CN110221085A - A kind of streptomysin and neomycin remain rapid fluorescence detection reagent and application simultaneously more - Google Patents
A kind of streptomysin and neomycin remain rapid fluorescence detection reagent and application simultaneously more Download PDFInfo
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Abstract
The present invention is the method for preparation and use that a kind of streptomysin and neomycin remain rapid fluorescence detection reagent simultaneously more.With to streptomysin and neomycin there are the aptamers of high specific recognition capability to be coupled respectively the two amounts point with identical material core and different emission, by the way that two kinds of conjugates are constituted fluorescence resonance energy transfer system with graphene oxide respectively, the reagent for remaining more to streptomysin and neomycin and carrying out rapid fluorescence detection simultaneously can be prepared.Using detection reagent of the present invention, quick, accurate and Sensitive Detection how remaining to milk sample streptomycin and neomycin can be realized independent of separate analytical technique, provides strong technical guarantee for condensed food security control.
Description
Technical field
The present invention relates to technical field of nanometer material preparation and detection of veterinary drugs in food technical field, and in particular to a kind of collection is suitable
Ligand high specific and the excellent fluorescent characteristic of quantum dot are in one, the fluorescence detection examination of alternative label streptomysin and neomycin
Agent, and be applied to rapid fluorescence while streptomysin and neomycin remain more and detect.
Background technique
Streptomysin and neomycin are the aminoglycoside antibiotics being widely used in animal husbandry, unreasonable use
It will lead to its residual in animal derived food, will lead to ototoxicity, Toxicity of Kidney, allergic reaction after being eaten for a long time by the mankind
Deng the adverse effect to health.Carrying out Accurate Determining to its residual in animal derived food is to prevent unqualified food to flow into
Last one of critical point in market had accurately both included quantitative accurate, and had also included qualitative accurate.In order to clearly learn in one test
The specific name of residual antibiotic in food, the method for establishing multiple antibiotic residues detection are very effective technological means,
And in multi-residue determination, the multi-residue determination of similar antibiotic (such as aminoglycoside antibiotics) is that difficulty is maximum again.
It is main sensitive and accurate qualitative and quantitative detection the method for progress can be remained to aminoglycoside antibiotics at present more
It is the method based on separation analysis principle, such as high performance liquid chromatography, Liquid Chromatography-Mass Spectrometry.Such methods use
Expensive equipment, for the professional more demanding of operator, it usually needs to sample carry out complicated and time consumption purification
Pre-treatment, it is difficult to which the quick detection for realizing scene limits quickly and effectively market surpervision.Fluorescence detection has very high spirit
Sensitivity, the antibiotics leftover detection being quite suitable for including streptomysin and neomycin, but do not have fluorescence for itself
For the streptomysin and neomycin of characteristic, fluorescent marker must be carried out to it using this method.Traditional organic fluorescent dye is not
Have the specific recognition ability to labeled object, it is necessary to and separate analytical technique combines, and can just reach the purpose of multi-residue determination.
The superior fluorescence that there are the novel nano fluorescent material quantum dot occurred in recent years many traditional organic fluorescent dyes not have is special
Property, be widely used in analytical chemistry field, but do not have the specific recognition ability to labeled object equally, usually and
Immunization is implemented in combination with the identification to labeled object.Though immunization can realize the quick detection of antibiotic residue, mostly single
Residue detection, can not achieve highly selective multi-residue determination, and antibody sheet is as protein, obtain need to through zoopery or
Cell culture, it is at high cost, it is easy to be affected by temperature and its stability is made to generate very big fluctuation, and not easy to maintain, it is difficult to
Detections universal and for batch samples.
Summary of the invention
The purpose of the present invention is to provide one kind can be realized independent of separate analytical technique to streptomysin and neomycin
More residuals carry out the preparation and application of rapid fluorescence detection reagent simultaneously, and are applied to strepto- in animal derived food
Element and the how remaining detection of neomycin.
