CN105300942B - A kind of method of AMP content in detection calf serum de-protein injection - Google Patents
A kind of method of AMP content in detection calf serum de-protein injection Download PDFInfo
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Abstract
The invention discloses a kind of method for detecting AMP content in calf serum de-protein injection.The inventive method comprises the following steps：(1) intensity of fluorescence emission peak of the FRET probe at 526nm is determined, is denoted as F0；(2) calf serum de-protein injection to be measured is added into the FRET probe to be reacted；The intensity of fluorescence emission peak of the reacted system at 526nm is determined, is denoted as F；(3) the Stern Volmer equations according to formula (1), the content of AMP in the calf serum de-protein injection can be calculated；The calf serum de-protein injection to be measured is by pre-treatment step.Detection method is stable, and easy to operate, high sensitivity, is adapted to do micro tracking, higher than the specificity of chromatogram；Operation and requirement ratio mass spectrum to detection device and environment are lower simultaneously, are particularly suited for the detection to producing product in batches.
Description
Technical field
The present invention relates to a kind of method for detecting AMP content, and in particular to a kind of Deporteinnized calf serum injection
The method of AMP content, belongs to detection technique field in liquid.
Background technology
Deporteinnized calf serum medicine is a kind of biological medicament that calf serum is prepared through techniques such as deproteinized, concentration, ultrafiltration,
Contain many biological activities such as micromolecule polypeptide, nucleotides, oligosaccharide in such medicine.Research shows that such medicine can
Stimulate under anaerobic conditions and increase absorption and utilization of the body to glucose and oxygen, improve metabolic balance.It can be stimulated carefully
Born of the same parents' respiratory activity, there is protective effect to the inducing nerve cell under anaerobic environment.Meanwhile it also have suppress platelet aggregation and
Thrombotic effect, play survival ability of the protection cell in the environment of hypoxic-ischemic.
Clinical trial finds that Deporteinnized calf serum medicine can be used for treatment brain blood dyshaemia and nutrition disorder
Neurologic impairment caused by disease and the various brain insufficiency diseases shown, such as ischemic cerebral infarction, cerebral hemorrhage；
The medicine is also proved can be by stimulating the synthesis accelerating wound healing process of collagen, and it also has to radio-induced damage
Good therapeutic effect.It is additionally operable to auxiliary treatment disease of digestive system, has protective effect to hepatic injury.Research also shows such
Medicine can play a part of neuroprotection to treat the nervous system disease by increasing neuron and cynapse quantity, be controlled in auxiliary
Treat senile dementia and also achieve certain effect.
The pharmacological effect of Deporteinnized calf serum medicine and its biological activity are closely related, thus study its activity into
Part detection method, can be to further appreciate that its pharmacological activity provides scientific basis.One of active ingredient of such medicine is single
AMP (Adenosine monophosphate, abbreviation AMP), it is a kind of nucleosides found in ribonucleic acid (RNA)
Acid.It is a kind of ester of phosphoric acid and nucleosides adenosine, and is made up of phosphate functional group, pentose nucleic acid sugar and bases adenine.
FRET (Fluorescence Resonance Energy Transfer, FRET) system, it is
Refer to when two fluorescence molecules distance close to when (being less than 10nm) generation energy transfer phenomenon, one of them as energy donor,
Another is energy acceptor.FRET systems can significantly increase the fluorescent emission intensity of energy acceptor, therefore can be obvious
Improve the detectability of the fluorescence probe sensor based on the design of this principle.In recent years, the fluorescence resonance based on quantum dot
Energy transfer probe receives much concern, be widely used in the identification of small molecule, the detection of metal ion, the detection of large biological molecule,
Cell imaging and medical diagnosis on disease etc..
The content of the invention
It is an object of the invention to provide a kind of side for detecting AMP (AMP) in calf serum de-protein injection
Method, specifically detected using a kind of FRET probe part, the inventive method is improved by the pre-treatment to sample
To the selectivity of AMP, the interference of other impurities in sample is eliminated.
