CN110221085B - Streptomycin and neomycin multi-residue simultaneous rapid fluorescence detection reagent and application - Google Patents

Streptomycin and neomycin multi-residue simultaneous rapid fluorescence detection reagent and application Download PDF

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CN110221085B
CN110221085B CN201910470326.3A CN201910470326A CN110221085B CN 110221085 B CN110221085 B CN 110221085B CN 201910470326 A CN201910470326 A CN 201910470326A CN 110221085 B CN110221085 B CN 110221085B
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neomycin
streptomycin
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宋尚红
陈冠华
郭欣
高志飞
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Jiangsu University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9446—Antibacterials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Abstract

The invention relates to a preparation and use method of a streptomycin and neomycin residue simultaneous rapid fluorescence detection reagent. Two quantum dots with the same material kernel and different emission wavelengths are respectively coupled with aptamers with high specificity recognition capability on streptomycin and neomycin, and the two conjugates and graphene oxide form a fluorescence resonance energy transfer system respectively, so that a reagent for simultaneously and rapidly carrying out fluorescence detection on multiple residues of streptomycin and neomycin can be prepared. By adopting the detection reagent, the rapid, accurate and sensitive detection of streptomycin and neomycin residue in milk samples can be realized without depending on a separation analysis technology, and a powerful technical guarantee is provided for strengthening food safety supervision.

Description

Streptomycin and neomycin multi-residue simultaneous rapid fluorescence detection reagent and application
Technical Field
The invention relates to the technical field of nano material preparation and the technical field of veterinary drug residue detection, in particular to a fluorescent detection reagent which integrates the high specificity of an aptamer and the excellent fluorescent characteristics of quantum dots, selectively marks streptomycin and neomycin, and is applied to the simultaneous rapid fluorescent detection of streptomycin and neomycin residue.
Background
Streptomycin and neomycin are aminoglycoside antibiotics which have been widely used in animal husbandry, and improper use of streptomycin and neomycin can cause residues in animal derived foods, and can cause adverse effects on health such as ototoxicity, renal toxicity and anaphylaxis after being eaten by human for a long time. The accurate measurement of the residue of the food in animal origin is the last pass for stopping unqualified food from flowing into the market, and the accuracy includes both quantitative accuracy and qualitative accuracy. In order to clearly know the specific name of the residual antibiotics in the food in one detection, the establishment of a method for detecting the residual antibiotics of multiple antibiotics is a very effective technical means, and in the detection of multiple residues, the detection of multiple residues of antibiotics (such as aminoglycoside antibiotics) of the same class is the most difficult.
At present, the method for sensitively, accurately, qualitatively and quantitatively detecting aminoglycoside antibiotic residues is mainly a method based on a separation analysis principle, such as high performance liquid chromatography and a liquid chromatography-mass spectrometry combined method. The method uses expensive instruments, has high requirements on the professional performance of operators, generally needs complex and time-consuming purification pretreatment on the tested sample, is difficult to realize on-site rapid detection, and limits rapid and effective market supervision. The fluorescence detection method has high sensitivity and is very suitable for detecting antibiotic residues including streptomycin and neomycin, but the method is used for carrying out fluorescence labeling on the streptomycin and neomycin which do not have the fluorescence characteristic per se. The traditional organic fluorescent dye does not have the specific recognition capability to a marked object, and can achieve the purpose of multi-residue detection only by combining with a separation analysis technology. The novel nano fluorescent material quantum dots appearing in recent years have excellent fluorescence characteristics which are not possessed by a plurality of traditional organic fluorescent dyes, are widely applied to the field of analytical chemistry, do not have specific recognition capability for a marked object, and are usually combined with an immunization method to realize the recognition of the marked object. Although the immunoassay can realize the rapid detection of antibiotic residues, the immunoassay can mostly realize single-residue detection and cannot realize high-selectivity multi-residue detection, and the antibody is protein, needs animal experiments or cell culture for obtaining the antibody, has high cost, is easily influenced by temperature to cause great fluctuation of the stability of the antibody, is not easy to store, and is difficult to popularize and use for the detection of mass samples.
