CN106706591A - Method for recognizing quantitative chiral amino acid by using reversible nano porphyrin fluorescence sensor - Google Patents
Method for recognizing quantitative chiral amino acid by using reversible nano porphyrin fluorescence sensor Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention discloses a method for recognizing quantitative chiral amino acid by using a reversible nano porphyrin fluorescence sensor, and belongs to the technical field of nano material preparation and chemical analysis detection. The reversible nano porphyrin fluorescence sensor for specifically recognizing quantitative proline, lysine and serine chirality adopts a CdTe quantum dot as a fluorescence probe, and self-assembled porphyrin prepared from a tetra-(4-pyridyl) zinc porphyrin tetrahydrofuran solution and cetyl trimethyl ammonium bromide (CTAB) as a fluorescence quencher, and with the specific combination of the florescence probe and the fluorescence quencher, an openable/closeable nano porphyrin fluorescence sensor can be prepared. The reversible (opening-closing-opening) nano porphyrin fluorescence sensor can be prepared under the action of the openable/closeable nano porphyrin fluorescence sensor with the chiral proline, lysine and serine. Compared with a conventional chromatographic method for recognizing amino acid chirality by separating, the method disclosed by the invention has multiple advantages.
Description
Technical field
Prepared the invention belongs to nano material and chemical analysis detection technique field, and in particular to a kind of reversible nanometer
The controllable preparation of porphyrin fluorescence sensor and its method for highly sensitive detection chiral amino acid.
Background technology
Chirality is a kind of critical nature that amino acid, sugar and heterocycle etc. construct living matter primitive molecule.Chiral molecules pair
The difference for reflecting micro-space configuration between isomers frequently can lead to the huge difference of its macroscopical physiological and pharmacological effect in vivo
It is different.Therefore, the chiral Recognition research of chiral material and large biological molecule is carried out, mechanism of action and rule to disclosing biomolecule
It is significant.Proline, lysine and serine play important regulation and make in biological physiology and pathologic process
With.Proline pharmaceutically can be used for the supplement of malnutrition, hypoproteinosis, gastrointestinal disease, scald and postoperative protein
Deng.Lysine is mainly used as feed addictive, food additive and pharmacy.Serine and fat and aliphatic acid metabolic and
The growth of muscle is relevant.Research shows that serine contributes to the generation of immunoglobulin and antibody, helps to maintain siberian crabapple
System.The physiological and pharmacological effect that their L-type and the different structure of D types are produced in vivo also has greatest differences.Therefore study
Effective proline, lysine and serine chiral Recognition and the detection technique of quantitative analysis, in chemistry, biology and medicine
Field is all extremely important.Due to nanometer porphyrin sensors have it is simple to operate, radiationless, high sensitivity and high stability it is excellent
Gesture, becomes the ideal chose of quantitative judge proline, lysine and serine.Conventional method has chromatography, electrochemistry side
Method and electron microscope technique etc., these methods have sensitivity high or the features such as strong separating power, but there is also some simultaneously not
Foot, is such as difficult to avoid that influence, the sample preparation preparation of complexity, detection time that derivative reagent recognize to amino acid chiral are long.
Therefore a kind of method of amino acid chiral identification and quantitative analysis of quick, high sensitivity and selectivity is studied to amino acid
Biological action has important very important meaning.
The present invention is the defect for overcoming existing method, there is provided a kind of new is known based on reversible nanometer porphyrin fluorescence sensor
Other quantitative determination chiral amino acid method.
The content of the invention
An object of the present invention provides a kind of gentle reversible nanometer porphyrin fluorescence of simple, reaction condition for preparing and passes
Sensor controllable method for preparing；The second purpose be to provide a kind of sensitivity it is high, selectivity it is good, based on fluorescence On-Off-On mode method
Fast quantification identification proline, lysine and the chiral reversible nanometer porphyrin fluorescence sensor of serine.
