL of Tris-HCl buffer solution (pH 8.5), then respectively adding diethylstilbestrol standard solutions with different concentrations to obtain detection solutions with different concentrations, after incubation, determining the characteristic value of a fluorescence intensity signal of the detection solution, performing linear fitting according to the diethylstilbestrol concentration and the characteristic value of the fluorescence intensity signal, and establishing a standard curve for detecting the diethylstilbestrol content; the specific positions are as follows: preparing DES solutions with different concentrations of 0.05, 0.1, 1, 5, 50, 100, 500ng/mL and the like, respectively adding 100uL into a specific detection system, and collecting characteristic values of fluorescence signals after incubating for 25min at 37 ℃; obtaining a standard curve (F-F) for DES content detection by linear fitting0)/F0＝1.1573log(CDES) +1.9582, coefficient of correlation R20.9882, the limit of detection LOD was 0.033ng/mL, and the linear range was 0.05-500 ng/mL.
(2) Detecting the content of diethylstilbestrol in fish meat: weighing 2g of fish samples, homogenizing, adding 6mL of acetonitrile-acetone mixed solution, shaking for 2min, and centrifuging at 15 ℃ for 10min at 4000 rmp; taking 3mL of supernatant, and drying the supernatant at 60 ℃ by using nitrogen; adding 0.5mL of chloroform, shaking for 20s, adding 2mL of sodium hydroxide solution (2mol/L), shaking for 30s, and centrifuging for 5min at 4000 rmp; taking 1mL of supernatant, adding 200uL of phosphoric acid solution (6mol/L), and shaking for 5 s; adding 3mL of acetonitrile for extraction, shaking for 2min, centrifuging at 4000rmp at room temperature for 10min, taking 1.5mL of upper organic phase, and drying by using nitrogen at 60 ℃; dissolving with 1mL of methanol solution (40%, V/V), shaking and mixing uniformly for 30 s; and (3) finally, taking 50uL for detection, after incubation with the flexible up-conversion luminescence biosensor for 25min, measuring a characteristic value of a fluorescence signal of the sensor, substituting the characteristic value into the standard curve obtained in the step (1), and calculating the content of diethylstilbestrol in the fish meat to be 4.22 ng/mL.
Example 3:
step one, preparing an oleic acid coated up-conversion nano material: 0.2412g of yttrium chloride hexahydrate, 0.0775g of ytterbium chloride hexahydrate and 0.0019g of thulium chloride hexahydrate are accurately weighed and ultrasonically dispersed in 10mL of methanol solvent, 6mL of oleic acid and 15mL of 1-octadecene are added, then the mixture is magnetically stirred and reacted for 30min at 160 ℃ under the protection of nitrogen, and after the mixture is cooled to room temperature, a mixed solution containing 0.1g of sodium hydroxide and 0.1482g of ammonium fluoride dissolved in 10mL of methanol is dropwise added. Sealing the flask, heating at 70 ℃ for 40min to volatilize methanol, magnetically stirring at 300 ℃ for 1h, cooling to room temperature, performing centrifugal separation to obtain an upconversion nanoparticle precipitate, cleaning with a mixed solution of cyclohexane and ethanol in a volume ratio of 1:1 for three times, and drying to obtain pure oleic acid-coated upconversion nanoparticles;
step two, preparing an amination up-conversion nano material: weighing 20mg of oleic acid-coated up-conversion nanoparticles, dispersing in 1mL of hydrochloric acid solution with the concentration of 0.1mol/L, and carrying out ultrasonic treatment to remove surface oleic acid groups; after the treatment is finished, dispersing the up-conversion nanoparticles in 60mL of ethanol and performing ultrasonic dispersion, then adding 10mL of deionized water and 2.5mL of ammonia water and uniformly stirring at 65 ℃; adding 20uL tetraethyl orthosilicate to react for 5h, and then adding 50uL 3-aminopropyltriethoxysilane to react for 1.5 h; after the reaction is finished, performing centrifugal separation, and collecting to obtain an amination up-conversion nano material;