Specific step is as follows:
1, a kind of streptomysin and neomycin remain rapid fluorescence detection reagent simultaneously more, it is characterised in that: the reagent is adopted
It is prepared with the following method:
(1) there are the aptamers of high specific and high-affinity to be used separately as streptomysin and new streptomysin and neomycin
The information identification agent of mycin, the nucleic acid sequence of streptomycin aptamers (SAPT) are 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 aptamers (NAPT) are 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 kinds of quantum dots that can emit different wave length fluorescence under identical excitation wavelength
QD1 and QD2 is used separately as the Information Conduction reagent of neomycin and streptomysin.
Measuring concentration is respectively 2.46 × 10-5With 1.62 × 10-5Each 5 μ L of the QD1 and QD2 of mol/L, is separately added into concentration
For the 10 μ L of 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 10g/L, 40 μ L are settled to deionized water, point
It is not activated；It is then respectively adding NAPT and SAPT through denaturation treatment, is settled to 400 μ with the phosphate buffer of pH7.4
L carries out coupling reaction；It uses 50kD super filter tube to filter respectively solution after reaction, solution is retained in pipe and uses phosphate-buffered respectively
Liquid is settled to 100 μ L, this SAPT-QD2 and NAPT-QD1 conjugate solution constitutes the composite fluorescence probe of streptomysin and neomycin
Solution is respectively 810 and 1230nmol/L with the conjugate concentration of QD2 and QD1 densimeter.
Graphene oxide (GO) is added after appropriate SAPT-QD2 and NAPT-QD1 is mixed, and is diluted with phosphate buffer
Concentration to wherein GO is 300mg/L, and the concentration with the SAPT-QD2 of QD2 and QD1 densimeter and NAPT-QD1 is respectively 81 Hes
Constituting after 123nmol/L, quenching reaction 30min can be used for the examination that streptomysin and neomycin remain rapid fluorescence detection simultaneously more
Agent SAPT-QD2/GO and NAPT-QD1/GO.
The high specific refers to SAPT in addition to streptomysin, to other classification antibiotic and aminoglycoside antibiotics, spy
It is not not have recognition reaction to neomycin, NAPT is in addition to neomycin, to other classification antibiotic and aminoglycoside antibiotics,
Especially do not have recognition reaction to streptomysin；The high-affinity is presented as that SAPT is 26.56 to the dissociation constant of streptomysin
± 6.57nmol/L, NAPT are 40.96 ± 11.56nmol/L to the dissociation constant of neomycin.The quantum dot QD1 and QD2 points
It is not the CdTe quantum modified with thioacetic acid and mercaptopropionic acid, excitation wavelength 370nm, launch wavelength is respectively 548 Hes
580nm。
The quantum dot QD1 is to prepare as follows: 0.0760g tellurium powder and 0.05g sodium borohydride are placed in note
In the bottle of emitter syringe needle, 2mL deionized water is added, is passed through nitrogen 5min immediately, covers bottle stopper, room temperature immediately after stopping ventilation
Lower reaction 4h obtains aubergine NaHTe solution；0.2200g caddy is placed in 250mL three-necked bottle, 100mL deionization is added
Water, is passed through nitrogen 30min, 206 μ L thioacetic acid is then added, and adjust pH to 11.2 with 1mol/L NaOH, is added freshly prepared
NaHTe solution, at 95 DEG C reflux condensation mode 2h obtain thioacetic acid modification CdTe quantum, excitation wavelength 370nm, transmitting
Wavelength 548nm.
The QD2 is to prepare as follows: 0.1276g tellurium powder and 0.0800g sodium borohydride are placed in syringe
In the bottle of syringe needle, 3mL deionized water is added, is passed through nitrogen 5min immediately, stops covering bottle stopper after ventilating immediately, it is anti-under room temperature
4h is answered to obtain aubergine NaHTe solution；0.4567g caddy is placed in 250mL three-necked bottle, 100mL deionized water is added, is led to
Enter nitrogen 30min, 420 μ L mercaptopropionic acids is then added, and adjust pH to 10 with 1mol/L NaOH, freshly prepd NaHTe is added
Solution transfers them in the stainless steel cauldron with polytetrafluoroethyllining lining, and 1h is heated in 160 DEG C of baking ovens and obtains sulfydryl
The CdTe quantum of propionic acid modification, excitation wavelength 370nm, launch wavelength 580nm.
