CN108918862A - A kind of method of the sulfa drug residue of five-ring heterocycles in quick detection animal food - Google Patents
A kind of method of the sulfa drug residue of five-ring heterocycles in quick detection animal food Download PDFInfo
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Abstract
The present invention relates to a kind of methods of the sulfa drug residue of five-ring heterocycles in quickly detection animal food, belong to field of food detection.For overcome the deficiencies in the prior art, the present invention provides a kind of method for quickly detecting the sulfa drug residue of five-ring heterocycles in animal food, and up-conversion nanoparticles NaYF is prepared by hydrothermal synthesis method in the present invention4：Yb, Er will be coupled using glutaraldehyde method with sulfamido antibody after its functional modification, obtain sulfamido antibody-NaYF4：Yb, Er；Gold nano-material is obtained by sodium citrate synthetic method and is coupled sulfamido antigen, then up-conversion nanoparticles-gold nano resonance energy transfer system is constructed, it is smaller that the present invention can obtain partial size, monodispersity is good, the strong up-conversion nanoparticles of fluorescence signal, the sulfa drugs of five-ring heterocycles class can be quantified by the change in fluorescence amount of nanoparticle, and determine the detection range of the sulfa drugs of five-ring heterocycles.
Description
Technical field
The present invention relates to a kind of methods of the sulfa drug residue of five-ring heterocycles in quickly detection animal food, belong to
Field of food detection.
Background technique
In food safety detection, it is frequently utilized that fluorescent dye and research object are combined to form compound, then leads to
Cross the information of the fluorescent characteristic image study object performance of compound.But the compound after these combinations is issued in ultraviolet band
When raw exciting light, it is possible to the background fluorescence that the generations such as biological tissue and its peptide, protein, nucleic acid can be made strong.Certain fluorescence
Element can also generate biology toxicity, lead to the sensitivity of antigen or antibody and selectivity decline, influence actually detected effect or tight
Detected organism is jeopardized again.Up-converting phosphor technology is exactly the up-conversion luminescent material and biomolecule using functionalization
In conjunction with good hypotoxicity, anti-light bleaching, convenient marker is formed, deleterious molecular is detected, to solve traditional
Learn the limitation of fluorescent dye.
Up-converting phosphor technology is used in food safety detection, it is necessary to amination, aldehyde first be carried out to up-conversion luminescent material
The surface modification of base, carboxylated is made it easier to enhancing the dispersibility and stability of up-conversion luminescent material in aqueous solution
It is combined with large biological molecule, convenient for being used in food safety detection.Using glutaraldehyde method, sulfamido antibody, shape are marked
At marker.
Up-converting phosphor technology is up-conversion luminescent material NaYF4：Yb, Er are as energy donor, gold nanoparticle
As energy acceptor, respectively in connection with the sulfamido antibody and antigen of five-ring heterocycles, due to the suction of the emission spectrum and receptor of donor
Spectra overlapping is received, antigen-antibody can be also immunoreacted, therefore can shorten the distance between donor and acceptor molecule, thus
Realize that energy is quenched.A certain amount of free antigen is then added, the antigen formation on free antigen and gold nano-material is allowed to exempt from
Epidemic disease competition, so that the fluorescence resonance energy transfer system of up-conversion luminescent material and gold nano is destroyed, fluorescent quenching disappears, glimmering
Light quantity increases, therefore can be quantified according to luminous variation to free antigen.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provides a kind of sulphur for quickly detecting five-ring heterocycles in animal food
Up-conversion nanoparticles NaYF is prepared by hydrothermal synthesis method in the method for sulfonamides residues, the present invention4：Yb, Er, by it
It is coupled with sulfamido antibody using glutaraldehyde method after functional modification, obtains sulfamido antibody-NaYF4：Yb, Er；Pass through lemon
Lemon acid sodium synthetic method obtains gold nano-material and is coupled sulfamido antigen, then constructs up-conversion nanoparticles-gold nano energy
Resonance transfer system is measured, the getable nano material partial size of the present invention is smaller, and monodispersity is good, and fluorescence signal is strong, can pass through
The change in fluorescence amount of up-conversion determines the detection range of the sulfa drugs of five-ring heterocycles.