Disclosure of Invention
The invention aims to provide a preparation and use method for simultaneously and rapidly detecting streptomycin and neomycin residue by fluorescence without depending on a separation analysis technology, and the preparation and use method is applied to the detection of streptomycin and neomycin residue in animal-derived food.
The method comprises the following specific steps:
1. a streptomycin and neomycin residue simultaneous rapid fluorescence detection reagent is characterized in that: the reagent is prepared by adopting the following method:
(1) aptamers having high specificity and high affinity for streptomycin and neomycin are used as information recognition reagents for streptomycin and neomycin, respectively, wherein the nucleic acid sequence of Streptomycin Aptamer (SAPT) is 5' -NH2-(CH2)6-TAG GGA ATT CGT CGA CGG ATC CGC TCT GGG AGG TGC GGC TCT TTA CTC CTC CAA CGA CCC GGC TGC AGG TCG ACG CAT GCG CCG-3', the nucleic acid sequence of Neomycin Aptamer (NAPT) is 5' -NH2-(CH2)6-TAG GGA ATT CGT CGA CGG ATC CGC GTG TAG TAG CCT GAC CAA GGC GCC CAC CTC GAT TTA GTC TGC AGG TCG ACG CAT GCG CCG-3'; two quantum dots QD1 and QD2, which can emit fluorescence at different wavelengths at the same excitation wavelength, are used as information transfer reagents for neomycin and streptomycin, respectively.
Measured concentration is 2.46 multiplied by 10 respectively-5And 1.62X 10-5Respectively adding 10 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with the concentration of 10g/L into 5 mu L of each of mol/L QD1 and QD2, and fixing the volume to 40 mu L by using deionized water for respectively activating; then adding the NAPT and SAPT after denaturation treatment respectively, using phosphate buffer solution with pH7.4 to fix the volume to 400 mu L, and carrying out coupling reaction; and filtering the reacted solution by using a 50kD ultrafiltration tube respectively, and metering the volume of the remained solution in the tube to 100 mu L by using phosphate buffer solution respectively, wherein the SAPT-QD2 and NAPT-QD1 conjugate solutions form a composite fluorescent probe solution of streptomycin and neomycin, and the conjugate concentrations of QD2 and QD1 are 810 and 1230nmol/L respectively.
Proper amounts of SAPT-QD2 and NAPT-QD1 are mixed and then added with Graphene Oxide (GO), the mixture is diluted by phosphate buffer solution until the GO concentration is 300mg/L, the SAPT-QD2 and NAPT-QD1 concentrations of QD2 and QD1 are 81 and 123nmol/L respectively, and reagents SAPT-QD2/GO and NAPT-QD1/GO which can be used for streptomycin and neomycin multi-residue simultaneous rapid fluorescence detection are formed after quenching reaction for 30 min.
The high specificity means that SAPT has no recognition effect on other antibiotics and aminoglycoside antibiotics except streptomycin, especially on neomycin, NAPT has no recognition effect on other antibiotics and aminoglycoside antibiotics except neomycin, especially on streptomycin; the high affinity is shown in that the dissociation constant of SAPT to streptomycin is 26.56 + -6.57 nmol/L, and the dissociation constant of NAPT to neomycin is 40.96 + -11.56 nmol/L. The quantum dots QD1 and QD2 are CdTe quantum dots modified by thioglycolic acid and mercaptopropionic acid, and have excitation wavelength of 370nm and emission wavelengths of 548nm and 580nm respectively.
The quantum dot QD1 was prepared as follows: placing 0.0760g of tellurium powder and 0.05g of sodium borohydride in a small bottle with a syringe needle, adding 2mL of deionized water, immediately introducing nitrogen for 5min, immediately covering a bottle stopper after stopping introducing the nitrogen, and reacting for 4h at normal temperature to obtain a mauve NaHTe solution; 0.2200g of cadmium chloride is placed in a 250mL three-necked bottle, 100mL of deionized water is added, nitrogen is introduced for 30min, 206 μ L of thioglycolic acid is added, the pH value is adjusted to 11.2 by 1mol/L of NaOH, a newly prepared NaHTe solution is added, the solution is refluxed and condensed for 2h at 95 ℃ to obtain the CdTe quantum dot modified by the thioglycolic acid, the excitation wavelength is 370nm, and the emission wavelength is 548 nm.