step three, preparing a flexible up-conversion luminescence sensor: cutting a polydimethylsiloxane film (1 multiplied by 1cm), soaking for 60s by using 2mL of mixed solution (1:3, V/V) of hydrogen peroxide and concentrated sulfuric acid, then soaking in 2mL of 3-aminopropyltriethoxysilane aqueous solution (5 percent, V/V) to react with 65 ℃ by heating, and drying by blowing nitrogen after the reaction is finished to obtain the amino functionalized polydimethylsiloxane film; activating the 10mg of upconversion nanoparticles prepared in the step two by using 1.25mL of glutaraldehyde aqueous solution for 2 h; dispersing the activated up-conversion nanoparticles in 2mL of phosphate buffer solution, and incubating with an amino-functionalized polydimethylsiloxane film for 13h to prepare a flexible up-conversion luminescence sensor;
step four, flexible up-conversion luminescence sensor biological functionalization: immersing the flexible up-conversion luminescence sensor into 1.25mL of glutaraldehyde aqueous solution to activate surface amino for 2 h; after the reaction is finished, washing the flexible up-conversion luminescence sensor by using deionized water, immersing the flexible up-conversion luminescence sensor into 2mL of phosphate buffer solution again, adding 10nmol/L of diethylstilbestrol aptamer complementary sequence solution, and incubating for 13h at the constant temperature of 37 ℃ of a shaking table at the rotating speed of 200 r; after the incubation is finished, adding 10nmol/L Dabcyl labeled diethylstilbestrol aptamer solution, and incubating for 2h at the constant temperature of 37 ℃ by a shaking table at the rotating speed of 200 r; after the reaction is finished, taking out the sensor, washing with deionized water, and drying with nitrogen to prepare the bio-functionalized flexible up-conversion luminescence sensor;
and (3) detecting the content of diethylstilbestrol in the river water:
(1) the establishment of a diethylstilbestrol content detection standard curve: immersing the bio-functionalized flexible up-conversion luminescence sensor obtained in the fourth step into 2mL of Tris-HCl buffer solution (pH 8.5), then respectively adding diethylstilbestrol standard solutions with different concentrations to obtain detection solutions with different concentrations, after incubation, determining the characteristic value of a fluorescence intensity signal of the detection solution, performing linear fitting according to the diethylstilbestrol concentration and the characteristic value of the fluorescence intensity signal, and establishing a standard curve for detecting the diethylstilbestrol content; the specific positions are as follows: preparing DES solutions with different concentrations of 0.05, 0.1, 1, 5, 50, 100, 500ng/mL and the like, respectively adding 100uL into a specific detection system, and collecting characteristic values of fluorescence signals after incubating for 25min at 37 ℃; obtaining a standard curve (F-F) for DES content detection by linear fitting0)/F0＝1.1554log(CDES) +1.9566, coefficient of correlation R20.9853, the limit of detection LOD is 0.038ng/mL, and the linear range is 0.05-500 ng/mL.
(2) And (3) detecting the content of diethylstilbestrol in the river water: and (3) centrifuging the river water sample for 5min at 6000rmp, filtering supernate with 0.45um, performing suction filtration, finally taking 50uL of filtrate for detection, after incubating the filtrate with the flexible up-conversion luminescence biosensor for 25min, measuring a characteristic value of a fluorescence signal of the sensor, substituting the characteristic value into the standard curve obtained in the step (1), and calculating the content of diethylstilbestrol in the fish meat to be 9.12 ng/mL.
Comparative example 1:
the difference from example 1 is that the bio-functionalized flexible up-conversion luminescence sensor prepared in example 1 is directly immersed into a standard solution containing 50ng/mL 17 β -estradiol for detection, and the characteristic value of fluorescence intensity signal is collected, as shown in B of FIG. 8, in a.
Comparative example 2:
the difference from example 1 is that the bio-functionalized flexible up-conversion luminescence sensor prepared in example 1 is directly immersed into a standard solution containing 50ng/mL bisphenol A for detection, and the characteristic value of fluorescence intensity signal is collected, as shown in B and c of FIG. 8.
Comparative example 3:
the difference from example 1 is that the bio-functionalized flexible up-conversion luminescence sensor prepared in example 1 is directly immersed into a standard solution containing 50ng/mL of estrene for detection, and the characteristic value of fluorescence intensity signal is collected, as shown in B of FIG. 8, e.