The present invention is achieved through the following technical solutions above-mentioned technical effect：
The method of the sulfa drug residue of five-ring heterocycles, is to pass through hydrothermal synthesis in a kind of quick detection animal food
Up-conversion nanoparticles NaYF is prepared in method4：Yb, Er will utilize glutaraldehyde side with sulfamido antibody after its functional modification
Method coupling, obtains sulfamido antibody-NaYF4：Yb, Er；Gold nano-material is obtained by sodium citrate synthetic method, is coupled sulfamido
Antigen obtains sulfamido antigen-BSA- gold nano solution, then constructs the resonance energy transfer of up-conversion nanoparticles-gold nano
System determines the concentration of sulfamido antigen by measuring fluorescence intensity.
The method of the sulfa drug residue of five-ring heterocycles in quick detection animal food of the present invention is specific to wrap
Include following step：
1)NaYF4：The preparation of Yb, Er nanoparticle
Measure YNO3Solution, YbNO3Solution and ErNO3Solution is uniformly mixed, and ultrasound is mixed after being slowly added to sodium citrate solution
It is even；NaF solution is slowly added dropwise thereto until having white precipitate appearance in mixed solution, the pH value of mixed solution is adjusted to 5, magnetic
It is transferred in homogeneous reactor after power stirring 1h, 180 DEG C of insulation reaction 4h are centrifuged after being cooled to room temperature, and sediment are washed dry
It is dry to get NaYF4：Yb, Er nanoparticle；
2) NaYF of functionalization4：The preparation of Yb, Er nanoparticle：
By NaYF4：Yb, Er nanoparticle are added in normal propyl alcohol, and 35 DEG C of water bath with thermostatic control is transferred to after ultrasonic magnetic agitation
In, ammonia spirit is instilled thereto, continues magnetic agitation 1h；The mixing for being mixed with normal propyl alcohol and EOS solution is slowly added dropwise thereto
Solution, the reaction was continued 4h；The mixed solution of APTES and normal propyl alcohol is added dropwise in mixed solution, centrifuging and taking after 1h is persistently stirred
Sediment, washing, the dry NaYF to get functionalization4：Yb, Er nanoparticle；
3) sulfamido antibody connects
Take the NaYF of functionalization4：The nanoparticle dissolution of Yb, Er are slowly added into glutaraldehyde solution and boron into PBS solution
Sodium hydride is centrifuged after slow oscillation reaction 1h at room temperature after mixing, and sediment is dispersed in again after being washed using PBS solution
In PBS solution, sulfamido antibody and sodium borohydride is added, Tris sealer is added after oscillating reactions 1h at room temperature, after persistent oscillation
It is centrifuged, is dispersed in PBS solution after sediment washing, 4 DEG C save backup, and obtain sulfamido antibody-NaYF after reaction 1h4：Yb,
Er solution；
4) gold nano is synthesized
Sodium citrate solution is added rapidly in chlorauric acid solution under magnetic agitation, stirred in water bath is until solution is presented
Claret is heated to reflux 30min, is cooled to room temperature to obtain cooling solution, PVP solution is added in cooling solution and is stirred at room temperature
For 24 hours to get gold nano solution, 4 DEG C are kept in dark place；
5) gold nano is connected with antigen
It is 8.0 that gold nano solution, which is transferred to pH with PBS solution, and sulfamido antigen-BSA is added in ice-water bath under stirring,
It is stirred to react 1h；Continue to be stirred to react 1h after BSA is added thereto, be centrifugated at 4 DEG C, after sediment is using PBS washing, point
It is dispersed in the PBS solution that pH is 8.0, obtains sulfamido antigen-BSA- gold nano solution, 4 DEG C save backup；
6) the sulfa drugs detection range of five-ring heterocycles is determined
It is separately added into the sulfamido antigenic solution of different content into test tube, sulfamido antibody-NaYF is added thereto4：
Sulfamido antigen-BSA- gold nano solution is added after reacting 0.5h in Yb, Er solution；PBS solution is added thereto and is placed on shaking table
40min is vibrated, the fluorescence intensity for then measuring mixed solution determines five yuan according to the relationship of fluorescence intensity and sulfamido antigen
The detection range of the sulfa drugs of heterocycle；
7) according to the detection range in step 6), the fluorescence containing other antibiotic in sample is measured within the scope of a certain concentration
Intensity, and according to the relationship of fluorescence intensity and sulfamido antigen concentration, it observes whether this method has specificity, sample can be detected
The five-ring heterocycles sulfa antibiotics contained in product.