The QD2 was prepared as follows: placing 0.1276g of tellurium powder and 0.0800g of sodium borohydride in a small bottle with a syringe needle, adding 3mL of deionized water, immediately introducing nitrogen for 5min, immediately covering a bottle stopper after stopping introducing the nitrogen, and reacting for 4h at normal temperature to obtain a mauve NaHTe solution; 0.4567g of cadmium chloride is placed in a 250mL three-necked bottle, 100mL of deionized water is added, nitrogen is introduced for 30min, then 420 μ L of mercaptopropionic acid is added, the pH value is adjusted to 10 by 1mol/L of NaOH, a newly prepared NaHTe solution is added, the solution is transferred to a stainless steel reaction kettle with a polytetrafluoroethylene lining, the stainless steel reaction kettle is heated in an oven at 160 ℃ for 1h to obtain the CdTe quantum dots modified by the mercaptopropionic acid, the excitation wavelength is 370nm, and the emission wavelength is 580 nm.
The activation refers to that QD1 and QD2 react with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride for 3.95-4.05min and 14.95-15.05min respectively under ultrasonic oscillation, and the fluorescence intensity of the quantum dots is reduced beyond the time range. The denaturation reaction refers to heating NAPT and SAPT at 95 deg.C for 10min respectively, and then ice-cooling for 10 min. The coupling reaction refers to that NAPT and SAPT are respectively reacted with activated QD1 and QD2 in a light-shielding manner for 2h according to the molar ratio of NAPT to QD1 being 1:3.8-4.2 and SAPT to QD2 being 1:4.8-5.2, and the surfaces of QD1 and QD2 can be fully coupled by NAPT and SAPT respectively in the range of the ratio. The excitation wavelength of the two composite fluorescent probes NAPT-QD1 and SAPT-QD2 is 370nm, and the emission wavelength is 550 nm and 586nm respectively.
The quenching reaction means that GO forms a fluorescence resonance energy transfer system with QD1 and QD2 through pi-pi stacking interaction, so that fluorescence of QD1 and QD2 is quenched.
2. A method for using streptomycin and neomycin residue simultaneous rapid fluorescence detection reagent is characterized by comprising the following steps:
(1) measuring 100 mu L of the fluorescence detection reagent, adding 1 mu L of streptomycin and neomycin mixed standard solution or sample solution, reacting for more than 30min, wherein the time below the reaction time can cause that the quenched quantum dot fluorescence can not be fully recovered, and the detection accuracy is influenced.
(2) And placing the reacted solution into an enzyme-labeling instrument for fluorescence determination.
The concentrations of streptomycin and neomycin in the streptomycin and neomycin mixed standard solution are respectively 10, 50, 100, 200, 500, 800, 1000 and 1000 mug/L.
The sample solution was prepared according to the following procedure:
(1) 10mL of blank milk (milk without streptomycin and neomycin residues) was measured and mixed standard solutions of different concentrations of streptomycin and neomycin were added.
(2) Adding 2mL of 15% (V/V) trichloroacetic acid, ultrasonically oscillating for 20min, centrifuging for 10min at 12000r/min, collecting supernatant, and diluting to 5mL with deionized water.
The fluorescence detection in the microplate reader is carried out according to the following steps:
(1) excitation wavelength for neomycin and streptomycin detection is 370 nm; emission wavelengths 550 and 586nm, corresponding to a fluorescence intensity of F550nmAnd F586nm(ii) a Corrected wavelengths 618 and 518nm, corresponding to a fluorescence intensity F618nmAnd F518nm(ii) a The net analytical signals for neomycin and streptomycin, respectively, are Δ F1＝F550nm-F618nm，ΔF2＝F586nm-F518nm。
(2) Measurement of Δ F of each standard solution1And Δ F2The values obtained for these and the neomycin concentration C in the respective standard solutionsNAnd streptomycin concentration CSThe origin software is used for regression, and a standard curve delta F of neomycin and streptomycin can be obtained1＝A1×CN+B1，ΔF2＝A2×CS+B2Wherein A is1、A2And B1、B2The slope and intercept are given in the regression process, respectively.