It is described activation refer to make under sonic oscillation QD1 and QD2 respectively with 1- (3- dimethylamino-propyl) -3- ethyl carbon two
Imide hydrochloride reactant salt 3.95-4.05min and 14.95-15.05min will lead to quantum dot fluorescence in addition in this time frame
Strength reduction.The reaction of degeneration (RD) refers to heats 10min for NAPT and SAPT at 95 DEG C respectively, then ice bath 10min.It is described
Coupling reaction, which refers to, makes NAPT according to the ratio of NAPT:QD1 molar ratio 1:3.8-4.2 and SAPT:QD2 molar ratio 1:4.8-5.2
With SAPT respectively with after activation QD1 and QD2 be protected from light 2h, in this proportional region, the surface of QD1 and QD2 can be divided
It is not coupled sufficiently by NAPT and SAPT.The excitation wavelength of described two composite fluorescence probe NAPT-QD1 and SAPT-QD2 is
370nm, launch wavelength are respectively 550 and 586nm.
The quenching reaction refers to that GO constitutes fluorescence resonance energy with QD1 and QD2 respectively by pi-pi accumulation interaction and turns
Shifting system makes the fluorescent quenching of QD1 and QD2.
2, a kind of streptomysin and neomycin remain the application method of rapid fluorescence detection reagent simultaneously more, it is characterised in that press
It is carried out according to following step:
(1) the 100 above-mentioned luciferase assay reagents of μ L are measured, 1 μ L streptomysin and neomycin mixed standard solution or sample is added
Solution reacts 30min or more, and the quantum dot fluorescence that will lead to quenching lower than this time cannot be thus capable of sufficiently recovering, and it is accurate to influence detection
Degree.
(2) solution after reaction is put into microplate reader and carries out fluoremetry.
The concentration of the streptomysin and neomycin mixed standard solution streptomycin and neomycin is respectively 10,50,50,
100,100,200,500,500,800,800,1000,1000 μ g/L.
The sample solution is prepared as steps described below:
(1) 10mL blank milk (without the milk of streptomysin and Detection of neomycin residues) is measured, various concentration streptomysin is added
With the mixed standard solution of neomycin.
(2) it is added 2mL 15% (V/V) trichloroacetic acid, sonic oscillation 20min, 12000r/min are centrifuged 10min, in collection
Clear liquid is settled to 5mL with deionized water.
The fluorescence detection carried out in microplate reader carries out as steps described below:
(1) the excitation wavelength 370nm of neomycin and streptomysin detection；Launch wavelength 550 and 586nm, corresponding fluorescence are strong
Degree is F550nmAnd F586nm；Tuning wavelength 618 and 518nm, corresponding fluorescence intensity are F618nmAnd F518nm；Neomycin and streptomysin
Only analyzing signal be respectively Δ F1=F550nm-F618nm, Δ F2=F586nm-F518nm。
(2) the Δ F of each standard solution is measured1With Δ F2, to the neomycin concentration C in these measured values and each standard solutionN
And streptomysin concentration CSIt is returned using origin software, the standard curve Δ F of neomycin and streptomysin can be obtained1=A1
×CN+B1, Δ F2=A2×CS+B2, wherein A1、A2And B1、B2The slope and intercept respectively provided in regression process.
(3) the Δ F of sample solution1With Δ F2, respective calibration curve equation is substituted into, neomycin and chain in sample are calculated
The concentration of mycin.