Preferably, in the quick detection animal food sulfa drug residue of five-ring heterocycles method, specifically
Include the following steps：
1)NaYF4：The preparation of Yb, Er nanoparticle
Y is taken respectively2O3, Yb2O3And Er2O3, excessive HNO is added thereto3Solution carries out heating reaction, evaporates to solution
Deionized water constant volume is used afterwards, obtains the YNO that concentration is respectively 1mol/L3Solution, the YbNO of 0.4mol/L3Solution and 0.05mol/L
ErNO3Solution；Measure YNO3Solution 2.5mL, YbNO3Solution 1.5mL and ErNO3Solution 1.2mL is uniformly mixed, and is slowly added to
Ultrasound is carried out after the sodium citrate solution of 1mmol, so that solution is uniformly mixed；It is slowly added dropwise 1.0mol/L's under magnetic agitation
The pH value of mixed solution is adjusted to 5 using NaOH solution, magnetic force stirs up to having white precipitate appearance in mixed solution by NaF solution
It is transferred in homogeneous reactor after mixing 1h, 180 DEG C of insulation reaction 4h are cooled to room temperature and are centrifuged mixed liquor, and centrifugal rotational speed is
10000rpm, centrifugation time 10min；Sediment washed once using dehydrated alcohol, then washed three times with distilled water；It washes
It washs to finish and is placed at 60 DEG C freeze-day with constant temperature to get NaYF4：Yb, Er nanoparticle；
2) NaYF of functionalization4：The preparation of Yb, Er nanoparticle：
By the NaYF of 40mg4：Yb, Er powder are added in the normal propyl alcohol solution of 60mL, ultrasonic magnetic agitation 40min, so
It is transferred in 35 DEG C of water bath with thermostatic control afterwards, instills 2.5mL ammonia spirit and 20mL water thereto, continue magnetic agitation 1h；Xiang Qi
In the mixed solution containing 20mL normal propyl alcohol and 25 μ LTEOS solution is slowly added dropwise, the reaction was continued 4h；Then it will contain 0.2mL
The mixed solution of APTES and 30mL normal propyl alcohol is added dropwise in above-mentioned solution, persistently stirs 1h；10000rpm is used after stirring
It is centrifuged 10min taking precipitate, first washed once with dehydrated alcohol, then washed three times with distilled water；It puts the precipitate at 60 DEG C
12h is dried to get the NaYF of functionalization4：Yb, Er nanoparticle；
3) sulfamido antibody connects
Take the NaYF of 20mg functionalization4：The PBS that the pH of the nanoparticle dissolution of Yb, Er to 5mL 0.01mol/L are 7.0
In solution, ultrasonic 15min；Then it is slowly added into the glutaraldehyde solution and 100mg sodium borohydride of 1.25mL 25%, is uniformly mixed
Slow oscillation reacts 1h at room temperature afterwards, and mixed solution is centrifuged 10min at 10000rpm, and the pH using 0.01mol/L is 7.0
PBS solution washing three times, then sediment is dispersed in the PBS solution that the 0.01mol/LpH of 5mL is 7.0 again, is added 25
The sulfamido antibody and 100mg sodium borohydride of the 1mg/mL of μ L, at room temperature after persistent oscillation 1h.Then, the Tris that 100mg is added is
Sealer continues oscillating reactions 1h；Resulting product is dispersed in 1mLPBS, 4 again after centrifugation, PBS solution are washed 3 times
It DEG C saves backup.
4) gold nano is synthesized
It weighs 0.0216g gold chloride and is dissolved in 237.5mL deionized water, be vigorously stirred and be heated to boiling；Weigh 0.125g
Sodium citrate is dissolved in 12.5mL deionized water, and sodium citrate solution is added rapidly to chlorauric acid solution under magnetic stirring
In, continue stirred in water bath up to solution presentation claret, continue to be heated to reflux 30min, is cooled to room temperature to obtain cooling solution.