(3) Measuring the Delta F of a sample solution1And Δ F2And substituting the standard curve equation into the standard curve equation to calculate the concentration of neomycin and streptomycin in the sample.
Advantageous effects
The existing fluorescent labeling materials of streptomycin and neomycin, no matter the traditional organic fluorescent dye or the nano fluorescent material quantum dot, do not have the selective labeling characteristic of streptomycin and neomycin. The aptamer has a specific recognition effect on the target substance, but does not have a signal transduction effect on the target substance, and needs to be combined with other materials or methods having a signal transduction effect. The invention combines two quantum dots with the same core material (CdTe) with aptamers capable of selectively identifying streptomycin and neomycin respectively, integrates the advantages of the two, and obtains the composite fluorescent reagent capable of carrying out fluorescence labeling on the streptomycin and neomycin simultaneously. The two quantum dots used by the reagent are made of the same core material, and can emit fluorescence with two different wavelengths under the same excitation wavelength only by utilizing the difference of the particle sizes of the two quantum dots, so that the signal conduction problem of different target substances is solved on a relatively simple substance basis. The reagent can carry out high-selectivity identification on streptomycin and neomycin, false positive results caused by mutual cross identification or false identification on other antibiotics can not occur, and the key point of achieving the excellent performance is that the aptamer used by the reagent is subjected to a large number of complex screening experiments and contains 1013-1015Highly specific aptamers from a random library of oligonucleotides. Once the sequence information of the aptamer is obtained, it can be chemically synthesized in large quantities with high purity and is easy to store, which is an advantage that antibodies do not have.
The existing detection methods for various antibiotic residues, especially the detection method for the similar antibiotic residues, are realized based on a separation analysis technology and depend on a large-scale analytical instrument. The detection reagent can get rid of the dependence on a separation analysis method, avoids the complicated and time-consuming sample purification pretreatment step, can directly detect the sample after simple treatment by using the reagent, has the characteristics of simplicity, rapidness, on-site accuracy and sensitivity, has the detection capability on streptomycin and neomycin reaching the level which can be reached by the liquid chromatogram-mass spectrum of the strongest detection method at present, does not need expensive instruments, and provides powerful technical guarantee for strengthening food safety supervision.
Drawings
FIG. 1 shows fluorescence emission spectra and selection of detection and calibration wavelengths for NAPT-QD1 and SAPT-QD 2.
FIG. 2 fluorescent detection reagents specific for neomycin and streptomycin in milk samples.
Detailed Description
Example 1
Measured concentration is 2.46 multiplied by 10 respectively-5And 1.62X 10-5mu.L of each of mol/L QD1 and QD2, 10. mu.L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with a concentration of 10g/L was added, the volume was adjusted to 40. mu.L with deionized water, and the QD1 and QD2 were reacted with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride for 4 and 15min, respectively, under ultrasonic oscillation. Then NAPT and SAPT denatured at 95 ℃ were added in a NAPT: QD1 molar ratio of 1:4 and SAPT: QD2 molar ratio of 1:5, respectively, and NAPT and SAPT were reacted with the activated QD1 and QD2, respectively, in the absence of light, to 400. mu.L with a phosphate buffer solution of pH 7.4. The reacted solution was filtered through 50kD ultrafilter tubes, and the remaining solution was diluted to 100. mu.L with phosphate buffer, respectively, and the concentrations of the conjugate in the SAPT-QD2 and NAPT-QD1 conjugate solutions were 810 and 1230nmol/L, respectively, in terms of the concentrations of QD2 and QD 1.
Mixing a proper amount of SAPT-QD2 and NAPT-QD1, adding GO, diluting with a phosphate buffer solution until the concentration of GO is 300mg/L, respectively setting the concentrations of SAPT-QD2 and NAPT-QD1 of QD2 and QD1 to be 81 and 123nmol/L, reacting for 30min, wherein the obtained solution is a streptomycin and neomycin residue simultaneous rapid fluorescence detection reagent, the excitation wavelength of the solution is 370nm, and the emission wavelengths are respectively 550 and 586 nm.