The method of AMP content in detection calf serum de-protein injection provided by the present invention, including it is as follows
Step：
(1) intensity of fluorescence emission peak of the FRET probe at 526nm is determined, is denoted as F0；
(2) calf serum de-protein injection to be measured is added into the FRET probe to carry out instead
Should；The intensity of fluorescence emission peak of the reacted system at 526nm is determined, is denoted as F；
(3) the Stern-Volmer equations according to formula (1), can calculate the calf serum de-protein injection
The content of middle AMP；
In formula (1), [Q] represents the molar concentration of AMP in calf serum de-protein injection to be measured, τ0Represent
In the mean fluorecence life-span of the FRET probe, it is 1.5 × 10-9S, kQFluorescent quenching speed constant is represented, is
1.2×1011L·mol-1·s-1, KDRepresent Stern-Volmer constants；
The energy donor and energy acceptor of the FRET probe are respectively the ZnS amounts of beta-cyclodextrin modified
Son point and trihydroxyflavone, the particle diameter of the ZnS quantum dot of the beta-cyclodextrin modified is 2~4nm；The fluorescence resonance energy turns
The pH value for moving probe is 4.5；
The calf serum de-protein injection to be measured is to carry out pre-treatment according to the method comprised the following steps：
1) the NaOH aqueous solution is added into the calf serum de-protein injection to be measured, and is cooked, by system
PH value be adjusted to 6.8~7.2；
2) activated carbon is included in into the system after step 1) processing to be adsorbed；
3) system after step 2) processing is filtered with microporous barrier；
4) SPE is carried out to the system after step 3) processing, eluant, eluent is methanol, collects eluent, i.e. completion pair
The pre-treatment of the calf serum de-protein injection.
In above-mentioned method, in step (2), the temperature of the reaction can be 20 DEG C~25 DEG C, and the time of the reaction can
For 10~20min.
In above-mentioned method, in step 1), the molar concentration of the NaOH aqueous solution can be 6mol/L, and the NaOH is water-soluble
The volume ratio of liquid and the calf serum de-protein injection to be measured can be 1：1；
Cooked under conditions of water-bath；The temperature of the cooking can be 59~61 DEG C, and the time can be 35~45min,
Such as 40min is cooked at 60 DEG C.
In above-mentioned method, in step 2), the quality addition of the activated carbon can be that the calf serum to be measured is gone
The 1.8%~2.0% of protein injection liquid quality, such as 2%.
In above-mentioned method, in step 3), the aperture of the microporous barrier can be 0.22 μm.
In above-mentioned method, in step 4), the solid absorbent of the SPE can be C18.
In above-mentioned method, in the FRET probe, the ZnS quantum dot of the beta-cyclodextrin modified
Molar concentration can be 5.0 × 10-5~7.5 × 10-5Mol/L, concretely 5.0 × 10-5Mol/L, the trihydroxyflavone rub
Your concentration can be 1.0 × 10-6~2.5 × 10-6Mol/L, concretely 2.5 × 10-6mol/L；
The mol ratio of the ZnS quantum dot of the beta-cyclodextrin modified and the trihydroxyflavone can be 20：1.
The ZnS quantum dot of beta-cyclodextrin modified used in the inventive method can be prepared as follows：
1) according to document (Freeman, R., Finder, T., Bahshi, L., Gill, R., Willner, I.,
Adv.Mater.2012,24(48),6416-6421.；Li-Yun Wang,Ling-Yu Dong,Luan Chen,Ya-Bing
Fan,Jing Wu,Xiang-Feng Wang and Meng-Xia Xie*New J.Chem.,2015,39(1),555-565)
In method prepare single -6- sulphur group-beta-cyclodextrin (single -6- sulfenyl-β-CD)；
2) zinc acetate, single -6- sulfenyl-β-CD and water are added in three-neck flask, stirring to dissolving；Again with 1.0mol/L's
The NaOH aqueous solution adjusts the pH of mixed solution to 10.0；At room temperature, N is led to215min is to remove air, then by mixture
It is heated to reflux.Under conditions of backflow, sodium sulfide solution is injected, and in N2Protection is lower to stir, then by solution in room temperature (25
DEG C) under aging 6h, obtain the ZnS quantum dot of beta-cyclodextrin modified.
The content of AMP is 10 × 10 in calf serum de-protein injection-5Mol/L~200 × 10-5mol/L
When, usable the inventive method is detected.