Beneficial effect
The fluorescence labeling material of existing streptomysin and neomycin, either traditional organic fluorescent dye or nano fluorescent material
Expect quantum dot, does not have the selected marker feature to streptomysin and neomycin.Aptamers, which have, knows target substance specificity
It does not act on, but does not have the conduction to target substance signal, need and other materials or method for having signal transduction effect
In conjunction with.The present invention by two kinds with inner nuclear material of the same race (CdTe) quantum dots respectively with streptomysin and neomycin can be selected
The aptamers of selecting property identification combine, and both integrate advantage, have obtained that streptomysin and neomycin while fluorescence can be carried out
The composite fluorescence reagent of label.Two amounts point used in the reagent is made of inner nuclear material of the same race, merely with the difference of its partial size,
It achieves that the fluorescence for emitting two kinds of different wave lengths under same excitation wavelength, solves difference with relatively simple material base
The signal transduction problem of target substance.The reagent can carry out highly selective identification to streptomysin and neomycin, be not in each other
Intersect identification or to false positive results caused by other antibiotic wrong identifications, the key that can reach such excellent properties exists
In aptamers used in this reagent are by large amount of complex screening experiment, from containing 1013-1015In the random library of a oligonucleotides
Obtained high specific aptamers.The sequence information of aptamers can largely high-purity chemicals be synthesized once acquisition, and be easy to
It saves, this is advantage not available for antibody.
Existing multiple antibiotic residues detection method, especially similar antibiotic relict detection method are all based on point
It is realized from analytical technology, depends on large-sized analytic instrument.Using detection reagent of the present invention, can get rid of to method for separating and analyzing
Dependence, avoid the sample purification pre-treatment step of complicated and time consumption therewith, by simply dealt sample can directly using this
Invention reagent is detected, and has simple, quick, live, accurate, sensitive feature, to the detection energy of streptomysin and neomycin
Power has reached the attainable level of current its most intensity detection method liquid chromatography-mass spectrography institute, without the instrument of such valuableness,
Strong technical guarantee is provided for condensed food security control.
Detailed description of the invention
The fluorescence emission spectrum of Fig. 1 NAPT-QD1 and SAPT-QD2 and the selection of detection and tuning wavelength.
Fig. 2 luciferase assay reagent is in milk sample to the specificity of neomycin and streptomysin.
Specific embodiment
Embodiment 1
Measuring concentration is respectively 2.46 × 10-5With 1.62 × 10-5Each 5 μ L of the QD1 and QD2 of mol/L, is separately added into concentration
For the 10 μ L of 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of 10g/L, 40 μ L are settled to deionized water,
Make under sonic oscillation QD1 and QD2 reacted respectively with 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride 4 and
15min.Then it is separately added into according to the ratio of NAPT:QD1 molar ratio 1:4 and SAPT:QD2 molar ratio 1:5 through at 95 DEG C of denaturation
The NAPT and SAPT of reason are settled to 400 μ L with the phosphate buffer of pH7.4, make NAPT and SAPT respectively with the QD1 after activation
2h is protected from light with QD2.It uses 50kD super filter tube to filter respectively solution after reaction, solution is retained in pipe and uses phosphate-buffered respectively
Liquid is settled to 100 μ L, with the conjugate concentration of QD2 and QD1 densimeter point in this SAPT-QD2 and NAPT-QD1 conjugate solution
It Wei not 810 and 1230nmol/L.
Will appropriate SAPT-QD2 and NAPT-QD1 mix after be added GO, and be diluted to the dense of wherein GO with phosphate buffer
Degree is 300mg/L, and the concentration with the SAPT-QD2 of QD2 and QD1 densimeter and NAPT-QD1 is respectively 81 and 123nmol/L, instead
30min is answered, acquired solution is streptomysin and neomycin remains more while rapid fluorescence detection reagent, excitation wavelength 370nm,
Launch wavelength is respectively 550 and 586nm.