Example 2
Measuring 10mL of blank milk, adding a streptomycin and neomycin mixed standard solution, adding 2mL of 15% trichloroacetic acid solution, and then samplingUltrasonic oscillation is carried out for 20min, centrifugation is carried out for 10min at 12000r/min, supernatant is collected and is made to be 5mL by deionized water, and the concentrations of streptomycin and neomycin are respectively 10 and 50 mug/L. The mixed standard solution series with streptomycin and neomycin concentrations of 10, 50, 100, 200, 500, 800, 1000 and 1000 mug/L and the standard added milk sample absorb 1 mug/L respectively, and are added into 100 mug/L of the fluorescence detection reagent for reaction for 30 min. The fluorescence intensities F at 550 and 618nm and at 586 and 518nm were measured for each of the post-reaction solutions with 370nm as the excitation wavelength550nmAnd F618nmAnd F586nmAnd F518nm. In terms of Δ F of each mixed standard solution1＝F550nm-F618nmAnd Δ F2＝F586nm-F518nmAs ordinate, the concentration C of neomycin and streptomycin inNAnd CSAs the abscissa, the standard curves for neomycin and streptomycin, respectively, are regressed to Δ F1＝603.48CN+1.1317(R20.9920) and Δ F2＝303.12CS-2.1867(R20.9986), linear response ranges from 50-1000 μ g/L and 10-1000 μ g/L, respectively, with detection limits (S/N3) of 10.02 and 6.65 μ g/L, respectively. The standard curve can be used for calculating the concentration of the neomycin and the streptomycin in the standard milk sample to be 52.33 and 10.47 mu g/L respectively, and the standard recovery rate to be 104.65 percent and 104.69 percent respectively.
Example 3
Weighing 10mL of blank milk, adding a streptomycin and neomycin mixed standard solution, adding 2mL of a 15% trichloroacetic acid solution, carrying out ultrasonic oscillation on the sample for 20min, centrifuging at 12000r/min for 10min, collecting supernatant, and fixing the volume to 5mL by using deionized water, wherein the concentrations of the streptomycin and the neomycin are 200 mug/L and 500 mug/L respectively. The mixed standard solution series with streptomycin and neomycin concentrations of 10, 50, 100, 200, 500, 800, 1000 and 1000 mug/L and the standard added milk sample absorb 1 mug/L respectively, and are added into 100 mug/L of the fluorescence detection reagent for reaction for 30 min. The fluorescence intensities F at 550 and 618nm and at 586 and 518nm were measured for each of the post-reaction solutions with 370nm as the excitation wavelength550nmAnd F618nmAnd F586nmAnd F518nm. In terms of Δ F of each mixed standard solution1＝F550nm-F618nmAnd Δ F2＝F586nm-F518nmAs ordinate, the concentration C of neomycin and streptomycin inNAnd CSAs the abscissa, the standard curves for neomycin and streptomycin, respectively, are regressed to Δ F1＝603.48CN+1.1317(R20.9920) and Δ F2＝303.12CS-2.1867(R20.9986), linear response ranges from 50-1000 μ g/L and 10-1000 μ g/L, respectively, with detection limits (S/N3) of 10.02 and 6.65 μ g/L, respectively. The standard curve can be used for calculating the concentration of the neomycin and the streptomycin in the standard milk sample to be 480.60 and 212.08 mu g/L respectively, and the standard recovery rate to be 96.12 percent and 106.04 percent respectively.