The inventive method is that the detection to AMP content is realized based on following mechanism：Trihydroxyflavone can enter
Enter into β-CD hydrophobic cavity, under excited state, the electronics that quantum dot occurs shifts to 3-hydroxyflavone alcohol, result in
Trihydroxyflavone emission peak at λ=526nm significantly increases；In the presence of AMP (AMP), it is with β-CD cavity bodies
Interior trihydroxyflavone combines, and hinders the generation of energy transfer, makes its fluorescence that being quenched for peak intensity occur；When will be to be measured
Product are added in fluorescence probe system, reacted, and the fluorescent intensity of system emission peak at λ=526nm, passes through after detection reaction
Stern-Volmer equations can obtain the content of AMP in product to be tested.
Detection method is stable, and easy to operate, high sensitivity, is adapted to do micro tracking, more exclusive than chromatogram
Property is higher；Operation and requirement ratio mass spectrum to detection device and environment are lower simultaneously, are particularly suited for producing product in batches
Detection.
Brief description of the drawings
Fig. 1 is that 3-HF and the β-CD of 80 μ L difference molar concentrations modification ZnS the QDs aqueous solution mix in the embodiment of the present invention 1
Fluorescent emission spectrogram after conjunction, wherein, from bottom to up, β-CD modify the ZnS QDs aqueous solution to the fluorescence emission peak at 526nm
Molar concentration is followed successively by 0,1.875 × 10-3、2.8125×10-3、3.75×10-3、5.625×10-3、9.375×10-3、1.5
×10-2With 2.8125 × 10-2mol/L。
Fig. 2 (a) is that the molar concentration of the AMP aqueous solution in the embodiment of the present invention 2 is followed successively by 10 × 10-5、25×10-5、50×
10-5、100×10-5、150×10-5With 200 × 10-5The fluorescence emission spectrogram of compound of reaction system during mol/L；According to Fig. 2 (b)
The linear regression curves that Stern-Volmer equations obtain.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Medicine and reagent used are as follows in following embodiments：Calf serum de-protein injection (10ml/ branch, Jinzhou letter difficult to understand
Medicine company Co., Ltd)；Beta-schardinger dextrin (β-CD, 98.0%, Beijing Suo Laibao Science and Technology Ltd)；Paratoluensulfonyl chloride
(98.0%, Tianjin great Mao chemical reagent factories, China)；Thiocarbamide (99.0%, Beijing Chemical Plant)；Trichloro ethylene (99.0%, Shan
Morning market Xi Long chemical industry Co., Ltd., Factory, China) zinc acetate dihydrate (Zn (Ac)2·2H2O, 99.0%) it is hydrated with vulcanized sodium nine
Thing (Na2S·9H2O, 98.0%) bought from Chinese medicines group chemical reagent Beijing Co., Ltd；(TCI (Shanghai) changes 3-hydroxyflavone
Into industrial development Co., Ltd)；Adenosine 5'- monophosphates (Aladdin Chemical Co., Ltd.)；Millipore ultra-pure waters (18.3M Ω
cm-1)。
Instrument is as follows in following embodiments：Fluorescence emission spectrum, FluoroMax-4fluorometer (HORIBA
JY, France)；Ultraviolet-visible absorption spectroscopy, SPECORD 200spectrophotometer (analytikjena, Germany)；It is red
External spectrum instrument, NEXUS 670FTIR spectrometer (Thermo Nicolet Co., the U.S.)；Transmission electron microscope,
Tecnai F20electron microscopy (FEI, Holland).
Embodiment 1, prepare FRET probe
First, the ZnS quantum dot of β-CD modifications is prepared
1) single -6- sulfenyl-β-CD are prepared：Single -6- sulfenyl-β-CD synthesis be according to document (Freeman, R.,
Finder,T.,Bahshi,L.,Gill,R.,Willner,I.,Adv.Mater.2012,24(48),6416-6421.；Li-
Yun Wang,Ling-Yu Dong,Luan Chen,Ya-Bing Fan,Jing Wu,Xiang-Feng Wang and Meng-
Xia Xie*New J.Chem., 2015,39 (1), 555-565) in the method for report prepared：At room temperature, by β-ring
Dextrin (60.0g, 52.8mmol) and water (471mL) are added in reaction flask, and stirring is in suspension；17.1mL NaOH is added dropwise
(5.67g, 142mmol) aqueous solution；Then the acetonitrile of paratoluensulfonyl chloride (8.67g, 45.6mmol) is added dropwise dropwise thereto
(25.8mL) solution；Terminating reaction after reaction 2h at room temperature, filters and removes sediment, and filtrate is stayed overnight in 4 DEG C；Filtrate is carried out
Filter, obtain white depositions three times with water recrystallization, pure white solid product β-CD-OTs are obtained after vacuum drying.