Embodiment 2
10mL blank milk is measured, streptomysin and neomycin mixed standard solution is added, adds the trichlorine of 2mL 15%
Then acetic acid solution vibrates 20min to the sample ultrasonic, 12000r/min is centrifuged 10min, and it is fixed with deionized water to collect supernatant
Appearance is respectively 10 and 50 μ g/L in concentration wherein to 5mL, streptomysin and neomycin.The concentration of streptomysin and neomycin is distinguished
The mixed standard solution for being 10,50,50,100,100,200,500,500,800,800,1000,1000 μ g/L series and mark-on
Milk sample respectively draws 1 μ L, is added separately in 100 μ L luciferase assay reagent of the invention, reacts 30min.It is sharp with 370nm
Wavelength is sent out, measures the fluorescence intensity F at 550 and 618nm and 586 and 518nm respectively to solution after each reaction550nmAnd F618nm
And F586nmAnd F518nm.With the Δ F of each mixed standard solution1=F550nm-F618nmWith Δ F2=F586nm-F518nmFor ordinate, with
Neomycin and streptomysin are in concentration C whereinNAnd CSFor abscissa, the standard curve difference of neomycin and streptomysin can be returned out
For Δ F1=603.48CN+1.1317(R2=0.9920) and Δ F2=303.12CS-2.1867(R2=0.9986), linear response
Range is respectively 50-1000 μ g/L and 10-1000 μ g/L, and detection limit (S/N=3) is respectively 10.02 and 6.65 μ g/L.Utilize mark
It is respectively 52.33 and 10.47 μ g/L that directrix curve, which can calculate the concentration of neomycin and streptomysin in mark-on milk sample, and mark-on returns
Yield is respectively 104.65% and 104.69%.
Embodiment 3
10mL blank milk is measured, streptomysin and neomycin mixed standard solution is added, adds the trichlorine of 2mL 15%
Then acetic acid solution vibrates 20min to the sample ultrasonic, 12000r/min is centrifuged 10min, and it is fixed with deionized water to collect supernatant
Appearance is respectively 200 and 500 μ g/L in concentration wherein to 5mL, streptomysin and neomycin.By the concentration of streptomysin and neomycin point
Not Wei 10,50,50,100,100,200,500,500,800,800,1000,1000 μ g/L mixed standard solution series and add
Mark milk sample respectively draws 1 μ L, is added separately in 100 μ L luciferase assay reagent of the invention, reacts 30min.It is with 370nm
Excitation wavelength measures the fluorescence intensity F at 550 and 618nm and 586 and 518nm to solution after each reaction respectively550nmWith
F618nmAnd F586nmAnd F518nm.With the Δ F of each mixed standard solution1=F550nm-F618nmWith Δ F2=F586nm-F518nmIt is sat to be vertical
Mark, with neomycin and streptomysin in concentration C whereinNAnd CSFor abscissa, the standard curve of neomycin and streptomysin can be returned out
Respectively Δ F1=603.48CN+1.1317(R2=0.9920) and Δ F2=303.12CS-2.1867(R2=0.9986), linearly
Response range is respectively 50-1000 μ g/L and 10-1000 μ g/L, and detection limit (S/N=3) is respectively 10.02 and 6.65 μ g/L.Benefit
It is respectively 480.60 and 212.08 μ g/L that the concentration of neomycin and streptomysin in mark-on milk sample, which can be calculated, with standard curve,
Recovery of standard addition is respectively 96.12% and 106.04%.