It weighs 0.0042g PVP to be dissolved in 1mL deionized water, then adds it in cooling solution, be stirred at room temperature for 24 hours, natural cooling
To room temperature to get gold nano, 4 DEG C are kept in dark place；
5) gold nano is connected with sulfamido antigen
10mL gold nano is taken, with 0.01mol/L, its pH value is adjusted to 8.0, is then placed in ice water by the PBS solution that pH is 9.0
It is stirred in bath；Sulfamido antigen-the BSA of 20 μ L 1mg/mL is added, stirs 1h.Then the BSA of 50mg is added as sealer,
Continue to stir 1h, be centrifugated at 4 DEG C, centrifugal rotational speed 10000rpm, centrifugation time 10min use the pH of 0.01mol/L
For 7.0 PBS solution washing precipitate three times, finally collected, obtained with the PBS solution that the pH of 10mL 0.01mol/L is 7.0
Sulfamido antigen-BSA- gold nano solution, 4 DEG C save backup；
6) the sulfa drugs detection range of five-ring heterocycles is determined
The small test tube of several 1.5mL is taken, each small test tube marks, and the sulfanilamide (SN) of different content is added in small test tube
Class antigenic solution, respectively 0.02,0.04,0.08,0.1,1,5,10,30,50,75,100,500,1000ng, are then added 25
Sulfamido antibody-the NaYF of μ g/mL4：20 μ g/mL sulfamido antigen-BSA- gold are added after reacting 0.5h in 100 μ L of Yb, Er solution
Appropriate nano-solution.Then successively the PBS solution that pH is 7.0 is added into each small test tube to add to volume is 200 μ L, then is put into
On shaking table after slow oscillation 40min, fluoremetry is carried out using sepectrophotofluorometer.
7) according to the detection range in step 6), measure in a certain range that the fluorescence containing other antibiotic is strong in sample
Degree, and according to the relationship of fluorescence intensity and sulfamido antigen concentration, observe whether this method has specificity, it can test sample
In the five-ring heterocycles sulfa antibiotics that contain.
Specific advantage is the present invention compared with prior art：
The present invention is on the basis of existing technology, miscellaneous respectively in connection with five yuan by up-conversion luminescent material and gold nano-material
The sulfamido antibody and antigen of ring construct the fluorescence resonance energy transfer body based on up-conversion luminescent material-gold nano
System, establishes the remaining rapid detection method of Sulfonamides, realizes the sulphur of the trace five-ring heterocycles in milk and Other Drinks
The detection of amine antibiotic, has broad application prospects.
Specific embodiment
The present invention is further described below by way of specific embodiment, but the embodiment does not limit this hair in any way
The range of bright patent protection.
The method of the sulfa drug residue of five-ring heterocycles in a kind of quickly detection animal food of the embodiment present invention
A kind of method of the sulfa drug residue of five-ring heterocycles in quick detection animal food comprising Xia Shubu
Suddenly：
1, up-conversion luminescent material NaYF4：The synthesis of Yb, Er
Preparation synthesis up-conversion nano material NaYF4：The condition of Yb, Er：LnNO is prepared first3Solution takes a certain amount of respectively
Y2O3, Yb2O3And Er2O3, excessive HNO is added3After solution heating evaporation, deionized water is added and is completely dissolved constant volume.Then
YNO is taken respectively3(1mol/L) 2.5mL, YbNO3(0.4mol/L) 1.5mL and ErNO3(0.05mol/L) 1.2mL, is slow added into
After the sodium citrate solution of 1mmol ultrasound and be uniformly mixed.Under magnetic stirring, slowly it is added dropwise suitable 1.0mol/L's
NaF solution is into mixed solution.It is gradually become cloudy to solution, there is white precipitate appearance, with the NaOH solution of 1mol/L by pH value
5 are adjusted to, magnetic agitation 1h.It is transferred in 50mL reaction kettle, is put into homogeneous reactor after stirring, keep 180 DEG C of reactions
4h.It is cooled to room temperature after reaction and is centrifuged and is washed.Centrifugal rotational speed is 10000rpm, is centrifuged 10min every time.First use nothing
Water-ethanol washed once, then three times using distilled water washing.After washing at 60 DEG C freeze-day with constant temperature to get upper conversion
NaYF4：Yb, Er nanoparticle.