β-CD-OTs (4.0g, 3.04mmol) and thiocarbamide (4.0g, 52.4mmol) are added in reaction bulb, add methanol
(about 160ml) and water (40ml) mixed solvent, are then heated to reflux 48h by mixture, by rotary evaporation by reaction dissolvent
Remove, obtain white solid, and be further purified, recrystallize, obtain list -6- sulfenyl-β-CD.
2) ZnS quantum dot of β-CD modifications is prepared：By zinc acetate (0.11g, 0.5mmol), single -6- sulfenyl-β-CD
(1.0g, 0.88mmol) and water (50mL) are added in three-neck flask, stirring to dissolving；Use the 1.0mol/L NaOH aqueous solution will again
The pH of mixed solution is adjusted to 10.0；At room temperature, N is led to2Then mixture is heated to reflux by 15min with removing air
20min.Under conditions of backflow, vulcanized sodium (0.12g, 0.5mmol) aqueous solution is injected, and in N2The lower stirring 20min of protection, so
Afterwards by solution under room temperature (25 DEG C) aging 6h, form the ZnS quantum dot of β-CD modification.Add into obtained quantum dot solution
Enter ethanol, centrifuge 3 times, be dried in a vacuum, be stored in 4 DEG C of refrigerators.
In above-mentioned preparation process, by β-CD hydroxyl with after tolysulfonyl chlorosulfonation, its hydroxyl is changed with thiocarbamide
Cyclodextrin is linked on the surface of zinc sulphide (ZnS) quantum dot into sulfydryl, then by sulfydryl, to the ZnS quantum of β-CD modifications
Point and β-CD do infrared spectrogram respectively, it is found that both spectrograms are close, illustrate that β-CD are successfully modified in quantum dot surface.
Principal band is as follows：In 3375.4cm-1The bands of a spectrum at place come from O-H absorption of vibrations；2925.7cm-1The bands of a spectrum at place are corresponding to C-H's
Nonsymmetrical vibration υa(C-H) absorb；1158.0cm-1The strong band at place vibrates corresponding to C-O-C denaturation；In 1080.9 Hes
1028.2.cm-1The bands of a spectrum at place correspond respectively to C-C and C-O denaturation vibration.
The transmission electron microscope picture of the ZnS quantum dot of β-CD manufactured in the present embodiment modification shows, its particle diameter 2~4nm it
Between, therefore there is good dispersiveness in the solution.
2nd, FRET probe is prepared
Take 20 μ L 1.5 × 10-4The ethanol solution of mol/L trihydroxyflavones alcohol (3-HF) and the ZnS of 80 μ L β-CD modifications
(concentration is followed successively by 0,1.875 × 10 to QDs (ZnS quantum dot of the β-CD modifications) aqueous solution-3、2.8125×10-3、3.75×10-3、5.625×10-3、9.375×10-3、1.5×10-2With 2.8125 × 10-2Mol/L) it is added separately to 2.9mL pH4.5's
In PBS cushioning liquid, the cumulative volume of reaction solution is held constant at 3.0mL.After reacting 1min at 20 DEG C, it is glimmering that its is measured respectively
Light spectrum.Excitation wavelength is set to：300nm, launch wavelength scope：310~700nm.
Corresponding test result is as shown in figure 1, modify the ZnS QDs aqueous solution by measuring 3-HF and different amounts of β-CD
Mixed fluorescent emission spectrogram, learns as drawn a conclusion：With the increase of β-CD modification ZnS QDs addition, 3-HF exists
Fluorescent emission peak intensity at 526nm gradually strengthens, and when the addition of quantum dot reaches to a certain degree, the intensity of emission peak is protected
Hold constant, illustrate to have reached balance between them, form resonance energy transfer fluorescence probe.