Claims (10)
1. a kind of streptomysin and neomycin remain rapid fluorescence detection reagent simultaneously more, which is characterized in that the reagent is using such as
Prepared by lower method: the Information Conduction reagent for being used separately as neomycin and streptomysin is had identical material core and difference
The two amounts point QD2 and QD1 of launch wavelength with to streptomysin and neomycin there is high specific and high-affinity to fit respectively
Ligand is coupled, by the way that two kinds of conjugates are constituted fluorescence resonance energy transfer system, preparation with graphene oxide respectively
The reagent for carrying out rapid fluorescence detection simultaneously is remained to streptomysin and neomycin out more；The quantum dot QD1 and QD2 be respectively with
The CdTe quantum of thioacetic acid and mercaptopropionic acid modification, excitation wavelength 370nm, launch wavelength is respectively 548 and 580nm；It is right
It is 5'-NH that streptomysin, which has the nucleic acid sequence of the aptamers SAPT of high specific and high-affinity,2-(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'；There is the nucleic acid sequence of the aptamers NAPT of high specific and high-affinity to neomycin
It is classified as 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. a kind of streptomysin as described in claim 1 and neomycin remain simultaneously more, rapid fluorescence detection reagent, feature exist
In specific step is as follows for the preparation method of reagent thereof:
Measuring concentration is respectively 2.46 × 10-5With 1.62 × 10-5Each 5 μ L of the QD1 and QD2 of mol/L, being separately added into concentration is 10g/
The 10 μ L of 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride of L, is settled to 40 μ L with deionized water, carries out respectively
Activation；It is then respectively adding NAPT and SAPT through denaturation treatment, is settled to 400 μ L with the phosphate buffer of pH7.4, is carried out
Coupling reaction；It uses 50kD super filter tube to filter respectively solution after reaction, solution is retained in pipe and uses phosphate buffer constant volume respectively
To 100 μ L, this SAPT-QD2 and NAPT-QD1 conjugate solution constitutes the composite fluorescence probe solution of streptomysin and neomycin, with
The conjugate concentration of QD2 and QD1 densimeter is respectively 810 and 1230nmol/L；
Graphene oxide GO is added after appropriate SAPT-QD2 and NAPT-QD1 is mixed, and is diluted to wherein with phosphate buffer
The concentration of GO is 300mg/L, and the concentration with the SAPT-QD2 of QD2 and QD1 densimeter and NAPT-QD1 is respectively 81 Hes
Constituting after 123nmol/L, quenching reaction 30min can be used for the examination that streptomysin and neomycin remain rapid fluorescence detection simultaneously more
Agent SAPT-QD2/GO and NAPT-QD1/GO.
3. a kind of streptomysin as described in claim 1 and neomycin remain simultaneously more, rapid fluorescence detection reagent, feature exist
In the high specific refers to SAPT in addition to streptomysin, to other classification antibiotic and aminoglycoside antibiotics, especially pair
Neomycin does not have recognition reaction, and NAPT is in addition to neomycin, to other classification antibiotic and aminoglycoside antibiotics, especially
Do not have recognition reaction to streptomysin；The high-affinity be presented as SAPT to the dissociation constant of streptomysin be 26.56 ±
6.57nmol/L, NAPT are 40.96 ± 11.56nmol/L to the dissociation constant of neomycin.
4. a kind of streptomysin as described in claim 1 and neomycin remain simultaneously more, rapid fluorescence detection reagent, feature exist
In the quantum dot QD1 is to prepare as follows: 0.0760g tellurium powder and 0.05g sodium borohydride are placed in syringe needle
In the bottle of head, 2mL deionized water is added, is passed through nitrogen 5min immediately, covers bottle stopper immediately after stopping ventilation, is reacted under room temperature
4h obtains aubergine NaHTe solution；0.2200g caddy is placed in 250mL three-necked bottle, 100mL deionized water is added, is passed through
Then nitrogen 30min is added 206 μ L thioacetic acid, and adjusts pH to 11.2 with 1mol/L NaOH, freshly prepd NaHTe is added
Solution, reflux condensation mode 2h obtains the CdTe quantum of thioacetic acid modification, excitation wavelength 370nm, launch wavelength at 95 DEG C
548nm。
5. a kind of streptomysin as described in claim 1 and neomycin remain simultaneously more, rapid fluorescence detection reagent, feature exist
In the quantum dot QD2 is to prepare as follows: 0.1276g tellurium powder and 0.0800g sodium borohydride are placed in syringe
In the bottle of syringe needle, 3mL deionized water is added, is passed through nitrogen 5min immediately, stops covering bottle stopper after ventilating immediately, it is anti-under room temperature
4h is answered to obtain aubergine NaHTe solution；0.4567g caddy is placed in 250mL three-necked bottle, 100mL deionized water is added, is led to
Enter nitrogen 30min, 420 μ L mercaptopropionic acids is then added, and adjust pH to 10 with 1mol/L NaOH, freshly prepd NaHTe is added
Solution transfers them in the stainless steel cauldron with polytetrafluoroethyllining lining, and 1h is heated in 160 DEG C of baking ovens and obtains sulfydryl
The CdTe quantum of propionic acid modification, excitation wavelength 370nm, launch wavelength 580nm.