Claims (10)

1. A streptomycin and neomycin residue simultaneous rapid fluorescence detection reagent is characterized in that the reagent is prepared by the following method: coupling two quantum dots QD2 and QD1 which are respectively used as information conduction reagents of streptomycin and neomycin and have the same material kernel and different emission wavelengths with aptamers with high specificity and high affinity to the streptomycin and the neomycin respectively, mixing the two conjugates, adding graphene oxide, and forming a fluorescence resonance energy transfer system by the two conjugates and the graphene oxide respectively to prepare a reagent for simultaneously and rapidly detecting the multi-residue of the streptomycin and the neomycin; the quantum dots QD1 and QD2 are CdTe quantum dots modified by thioglycolic acid and mercaptopropionic acid, the excitation wavelength is 370nm, and the emission wavelengths are 548nm and 580nm respectively; the nucleic acid sequence of aptamer SAPT with high specificity and high affinity to streptomycin is 5' -NH2-(CH2)6-TAG GGA ATT CGT CGA CGG ATC CGC TCT GGG AGG TGC GGC TCT TTA CTC CTC CAA CGA CCC GGC TGC AGG TCG ACG CAT GCG CCG-3'; the nucleic acid sequence of aptamer NAPT having high specificity and high affinity for neomycin is 5' -NH2-(CH2)6-TAG GGA ATT CGT CGA CGG ATC CGC GTG TAG TAG CCT GAC CAA GGC GCC CAC CTC GAT TTA GTC TGC AGG TCG ACG CAT GCG CCG-3'。
2. The reagent for simultaneously and rapidly detecting streptomycin and neomycin remains and fluorescence according to claim 1, which is prepared by the following steps:
measured concentration is 2.46 multiplied by 10 respectively-5And 1.62X 10-5Respectively adding 10 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with the concentration of 10g/L into 5 mu L of each of mol/L QD1 and QD2, and fixing the volume to 40 mu L by using deionized water for respectively activating; then adding the NAPT and SAPT after denaturation treatment respectively, using phosphate buffer solution with pH7.4 to fix the volume to 400 mu L, and carrying out coupling reaction; filtering the reacted solution by using a 50kD ultrafiltration tube respectively, and metering the volume of the remained solution in the tube to 100 mu L by using a phosphate buffer solution respectively, wherein the SAPT-QD2 and NAPT-QD1 conjugate solutions form a streptomycin and neomycin composite fluorescent probe solution, and the conjugate concentrations of QD2 and QD1 concentration meters are 810 and 1230nmol/L respectively;
proper amounts of SAPT-QD2 and NAPT-QD1 are mixed, graphene oxide GO is added, the mixture is diluted by phosphate buffer solution until the GO concentration is 300mg/L, the concentrations of SAPT-QD2 and NAPT-QD1 of QD2 and QD1 are 81 and 123nmol/L respectively, and reagents SAPT-QD2/GO and NAPT-QD1/GO which can be used for streptomycin and neomycin multi-residue simultaneous rapid fluorescence detection are formed after quenching reaction for 30 min.
3. The reagent for simultaneous rapid fluorescence detection of streptomycin and neomycin multiresidue as claimed in claim 1, wherein the high specificity means that SAPT has no recognition effect on other antibiotics including neomycin except streptomycin, NAPT has no recognition effect on other antibiotics including streptomycin except neomycin; the high affinity is shown in that the dissociation constant of SAPT to streptomycin is 26.56 + -6.57 nmol/L, and the dissociation constant of NAPT to neomycin is 40.96 + -11.56 nmol/L.
4. The reagent for simultaneously and rapidly detecting streptomycin and neomycin multiresidue by fluorescence according to claim 1, wherein the quantum dot QD1 is prepared by the following steps: placing 0.0760g of tellurium powder and 0.05g of sodium borohydride in a small bottle with a syringe needle, adding 2mL of deionized water, immediately introducing nitrogen for 5min, immediately covering a bottle stopper after stopping introducing the nitrogen, and reacting for 4h at normal temperature to obtain a mauve NaHTe solution; 0.2200g of cadmium chloride is placed in a 250mL three-necked bottle, 100mL of deionized water is added, nitrogen is introduced for 30min, 206 μ L of thioglycolic acid is added, the pH value is adjusted to 11.2 by 1mol/L of NaOH, a newly prepared NaHTe solution is added, the solution is refluxed and condensed for 2h at 95 ℃ to obtain the CdTe quantum dot modified by the thioglycolic acid, the excitation wavelength is 370nm, and the emission wavelength is 548 nm.