Comparative example 4:
the difference from example 1 is that the bio-functionalized flexible up-conversion luminescence sensor prepared in example 1 is directly immersed into a standard solution containing 50ng/mL dienestrol for detection, and the characteristic value of fluorescence intensity signal is collected, as shown in B in FIG. 8, in g.
Comparative example 5:
the difference from example 1 is that the bio-functionalized flexible up-conversion luminescence sensor prepared in example 1 is directly immersed into a standard solution containing a mixed solution of 50ng/mL 17 β -estradiol and 5ng/mL diethylstilbestrol for detection, and a characteristic value of a fluorescence intensity signal is collected, as shown in B of FIG. 8.
Comparative example 6:
the difference from example 1 is that the bio-functionalized flexible up-conversion luminescence sensor prepared in example 1 is directly immersed into a standard solution containing 50ng/mL bisphenol A and 5ng/mL diethylstilbestrol for detection, and the characteristic value of fluorescence intensity signal is collected, as shown in B in FIG. 8, d.
Comparative example 7:
the difference from example 1 is that the bio-functionalized flexible up-conversion luminescence sensor prepared in example 1 is directly immersed into a standard solution containing a mixed solution of 50ng/mL of diethylstilbestrol and 5ng/mL of diethylstilbestrol for detection, and a characteristic value of a fluorescence intensity signal is collected, as shown by f in B of FIG. 8.
Comparative example 8:
the difference from example 1 is that the bio-functionalized flexible up-conversion luminescence sensor prepared in example 1 is directly immersed into a standard solution containing a mixed solution of 50ng/mL dienestrol and 5ng/mL diethylstilbestrol for detection, and a characteristic value of a fluorescence intensity signal is collected, as shown in B in FIG. 8, i.e. h.
The characteristic values of the fluorescence signals of comparative examples 1 to 8 are shown in FIG. 5B. As can be seen in fig. 5B, the constructed detection method was used for other structural analogs: in the detection of 17 beta-estradiol, bisphenol A, estradiol and dienestrol, even if the concentration of 10 times of DES is used, the obvious fluorescence signal change of the system can not be caused, and the fluorescence intensity of the system can be obviously changed only when DES solution is added, so that the detection method constructed by the invention has high specificity and high sensitivity to DES.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (10)
1. A preparation method of an up-conversion luminescence flexible biosensor for detecting diethylstilbestrol is characterized by comprising the following steps:
step one, preparing an oleic acid coated up-conversion nano material: weighing yttrium chloride hexahydrate, ytterbium chloride hexahydrate and thulium chloride hexahydrate in proportion, dispersing the weighed materials in a methanol solvent A, adding oleic acid and 1-octadecene, stirring for the first time under a certain temperature condition, cooling to room temperature after stirring, dropwise adding a mixed solution of sodium hydroxide, ammonium fluoride and the methanol solvent B, heating for a period of time after sealing, stirring for the second time after heating, cooling to room temperature after stirring, carrying out centrifugal separation to obtain an upconversion nano particle precipitate, washing with a mixed solution of cyclohexane and ethanol, and drying to obtain pure oleic acid-coated upconversion nano particles;
step two, preparing an amination up-conversion nano material: weighing the oleic acid-coated up-conversion nanoparticles prepared in the step one, and dispersing the oleic acid-coated up-conversion nanoparticles in a hydrochloric acid solution for ultrasonic treatment; after the treatment is finished, adding ethanol, performing ultrasonic dispersion uniformly, and then adding deionized water and ammonia water, and stirring uniformly at a certain temperature; after stirring uniformly, adding tetraethyl orthosilicate for carrying out a first reaction, and then adding 3-aminopropyltriethoxysilane for carrying out a second reaction; after the reaction is finished, performing centrifugal separation, and cleaning the obtained product with distilled water to obtain an amination up-conversion nano material;