6. a kind of streptomysin as claimed in claim 2 and neomycin remain simultaneously more, rapid fluorescence detection reagent, feature exist
In, it is described activation refer to make under sonic oscillation QD1 and QD2 respectively with 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide
Hydrochloric acid reactant salt 3.95-4.05min and 14.95-15.05min will lead to quantum dot fluorescence intensity in addition in this time frame
It reduces；The reaction of degeneration (RD) refers to heats 10min for NAPT and SAPT at 95 DEG C respectively, then ice bath 10min；The coupling
Reaction refer to according to the ratio of NAPT:QD1 molar ratio 1:3.8-4.2 and SAPT:QD2 molar ratio 1:4.8-5.2 make NAPT and
SAPT respectively with after activation QD1 and QD2 be protected from light 2h, in this proportional region, the surface of QD1 and QD2 can be distinguished
It is sufficiently coupled by NAPT and SAPT；The excitation wavelength of described two composite fluorescence probe NAPT-QD1 and SAPT-QD2 are 370nm,
Launch wavelength is respectively 550 and 586nm；Quenching reaction refer to GO by pi-pi accumulation interact respectively with QD1 and QD2
Fluorescence resonance energy transfer system is constituted, the fluorescent quenching of QD1 and QD2 are made.
7. the user that a kind of streptomysin as described in claim 1 and neomycin remain rapid fluorescence detection reagent simultaneously more
Method, it is characterised in that carry out as steps described below:
(1) the 100 above-mentioned luciferase assay reagents of μ L are measured, 1 μ L streptomysin and neomycin mixed standard solution or sample solution is added,
30min or more is reacted, the quantum dot fluorescence that will lead to quenching lower than this time cannot be thus capable of sufficiently recovering, and influence accuracy in detection；
(2) solution after reaction is put into microplate reader and carries out fluoremetry.
8. application method as claimed in claim 7, which is characterized in that chain in the streptomysin and neomycin mixed standard solution
The concentration of mycin and neomycin is respectively 10,50,50,100,100,200,500,500,800,800,1000,1000 μ g/L.
9. application method as claimed in claim 7, which is characterized in that the sample solution is prepared as steps described below:
(1) 10mL blank milk is measured, i.e., various concentration streptomysin and new is added in the milk of no streptomysin and Detection of neomycin residues
The mixed standard solution of mycin；
(2) 2mL15% (V/V) trichloroacetic acid is added, sonic oscillation 20min, 12000r/min are centrifuged 10min, collect supernatant,
5mL is settled to deionized water.
10. application method as claimed in claim 7, which is characterized in that the fluorescence detection carried out in microplate reader according to
Following step carries out:
(1) the excitation wavelength 370nm of neomycin and streptomysin detection；Launch wavelength 550 and 586nm, corresponding fluorescence intensity are
F550nmAnd F586nm；Tuning wavelength 618 and 518nm, corresponding fluorescence intensity are F618nmAnd F518nm；Neomycin and streptomysin it is net
Analyzing signal is respectively Δ F1=F550nm-F618nm, Δ F2=F586nm-F518nm；
(2) the Δ F of each standard solution is measured1With Δ F2, to the neomycin concentration C in these measured values and each standard solutionNAnd
Streptomysin concentration CSIt is returned using origin software, the standard curve Δ F of neomycin and streptomysin can be obtained1=A1×CN+
B1, Δ F2=A2×CS+B2, wherein A1、A2And B1、B2The slope and intercept respectively provided in regression process；
(3) the Δ F of sample solution1With Δ F2, respective calibration curve equation is substituted into, neomycin and streptomysin in sample are calculated
Concentration.
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