In FRET probe manufactured in the present embodiment, trihydroxyflavone alcohol (3-HF) molecule is repaiied into β-CD
In the hydrophobic cavity body of beta cyclodextrin (β-CD) in the ZnS quantum dot of decorations, and then construct using ZnS quantum dot and be used as fluorescence
Energy donor, novel fluorescence probe of the trihydroxyflavone alcohol as energy acceptor, wherein, the ZnS quantum dot of the β-CD modifications
Particle diameter be 2~4nm.
In FRET probe manufactured in the present embodiment, 3-hydroxyflavone can enter β-CD hydrophobicity
In cavity, under excited state, the electronics of quantum dot occurs and is shifted to 3-hydroxyflavone alcohol, result in 3-hydroxyflavone λ=
Emission peak significantly increases at 526nm.In the presence of AMP (AMP), itself and the 3-hydroxyflavone in β-CD cavity bodies
With reference to hindering the generation of energy transfer, make its fluorescence that peak intensity occur and be quenched.The ZnS quantum dots of β-CD modifications
Ultraviolet absorption peak and 3-HF fluorescence emission peak between have it is overlapping, also illustrate resonance energy transfer can occur between the two
Phenomenon (Elmlinger MW, Kriebel M, Ziegler D.Neuromolecular Med2011Dec；13(4):266-
74.；Li-Yun Wang,Ling-Yu Dong,Luan Chen,Ya-Bing Fan,Jing Wu,Xiang-Feng).
Embodiment 2, utilize FRET probe (FRET fluorescence probes) detection AMP
It is used for detecting the feasible of AMP to study the FRET fluorescence probes formed between β-CD modification ZnS QDs and 3-HF
Property, it have studied influence of the various concentrations AMP aqueous solution to FRET fluorescence probe fluorescent emission intensities.
Take 20 μ L 3.75 × 10-4Mol/L 3-HF ethanol solution and 80 μ L 1.875 × 10-3Mol/L β-CD are repaiied
The ZnS QDs aqueous solution of decorations is added in 2.8mL pH 4.5 PBS cushioning liquid, and the cumulative volume of reaction solution is held constant at
3.0mL.After reacting 1min at 20 DEG C, being then separately added into the 100 μ L AMP aqueous solution thereto, (concentration is followed successively by 10 × 10-5、25×10-5、50×10-5、100×10-5、150×10-5With 200 × 10-5Mol/L), its fluorescence spectrum, fluorescence are measured respectively
Shown in spectrogram such as Fig. 2 (a).
Above-mentioned experimental data is calculated into (Xie, M X with Stern-Volmer equations (shown in formula (1))；Long,M；
Liu,Y；Qin,C；Wang,Y D.Biochim.Biophys.Acta 2006,1760：1184-1193.；Xie M X, Xu X
Y,Wang Y D.Biochim.Biophys.Acta 2005,1724,215-224.)：
Wherein, F0With the intensity of the fluorescence emission peak of FRET probe before and after F respectively addition AMP；[Q]
For AMP molar concentration；τ0For the mean fluorecence life-span of the FRET probe, τ0≈1.5×10-9s；KDFor
Stern-Volmer constants；kQFor fluorescent quenching speed constant.
According to linear regression curves such as Fig. 2 (b) that Stern-Volmer equations obtain, it can be learnt from Fig. 2 (b)：
Be presented good linear relationship between the two, linear regression coeffficient 0.996, equation of linear regression be Y=160.2 [Q]+
0.006；K can obtain by the slope of the linear regression curvesQAbout 1.2 × 1011L·mol-1·s-1, expand much larger than maximum dynamic
Dissipate quenching constant 2.0 × 1010L·mol-1·s-1, it is mainly that static state is quenched to the quenching mechanism of fluorescence probe to illustrate AMP.
Embodiment 3, the selectivity of detection method (exclusive PCR)
(1) the exclusive PCR effect of the purification step in detection method is investigated
Using the method for basic hydrolysis, charcoal absorption and C18 SPE column purifications to calf serum de-protein injection
Purified, with exclusive PCR；Because calf serum de-protein injection comparison of ingredients is complicated, Multiple components are included, it is therein
Some biomolecule are likely to produce quenching effect to fluorescence probe such as polypeptide, polysaccharide, nucleotides, ATP.