2, up-conversion luminescent material NaYF4：Yb, Er modification
Prepare NaYF4：NaYF is obtained after Yb, Er are dry at 60 DEG C4：The powder of Yb, Er, then by NaYF4：Yb, Er into
Row functional modification.Take the NaYF of 40mg4：Yb, Er powder, are added in the beaker of 250mL, and 60mL is being added just into beaker
Propanol solution, ultrasound, then magnetic agitation 40min, are then placed in 35 DEG C of water bath with thermostatic control and continue to stir, instill the ammonia of 2.5mL
Aqueous solution and 20mL water.After magnetic agitation 1h, 20mL normal propyl alcohol will be mixed with and 25 μ L TEOS solution are slowly dropped into above-mentioned solution
In, the reaction was continued 4h.Then the mixed solution for instilling APTES the and 30mL normal propyl alcohol containing 0.2mL, persistently stirs 1h.Stirring
After, it puts into a centrifuge and is centrifuged 10min under 10000rpm, taking precipitate washed once with dehydrated alcohol, then use distilled water
Washing is three times.60 DEG C of dry 12h are finally put the precipitate in, the NaYF of functionalization can be obtained4：Yb, Er nanoparticle.
3, anti-sulfamido antibody repairs connection
Take the NaYF of 20mg functional modification4：It is molten that Yb, Er powder are dissolved into the PBS that the pH of 5mL 0.01mol/L is 7.0
In liquid, ultrasonic 15min.Then the glutaraldehyde solution and 100mg sodium borohydride of 1.25mL 25% are added slowly, mixes rear chamber
The lower slow oscillation 1h of temperature.It reacts obtained product and is centrifuged 10min at 10000rpm, the PBS for being 7.0 with the pH of 0.01mol/L
Solution washs three times, removes upper solution.It obtains sediment and is dispersed in the PBS solution that the 0.01mol/L pH of 5mL is 7.0 again
In, the sulfamido antibody of 25 μ L1mg/mL and the sodium borohydride of 100mg are added, at room temperature after persistent oscillation 1h.Then it is added
The Tris of 100mg is to make sealer, continues oscillating reactions 1h.After the centrifugation of resulting product, washing 3 times with PBS solution, then weigh
It is newly dispersed in 1mLPBS, 4 DEG C save backup.
4, gold nano is synthesized
Gold nano is prepared using citric acid reduction method.Vessel used in all preparation gold nanos are soaked with wang aqueous solution first
Bubble is overnight.Glass apparatus after immersion was cleaned up in second day, is put into baking oven and dries.0.0216g gold chloride is weighed to be dissolved in
237.5mL deionized water, three necks for adding it to the 500mL of aqueduct, reflux condensing tube, vacuum stopper and magneton device are burnt
In bottle, it is vigorously stirred, is heated to reflux with magneton, be heated to boiling.0.125g sodium citrate is weighed to be dissolved in 12.5mL deionized water,
Sodium citrate solution is added in chlorauric acid solution rapidly under magnetic stirring, continues stirred in water bath.After 5min, solution face
Color starts to deepen, and gradually appears grey, blue and purple, eventually becomes claret, continues to be heated to reflux 30min, is cooled to room
Temperature.After cooling, weighs 0.0042g PVP and be dissolved in 1mL deionized water, be then added in the solution of above-mentioned cooling, stir at room temperature
It mixes for 24 hours, then cooled to room temperature, is transferred in brown reagent bottle, 4 DEG C save to get gold nano.
5, gold nano connects antigen
10mL gold nano is taken, with 0.01mol/L, its pH is adjusted to 8.0, is then placed in ice-water bath by the PBS solution that pH is 9.0
Middle stirring.Sulfamido antigen-the BSA of 20 μ L 1mg/mL is added, stirs 1h.Then the BSA of 50mg is added as sealer, after
Continuous stirring 1h.It is centrifugated at 4 DEG C, is centrifuged 10min under 10000rpm, washed with the PBS solution that the pH of 0.01mol/L is 7.0
It washs sediment three times, removes upper solution, finally collected with the PBS solution that the pH of 10mL 0.01mol/L is 7.0,4 DEG C of preservations
It is spare.
6, detection sulfa drugs takes the small test tube of several 1.5mL, and each small test tube marks, and adds in small test tube
Enter different content sulfamido antigenic solution, respectively 0.02,0.04,0.08,0.1,1,5,10,30,50,75,100,500,
Then the sulfamido antibody-NaYF of 25 μ g/mL is added in 1000ng4：100 μ L of Yb, Er solution is added in right amount after reacting 0.5h
20 μ g/mL sulfamido antigen-BSA- gold nano solutions.Then the volume for making final solution with the PBS solution that pH is 7.0 is
200μL.Then it is put on shaking table after slow oscillation reaction 40min, carries out fluoremetry using sepectrophotofluorometer, obtain five
The detection range of the sulfamido of circle heterocyclic ring.