step three, preparing a flexible up-conversion luminescence sensor: cutting a polydimethylsiloxane film according to requirements, soaking the polydimethylsiloxane film in a mixed solution of hydrogen peroxide solution and concentrated sulfuric acid, taking out the polydimethylsiloxane film, washing the polydimethylsiloxane film with distilled water, soaking the polydimethylsiloxane film in a 3-aminopropyltriethoxysilane aqueous solution for heating reaction, and drying the polydimethylsiloxane film with nitrogen after the reaction is finished to obtain an amino-functionalized polydimethylsiloxane film;
activating the upconversion nanoparticles prepared in the step two by using a glutaraldehyde aqueous solution; separating the activated up-conversion nano particles, dispersing the up-conversion nano particles in a phosphate buffer solution, and immersing the amino-functionalized polydimethylsiloxane film into the buffer solution for incubation to prepare the flexible up-conversion luminescence sensor;
step four, preparing a bio-functionalized flexible up-conversion luminescence sensor: immersing the flexible up-conversion luminescence sensor obtained in the third step into a glutaraldehyde aqueous solution for activation; after the reaction is finished, taking out the flexible up-conversion luminescence sensor, washing the flexible up-conversion luminescence sensor by using distilled water, immersing the flexible up-conversion luminescence sensor into a phosphate buffer solution, adding a diethylstilbestrol aptamer complementary sequence solution, and performing primary constant-temperature incubation in a shaking table; after the incubation is finished, adding a 4- ((4- (dimethylamino) phenyl) azo) benzoic acid marked diethylstilbestrol aptamer solution, and performing second constant-temperature incubation on a shaking table; and taking out the sensor after the reaction is finished, washing with deionized water, and blow-drying with nitrogen to prepare the bio-functionalized flexible up-conversion luminescence sensor.
2. The method for preparing an upconversion luminescence flexible biosensor for diethylstilbestrol detection according to claim 1, wherein the dosage ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, thulium chloride hexahydrate and methanol solvent A in step one is 0.2412 g: 0.0775 g: 0.0019 g: 10 mL; the dosage ratio of the methanol solvent A to the oleic acid to the 1-octadecene is 10 mL: 6mL of: 15 mL; the dosage ratio of the methanol solvent A to the sodium hydroxide to the ammonium fluoride to the methanol solvent B is 10 mL: 0.1 g: 0.1482 g: 10 mL.
3. The method for preparing an upconversion luminescence flexible biosensor for detecting diethylstilbestrol according to claim 1, wherein the certain temperature in the first step is 160 ℃; the first stirring is magnetic stirring for 30 min; the heating temperature after sealing is 50-80 ℃, and the heating time is 40 min; the temperature of the second stirring is 300 ℃, and the stirring time is 1 h.
4. The method for preparing the up-conversion luminescence flexible biosensor for detecting diethylstilbestrol according to claim 1, wherein in the second step, the proportions of the up-conversion nanoparticles, the hydrochloric acid solution, the ethanol, the ammonia water, the tetraethyl orthosilicate and the 3-aminopropyltriethoxysilane are 20mg:0.5mL:60mL:2.5mL:20uL:50 uL; the first reaction time is 4-6h, and the second reaction time is 1-2 h; the concentration of the hydrochloric acid solution is 0.1 mol/L; the certain temperature condition is 65 ℃.
5. The method for preparing an upconversion luminescence flexible biosensor for diethylstilbestrol detection according to claim 1, wherein in step three, the polydimethylsiloxane film has a size of 1 x 0.05 cm; the volume ratio of the hydrogen peroxide solution to the concentrated sulfuric acid mixed solution is 1:3, and the soaking time is 60 s; the volume fraction of the 3-aminopropyltriethoxysilane solution is 5%, the heating reaction temperature is 60-70 ℃, and the reaction time is 3 h; the mass fraction of the hydrogen peroxide solution is 30 percent, and the mass fraction of the concentrated sulfuric acid is 98 percent.
6. The preparation method of the up-conversion luminescence flexible biosensor for detecting diethylstilbestrol according to claim 1, wherein the mass fraction of the glutaraldehyde aqueous solution is 25%, the ratio of the up-conversion nanoparticles to the glutaraldehyde aqueous solution is 10mg:1.25mL, and the activation time is 2 h; the pH value of the phosphate buffer solution is 7.4, and the molar concentration is 10 mmol/L; the dosage ratio of the up-conversion nano particles to the phosphate buffer solution is 10mg:5 mL; the incubation time of the up-conversion nano particle solution and the polydimethylsiloxane film is 10-14 h.