Above-mentioned purification step is：
Basic hydrolysis：The isometric 6mol/L NaOH aqueous solution is added into calf serum de-protein injection to be measured,
40min is boiled in 60 DEG C of water-baths, and pH value is transferred into 7.0.
Charcoal absorption：Activated carbon is added into the system after upper step process, by 0.22 μm of microporous barrier after 10min
Filtration, wherein, the addition of activated carbon is the 2% of calf serum de-protein injection quality.
C18 SPE column purifications：Use C18The solid-phase extraction column of filler is extracted, and is eluted with methanol, collects eluent.
Carry out mass spectral analysis by above-mentioned sample after purification, from mass spectrogram, it is purified after sample in except AMP
Outside, substantially without above-mentioned chaff interference.
(2) influence of the purification step to AMP contents in product to be tested in detection method is investigated
Four parts of 2mL calf serum de-protein injections (10mL/ branch, Jinzhou Aohong Pharmaceutical Co., Ltd's life are taken respectively
Production, lot number 20141011, AMP concentration are 11.2 × 10-5Mol/L), sample 1, sample 2, sample 3 and sample 4 are respectively labeled as,
It is handled as follows respectively：
A) sample 1 plus water constant volume are standby to 10ml.
B) 6mol/L NaOH aqueous solution 2mL is added into sample 2,60 DEG C of water-baths boil 40min, pH value is transferred into 7.0, fixed
Hold 10mL, it is standby.
C) sample 3 adds 40mg activated carbons on the basis of after method a) processing, by 0.22 μm of microporous barrier after 10min
Filtration, constant volume are standby to 10mL.
D) 6mol/L NaOH aqueous solution 2mL is added into sample 4,60 DEG C of water-baths boil 40min, pH value is transferred into 7.0；To
40mg activated carbons are wherein added, are filtered after 10min by 0.22 μm of microporous barrier；It is loaded to and 3g C is housed18The SPE of filler
Post, slowly eluted with 10mL methanol, collect eluent, constant volume (add water or be concentrated under reduced pressure) is standby to 10mL.
The AMP in sample 1- samples 4 is detected respectively：
1) FRET probe is prepared
Take 20 μ L 3.75 × 10-4Mol/L 3-HF ethanol solution and 80 μ L 1.875 × 10-3Mol/L β-CD are repaiied
The ZnS QDs aqueous solution of decorations is added in 2.8mL pH 4.5 PBS cushioning liquid, and the cumulative volume of reaction solution is held constant at
3.0mL。
The intensity of fluorescence emission peak of the FRET probe at 526nm is detected, is denoted as F0；
2) sample 1, sample 2, sample 3 and sample 4 are added into FRET probe respectively, it is anti-at 20 DEG C
After answering 1min, the intensity of fluorescence emission peak of the measurement system at 526nm, is denoted as F respectively.
3) the Stern-Volmer equations according to formula (1), can converse AMP in calf serum de-protein injection
Content, as a result as shown in table 1；
In formula (1), [Q] represents the molar concentration of AMP in calf serum de-protein injection to be measured, τ0Represent
The mean fluorecence life-span of the FRET probe, about 1.5 × 10-9S, kQFluorescent quenching speed constant is represented,
For 1.2 × 1011L·mol-1·s-1, KDRepresent Stern-Volmer constants.
AMP content in the sample 1- samples 4 of table 1
| Sample | AMP concentration (× 10-5mol/L) |
| 1 | 20.8 |
| 2 | 17.6 |
| 3 | 14.3 |
| 4 | 11.4 |
Each purified treatment can exclude some interference it can be seen from the data in table 1, make testing result closer
Actual value.
(3) consumption of the purification step to AMP in product to be tested in detection method is investigated
Compound concentration is 20 × 10-5Two parts of 2mL of mol/L adenosine 5'- monophosphates (AMP) are labeled as sample A and sample B, sample
Product A is standby to 10mL with water constant volume, and sample B is handled according to the method for above-mentioned sample 4.
Sample A and sample B are detected respectively according to above-mentioned detection method, AMP concentration is conversed by volume, as a result
As shown in table 2：
AMP content in the sample A of table 2 and sample B
| Sample | AMP concentration (× 10-5mol/L) |
| A | 18.2 |
| B | 17.8 |
Sample A and sample B testing result approach it can be seen from the data in table 2, show in detection method
Purified treatment step the AMP in sample is lost seldom, testing result is influenceed little.