7, according to the detection range of the sulfamido of five-ring heterocycles, the antibiotic compound of doping difference same concentrations in milk
Sulfamethoxazole, methoxybenzyl aminopyrimidine, sulfamido antigen and BSA form various mixture milk solns.Then exist respectively
It is added that the milk soln that equivalent contains different mixtures is appropriate, and the sulfamido for being subsequently added into 25 μ g/mL is anti-in the small test tube of 1.5mL
Body-NaYF4：Appropriate 20 μ g/mL sulfamido antigen-BSA- gold nano solution is added after reacting 0.5h in 100 μ L of Yb, Er solution.So
It is 200 μ L that PBS solution is successively added into each small test tube afterwards and adds to volume, so that the contained mixture (compound in test tube
Sulfamethoxazole, methoxybenzyl aminopyrimidine, sulfamido antigen and BSA) concentration be 20ng/ml.Then it is put on shaking table and slowly shakes
After 40min shaking, fluoremetry is carried out.
Claims (4)
1. the method for the sulfa drug residue of five-ring heterocycles, is to pass through hydrothermal synthesis method in a kind of quickly detection animal food
Up-conversion nanoparticles NaYF is prepared4：Yb, Er will utilize glutaraldehyde method with sulfamido antibody after its functional modification
Coupling, obtains sulfamido antibody-NaYF4：Yb, Er；Gold nano-material is obtained by sodium citrate synthetic method, coupling sulfamido is anti-
Original obtains sulfamido antigen-BSA- gold nano solution, then constructs up-conversion nanoparticles-gold nano resonance energy transfer body
System determines the concentration of sulfamido antigen by measuring fluorescence intensity.
2. the method for the sulfa drug residue of five-ring heterocycles in quick detection animal food according to claim 1,
It is characterized in that, it specifically comprises the following steps：
1)NaYF4：The preparation of Yb, Er nanoparticle：
Measure YNO3Solution, YbNO3Solution and ErNO3Solution is uniformly mixed, and ultrasound mixes after being slowly added to sodium citrate solution；
NaF solution is slowly added dropwise thereto until having white precipitate appearance in mixed solution, the pH value of mixed solution is adjusted to 5, magnetic force
It being transferred in homogeneous reactor after stirring 1h, 180 DEG C of insulation reaction 4h are centrifuged after being cooled to room temperature, sediment are washed drying,
Up to NaYF4：Yb, Er nanoparticle；
2) NaYF of functionalization4：The preparation of Yb, Er nanoparticle：
By NaYF4：Yb, Er nanoparticle are added in normal propyl alcohol, are transferred in 35 DEG C of water bath with thermostatic control after ultrasonic magnetic agitation, to
Ammonia spirit is wherein instilled, magnetic agitation 1h is continued；The mixed solution for being mixed with normal propyl alcohol and TEOS solution is slowly added dropwise thereto,
The reaction was continued 4h；The mixed solution of APTES and normal propyl alcohol is added dropwise in mixed solution, centrifuging and taking precipitates after persistently stirring 1h
Object, washing, the dry NaYF to get functionalization4：Yb, Er nanoparticle；
3) sulfamido antibody connects：
Take the NaYF of functionalization4：The nanoparticle dissolution of Yb, Er are slowly added into glutaraldehyde solution and hydroboration into PBS solution
Sodium is centrifuged after slow oscillation reaction 1h at room temperature after mixing, and sediment is dispersed in PBS after washing using PBS solution again
In solution, sulfamido antibody and sodium borohydride is added, Tris sealer is added after oscillating reactions 1h at room temperature, continues oscillating reactions
It is centrifuged after 1h, is dispersed in PBS solution after sediment is washed, 4 DEG C save backup, and obtain sulfamido antibody-NaYF4：Yb, Er
Solution；
4) gold nano is synthesized：
Sodium citrate solution is added rapidly in chlorauric acid solution under magnetic agitation, stirred in water bath is until solution presentation wine is red
Color is heated to reflux 30min, is cooled to room temperature to obtain cooling solution, PVP solution is added in cooling solution and is stirred at room temperature for 24 hours, i.e.,
Gold nano solution is obtained, 4 DEG C are kept in dark place；
5) gold nano is connected with antigen：
Gold nano solution pH value is adjusted to 8.0 with PBS solution, sulfamido antigen-BSA is added in ice-water bath under stirring, stirs
Mix reaction 1h；BSA is added thereto again, continues to be stirred to react 1h, is centrifugated at 4 DEG C, after sediment is using PBS washing, point
It is dispersed in the PBS solution that pH is 8.0, obtains sulfamido antigen-BSA- gold nano solution, 4 DEG C save backup；
6) the sulfa drugs detection range of five-ring heterocycles is determined：
It is separately added into the sulfamido antigenic solution of different content into test tube, sulfamido antibody-NaYF is added thereto4：Yb, Er
Sulfamido antigen-BSA- gold nano solution is added after reacting 0.5h in solution；It is vibrated after PBS solution is added thereto as shaking table
Mixed liquor fluoremetry is determined into the sulfanilamide (SN) of five-ring heterocycles according to the relationship of fluorescence intensity and sulfamido antigen concentration after 40min
The detection range of class drug；
7) according to the detection range in step 6), the fluorescence intensity containing other antibiotics samples in a certain range is detected, and
It according to the relationship of fluorescence intensity and sulfamido antigen concentration, observes whether this method has specificity, can detect containing five yuan
The sample of the sulfa antibiotics of heterocycle.