5. The reagent for simultaneously and rapidly detecting streptomycin and neomycin multiresidue by fluorescence according to claim 1, wherein the quantum dot QD2 is prepared by the following steps: placing 0.1276g of tellurium powder and 0.0800g of sodium borohydride in a small bottle with a syringe needle, adding 3mL of deionized water, immediately introducing nitrogen for 5min, immediately covering a bottle stopper after stopping introducing the nitrogen, and reacting for 4h at normal temperature to obtain a mauve NaHTe solution; 0.4567g of cadmium chloride is placed in a 250mL three-necked bottle, 100mL of deionized water is added, nitrogen is introduced for 30min, then 420 μ L of mercaptopropionic acid is added, the pH value is adjusted to 10 by 1mol/L of NaOH, a newly prepared NaHTe solution is added, the solution is transferred to a stainless steel reaction kettle with a polytetrafluoroethylene lining, the stainless steel reaction kettle is heated in an oven at 160 ℃ for 1h to obtain the CdTe quantum dots modified by the mercaptopropionic acid, the excitation wavelength is 370nm, and the emission wavelength is 580 nm.
6. The reagent for simultaneously and rapidly detecting streptomycin and neomycin multiresidue fluorescence as claimed in claim 2, wherein the activation means that QD1 and QD2 react with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride for 3.95-4.05min and 14.95-15.05min respectively under ultrasonic oscillation, and the fluorescence intensity of the quantum dots is reduced when the time is out of the time range; the denaturation reaction refers to heating NAPT and SAPT at 95 deg.C for 10min, and ice-cooling for 10 min; the coupling reaction refers to that NAPT and SAPT are respectively reacted with activated QD1 and QD2 in a light-shielding manner for 2h according to the molar ratio of NAPT to QD1 to 1:3.8-4.2 and the molar ratio of SAPT to QD2 to 1:4.8-5.2, and in the range of the ratio, the surfaces of QD1 and QD2 can be respectively and fully coupled by NAPT and SAPT; the excitation wavelength of the two composite fluorescent probes NAPT-QD1 and SAPT-QD2 is 370nm, and the emission wavelength is 550 nm and 586nm respectively; the quenching reaction means that GO forms a fluorescence resonance energy transfer system with QD1 and QD2 through pi-pi stacking interaction, so that fluorescence of QD1 and QD2 is quenched.
7. The method for using the reagent for simultaneously and rapidly detecting the multi-residue of streptomycin and neomycin as claimed in claim 1, which is characterized by comprising the following steps:
(1) measuring 100 mu L of the fluorescence detection reagent, adding 1 mu L of streptomycin and neomycin mixed standard solution or sample solution, reacting for more than 30min, wherein the time below the reaction time can cause that the quenched quantum dot fluorescence can not be fully recovered, and the detection accuracy is influenced;
(2) and placing the reacted solution into an enzyme-labeling instrument for fluorescence determination.
8. The use of claim 7, wherein the concentrations of streptomycin and neomycin in the standard mixed streptomycin and neomycin solution are 10, 50, 100, 200, 500, 800, 1000 μ g/L, respectively.
9. The method of use of claim 7, wherein the sample solution is prepared by:
(1) measuring 10mL of blank milk, namely milk without streptomycin and neomycin residues, and adding mixed standard solutions of streptomycin and neomycin with different concentrations;
(2) adding 2mL of 15% (V/V) trichloroacetic acid, ultrasonically oscillating for 20min, centrifuging for 10min at 12000r/min, collecting supernatant, and diluting to 5mL with deionized water.
10. The use according to claim 7, wherein the fluorescence detection in the microplate reader is carried out according to the following steps:
(1) excitation wavelength for neomycin and streptomycin detection is 370 nm; emission wavelength 550 and586nm, corresponding fluorescence intensity F550nmAnd F586nm(ii) a Corrected wavelengths 618 and 518nm, corresponding to a fluorescence intensity F618nmAnd F518nm(ii) a The net analytical signals for neomycin and streptomycin, respectively, are Δ F1＝F550nm-F618nm，ΔF2＝F586nm-F518nm；
(2) Measurement of Δ F of each standard solution1And Δ F2The values obtained for these and the neomycin concentration C in the respective standard solutionsNAnd streptomycin concentration CSThe origin software is used for regression, and a standard curve delta F of neomycin and streptomycin can be obtained1＝A1×CN+B1，ΔF2＝A2×CS+B2Wherein A is1、A2And B1、B2Respectively representing the slope and intercept given in the regression process;
(3) measuring the Delta F of a sample solution1And Δ F2And substituting the standard curve equation into the standard curve equation to calculate the concentration of neomycin and streptomycin in the sample.
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