Claims (10)
1. a kind of method for detecting AMP content in calf serum de-protein injection, comprises the following steps：
(1) intensity of fluorescence emission peak of the FRET probe at 526nm is determined, is denoted as F0；
(2) calf serum de-protein injection to be measured is added into the FRET probe to be reacted；Survey
The intensity of fixed fluorescence emission peak of the reacted system at 526nm, is denoted as F；
(3) the Stern-Volmer equations according to formula (1), can calculate single in the calf serum de-protein injection
The content of AMP；
<mrow>
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<mi>F</mi>
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</msub>
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<mi>k</mi>
<mi>Q</mi>
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<mi>&tau;</mi>
<mn>0</mn>
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<mi>Q</mi>
<mo>&rsqb;</mo>
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In formula (1), [Q] represents the molar concentration of AMP in calf serum de-protein injection to be measured, τ0Represent described glimmering
In the mean fluorecence life-span of photoresonance energy transfer probe, it is 1.5 × 10-9S, kQRepresent fluorescent quenching speed constant, be 1.2 ×
1011L·mol-1·s-1, KDRepresent Stern-Volmer constants；
The energy donor and energy acceptor of the FRET probe are respectively the ZnS quantum dot of beta-cyclodextrin modified
And trihydroxyflavone, the particle diameter of the ZnS quantum dot of the beta-cyclodextrin modified is 2~4nm；The FRET is visited
The pH value of pin is 4.5；
The calf serum de-protein injection to be measured is to carry out pre-treatment according to the method comprised the following steps：
A) the NaOH aqueous solution is added into the calf serum de-protein injection to be measured, and is cooked, by the pH of system
Value is adjusted to 6.8~7.2；
B) activated carbon is added into the system after step a) processing to be adsorbed；
C) system after step b) processing is filtered with microporous barrier；
D) SPE is carried out to the system after step c) processing, eluant, eluent is methanol, collects eluent, that is, completes to described
The pre-treatment of calf serum de-protein injection.
2. according to the method for claim 1, it is characterised in that：In step (2), the temperature of the reaction is 20 DEG C~25
DEG C, the time of the reaction is 10~20min.
3. method according to claim 1 or 2, it is characterised in that：In step a), the molar concentration of the NaOH aqueous solution
For 6mol/L, the volume ratio of the NaOH aqueous solution and the calf serum de-protein injection to be measured is 1：1；
Cooked under conditions of water-bath；The temperature of the cooking is 59~61 DEG C, and the time is 35~45min.
4. according to the method for claim 3, it is characterised in that：In step b), the quality addition of the activated carbon is institute
State the 1.8%~2.0% of calf serum de-protein injection quality to be measured.
5. according to the method for claim 4, it is characterised in that：In step c), the aperture of the microporous barrier is 0.22 μm.
6. according to the method for claim 5, it is characterised in that：In step d), the solid absorbent of the SPE is
C18。
7. according to the method for claim 6, it is characterised in that：In the FRET probe, the β-ring
The molar concentration of the ZnS quantum dot of dextrin modification is 5.0 × 10-5~7.5 × 10-5Mol/L, mole of the trihydroxyflavone
Concentration is 1.0 × 10-6~2.5 × 10-6Mol/L, the ZnS quantum dot of the beta-cyclodextrin modified and the trihydroxyflavone
Mol ratio is 20：1.
8. according to the method for claim 7, it is characterised in that：In the FRET probe, the β-ring
The molar concentration of the ZnS quantum dot of dextrin modification is 5.0 × 10-5Mol/L, the molar concentration of the trihydroxyflavone for 2.5 ×
10-6mol/L。
9. application of the FRET probe in AMP in detecting medicine；
The energy donor and energy acceptor of the FRET probe are respectively the ZnS quantum dot of beta-cyclodextrin modified
And trihydroxyflavone, the particle diameter of the ZnS quantum dot of the beta-cyclodextrin modified is 2~4nm；The FRET is visited
The pH value of pin is 4.5.
10. application according to claim 9, it is characterised in that：The medicine is calf serum de-protein injection.
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