3. the method for the sulfa drug residue of five-ring heterocycles in quick detection animal food according to claim 1,
It is characterized in that, the method specifically includes following step：
1)NaYF4：The preparation of Yb, Er nanoparticle
Y is taken respectively2O3, Yb2O3And Er2O3, excessive HNO is added thereto3Solution heating reaction, after solution evaporation after spend from
Sub- water constant volume obtains the YNO that concentration is respectively 1mol/L3Solution, the YbNO of 0.4mol/L3The ErNO of solution and 0.05mol/L3
Solution；Measure YNO3Solution 2.5mL, YbNO3Solution 1.5mL and ErNO3Solution 1.2mL is uniformly mixed, and is slowly added to 1mmol's
Sodium citrate solution carries out ultrasonic vibration, is uniformly mixed solution；The NaF solution of 1.0mol/L is slowly added dropwise under magnetic agitation
Until having white precipitate appearance in mixed solution, the pH value of mixed solution is adjusted to 5 using NaOH solution, is turned after magnetic agitation 1h
It moving in homogeneous reactor, 180 DEG C of insulation reaction 4h are cooled to room temperature and are centrifuged mixed liquor, centrifugal rotational speed 10000rpm,
Centrifugation time is 10min；Sediment washed once using dehydrated alcohol, then washed three times with distilled water again；Washing finishes
Freeze-day with constant temperature is placed at 60 DEG C to get NaYF4：Yb, Er nanoparticle；
2) NaYF of functionalization4：The preparation of Yb, Er nanoparticle：
By the NaYF of 40mg4：Yb, Er powder are added in the normal propyl alcohol solution of 60mL, then ultrasonic magnetic agitation 40min is shifted
Into 35 DEG C of waters bath with thermostatic control, 2.5mL ammonia spirit and 20mL ultrapure water are instilled thereto, continue magnetic agitation 1h；Thereto
The mixed solution containing 20mL normal propyl alcohol and 25 μ L TEOS solution is slowly added dropwise, the reaction was continued 4h；Then it will contain 0.2mL
The mixed solution of APTES and 30mL normal propyl alcohol is added dropwise in solution, persistently stirs 1h；It is centrifuged after stirring using 10000rpm
10min, taking precipitate washed once with dehydrated alcohol, then be washed three times with distilled water；It puts the precipitate in dry at 60 DEG C
12h to get functionalization NaYF4：Yb, Er nanoparticle；
3) sulfamido antibody connects
Take the NaYF of 20mg functionalization4：The PBS solution that the pH of the nanoparticle dissolution of Yb, Er to 5mL 0.01mol/L are 7.0
In, ultrasonic 15min；Then it is slowly added into the glutaraldehyde solution and 100mg sodium borohydride of 1.25mL 25%, is existed after mixing
Slow oscillation reacts 1h at room temperature, and mixed solution is centrifuged 10min at 10000rpm, is washed using the PBS solution of 0.01mol/L
It washs three times, then sediment is dispersed in again in the PBS that the 0.01mol/LpH of 5mL is 7.0, the sulphur of the 1mg/mL of 25 μ L is added
The sodium borohydride of amine antibody and 100mg, at room temperature after persistent oscillation 1h；Then the Tris that 100mg is added is sealer, continues to shake
Swing reaction 1h；Resulting product centrifugation after washing 3 times with PBS solution, is dispersed in 1mL PBS solution, 4 DEG C of preservations are standby again
With；
4) gold nano is synthesized
It weighs 0.0216g gold chloride and is dissolved in 237.5mL deionized water, be vigorously stirred and be heated to boiling；Weigh 0.125g lemon
Sour sodium is dissolved in 12.5mL deionized water, and sodium citrate solution is added rapidly in chlorauric acid solution under magnetic stirring, after
Continuous stirred in water bath continues to be heated to reflux 30min until solution presentation claret, is cooled to room temperature to obtain cooling solution.It weighs