7. The preparation method of the up-conversion luminescence flexible biosensor for detecting diethylstilbestrol according to claim 1, wherein in the fourth step, the mass fraction of the glutaraldehyde aqueous solution is 25%, and the activation time is 2 h; the pH value of the phosphate buffer solution is 7.4, and the molar concentration is 10 mmol/L; the phosphate buffer solution, the diethylstilbestrol aptamer complementary sequence solution and the diethylstilbestrol aptamer solution are mixed according to the proportion of 2 mL: 30 uL: 30 uL; the concentration of the diethylstilbestrol aptamer solution is 10nmol/L, the temperature of the first constant-temperature incubation is 37 ℃, the incubation time is 10-14h, and the rotating speed of a shaking table is 200 r; the concentration of the 4- ((4- (dimethylamino) phenyl) azo) benzoic acid labeled diethylstilbestrol aptamer complementary sequence solution is 10nmol/L, the temperature of the second constant-temperature incubation is 37 ℃, the incubation time is 2h, and the rotating speed of a shaking table is 200 r.
8. Use of a sensor prepared according to any one of claims 1 to 7 for the detection of diethylstilbestrol, characterized by the following steps:
(1) the establishment of a diethylstilbestrol content detection standard curve: immersing a biological functionalized flexible up-conversion luminescence sensor into a Tris-HCl buffer solution, and then respectively adding diethylstilbestrol standard solutions with different concentrations, wherein one diethylstilbestrol standard solution corresponds to one sensor, and the concentrations and the sensors are in one-to-one correspondence; obtaining detection solutions with different concentrations, determining the characteristic value of a fluorescence intensity signal of the detection solution after incubation, and performing linear fitting by using the concentration of diethylstilbestrol and the characteristic value of the fluorescence intensity signal to establish a standard curve for detecting the content of diethylstilbestrol;
(2) detection of diethylstilbestrol content in food and environmental samples: pretreating the food and environment samples, extracting to obtain a sample solution containing diethylstilbestrol, immersing a biological functionalized flexible up-conversion luminescence sensor in the sample solution, incubating, measuring the characteristic value of a fluorescence intensity signal of the solution, substituting the characteristic value into the standard curve obtained in the step (1), and calculating the content of the diethylstilbestrol in the food and environment samples.
9. The use according to claim 8, wherein in step (1), the Tris-HCl buffer solution has a pH of 8.5 and a molarity of 10 mmol/L; the volume ratio of the Tris-HCl buffer solution to the diethylstilbestrol standard solution is 20: 1; the concentration range of the diethylstilbestrol standard solution is 0.05-500 ng/mL; the incubation temperature is 37 ℃, and the incubation time is 20-25 min;
the step of measuring the fluorescence intensity signal characteristic value of the detection solution comprises the following steps: measuring the fluorescence intensity value of the flexible sensor at 477nm under the excitation of 980nm exciting light, and recording the fluorescence intensity value F of the flexible sensor without diethylstilbestrol0Adding diethylstilbestrol standard solution to react, and then obtaining the fluorescence intensity value F of the flexible sensor; (F-F)0)/F0The fluorescence intensity signal characteristic value of the detection solution is obtained.
10. The use according to claim 8, wherein in the step (2), the sample is pretreated by: homogenizing solid food, adding into acetonitrile-acetone mixed solution, oscillating, blowing with nitrogen, centrifuging, extracting with acetonitrile, collecting supernatant, blowing with nitrogen, and re-dissolving with methanol solution;
the volume ratio of acetonitrile to acetone in the acetonitrile-acetone mixed solution is 4: 1; the dosage ratio of the solid food to the acetonitrile-acetone mixed solution is 1 g: 3 mL; the mass fraction of the methanol solution is 40 percent; the relationship between the use amount of the methanol solution during redissolution and the use amount of solid food is 1-2 ml: 2g of the total weight of the mixture; the incubation temperature is 37 ℃, and the incubation time is 20-25 min.
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