0.0042g PVP is dissolved in 1mL deionized water, then is added it in cooling solution, is stirred at room temperature for 24 hours, is naturally cooled to room
To get gold nano, 4 DEG C are kept in dark place temperature；
5) gold nano is connected with sulfamido antigen
10mL gold nano solution is taken, with 0.01mol/L, its pH is adjusted to 8.0, is then placed in ice-water bath by the PBS solution that pH is 9.0
Middle stirring；Sulfamido antigen-the BSA of 20 μ L 1mg/mL is added, stirs 1h；Then the BSA of 50mg is added as sealer, after
Continuous stirring 1h, 4 DEG C of centrifuge separations, centrifugal rotational speed 10000rpm, centrifugation time 10min are using the pH of 0.01mol/L
7.0 PBS solution washing precipitating three times, removes upper solution, the PBS solution for being finally 8.0 with the pH of 10mL 0.01mol/L
It collects, obtains sulfamido antigen-BSA- gold nano solution, 4 DEG C save backup；
6) the sulfa drugs detection range of five-ring heterocycles is determined
The small test tube of several 1.5mL is taken, each small test tube marks, and the sulfamido that different content is added in small test tube is anti-
Then 25 μ g/ are added in original solution, respectively 0.02,0.04,0.08,0.1,1,5,10,30,50,75,100,500,1000ng
Sulfamido antibody-the NaYF of mL4：100 μ L of Yb, Er solution adds appropriate 20 μ g/mL sulfamido antigen-after reacting 0.5h
BSA- gold nano solution；Then PBS solution successively is added into each small test tube, makes 200 μ L of its final volume, then puts again
Slow oscillation 40min on to shaking table, is put on sepectrophotofluorometer after oscillation and carries out fluoremetry.
4. the side of the sulfa drug residue of five-ring heterocycles in quick detection animal food according to claim 1 or 2
Method, which is characterized in that the sulfamido antigen-antibody can be to sulphathiazole, sulfamethoxazole, bacteresulf, sulfalene
The pentacyclic sulfa drugs such as diazole has preferable cross reactivity, is able to detect the sulfanilamide (SN) of the five-ring heterocycles such as sulfamethoxazole
Class antibiotic.
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| CN109856099A (en) * | 2019-03-15 | 2019-06-07 | 浙江工业大学 | A method of based on alcoholic strength in upconverting fluorescent material detection wine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940792A (en) * | 2014-02-20 | 2014-07-23 | 江南大学 | Method used for simultaneous detection of three food-borne pathogenic bacteria based on multicolor upconversion fluorescence labeling |
| WO2017044494A1 (en) * | 2015-09-08 | 2017-03-16 | BellBrook Labs | High-throughput split aptamer screening assay |
-
2018
- 2018-05-14 CN CN201810460807.1A patent/CN108918862A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103940792A (en) * | 2014-02-20 | 2014-07-23 | 江南大学 | Method used for simultaneous detection of three food-borne pathogenic bacteria based on multicolor upconversion fluorescence labeling |
| WO2017044494A1 (en) * | 2015-09-08 | 2017-03-16 | BellBrook Labs | High-throughput split aptamer screening assay |
Non-Patent Citations (2)
| Title |
|---|
| HONGQI CHEN ET AL.: "Aptamer-based sensing for thrombin in red region via fluorescence resonant energy transfer between NaYF4:Yb,Er upconversion nanoparticles and gold nanorods", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 王莉平: "上转换纳米粒子的制备及其在毒素快速检测中的应用研究", 《中国优秀硕士学位论文全文数据(电子期刊)工程科技I辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109856099A (en) * | 2019-03-15 | 2019-06-07 | 浙江工业大学 | A method of based on alcoholic strength in upconverting fluorescent material detection wine |
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