The quantitative proline of specific recognition, lysine and the chiral reversible nanometer porphyrin fluorescence of serine that the present invention is used
Sensor, using CdTe quantum as fluorescence probe, four-(4- pyridine radicals) zinc protoporphyrin tetrahydrofuran solution and cetyl three
The self-assembled nanometer porphyrin that methyl bromide ammonium (CTAB) is prepared is fluorescence quencher, and both specific bindings are switched
Nanometer porphyrin fluorescence sensor.Switch nanometer porphyrin fluorescence sensor is obtained with the effect of chiral proline, lysine and serine
Reversible (On-Off-On) nanometer porphyrin fluorescence sensor.
The technical scheme that solve problem of the present invention is taken is, quantitative judge chiral proline, lysine and serine are received
The preparation method of rice porphyrin fluorescence sensor, comprises the following steps：
(1) dichloride cadmium and N-acetyl-L-cysteine are dissolved in ultra-pure water, are stirred 15 minutes under normal temperature, normal pressure
PH value of solution is adjusted to 8.00 with sodium hydroxide solution afterwards, then inflated with nitrogen ice bath is stirred 20 minutes；Add sodium tellurite, stirring 15
Minute；Sodium borohydride is added, is stirred 15 minutes；Finally this solution is put into reactor, 50 are reacted in 200 DEG C of baking oven
Minute, obtain the feux rouges CdTe quantum fluorescence probe that launch wavelength is 641nm；
(2) four-(4- pyridine radicals) zinc protoporphyrins are dissolved in tetrahydrofuran solution, to cetyl trimethylammonium bromide
(CTAB) in the aqueous solution add four-(4- pyridine radicals) zinc protoporphyrin tetrahydrofuran solutions, normal temperature, atmospheric agitation 10 minutes, solution by
Muddiness becomes clarification, and reaction stops, and obtains four-(4- pyridine radicals) zinc protoporphyrin self-assembly for nanosphere solution；
(3) four-(4- pyridine radicals) zinc protoporphyrin self-assembly for nanosphere solution are added in CdTe quantum fluorescence probe, then
The Tris-HCl cushioning liquid of pH=4.02, four-(4- pyridine radicals) zinc protoporphyrin self-assembly for nanosphere solution is added to turn by electronics
Move and FRET effect, be quenched quantum dot fluorescence, the compound obtained by specific binding, there is provided quantum dot
One state of " Turn-off "；
(4) quantum dot fluorescence that step (3) is obtained is added suitably to be quenched on the D types of various concentrations scope and L-type amino acid
Compound in, quantum dot fluorescence recovers, and it is obvious poor that D types and L-type amino acid cause the phenomenon that quantum dot fluorescence recovers to produce
It is different, realize identification in reversible nanometer porphyrin fluorescence sensing modes assistant's acidic amino acid with it is quantitative；So as to obtain it is reversible (open-
On/off) nanometer porphyrin fluorescence sensor.
Or directly by step (3) and (4) merging：By synthesis in the D types and L-type amino acid of various concentrations scope, step (2)
Four-(4- pyridine radicals) zinc protoporphyrin self assembly solution, PH=4.02 Tris-HCl cushioning liquid mixing, stand 5 minutes；Again plus
Enter the CdTe quantum of step (1) synthesis, fluorescence spectrometry is carried out at 540-720nm, measure its spectrum after 5 minutes.
Further preferably：
Dichloride cadmium, N-acetyl-L-cysteine, the ratio of the amount of the material of sodium tellurite are in the present invention：1.0:(1.2
~1.5):0.2, general step (1) CdTe quantum fluorescence probe launch wavelength is 620~640nm；
Four in step (2) of the present invention-(4- pyridine radicals) zinc protoporphyrin and cetyl trimethylammonium bromide be in mixed solution
The amount ratio of material is 1:(1.3~1.5)；
Four-(4- pyridine radicals) zinc protoporphyrin is measured with CdTe in step (3) four-(4- pyridine radicals) zinc protoporphyrin nanosphere in the present invention
The amount ratio of the material of son point solution is 40~42:1；
In the present invention four in step (3) mixed solution-concentration of (4- pyridine radicals) zinc protoporphyrin nanosphere, CdTe quantum be dense
Degree is respectively 8.0 × 10-9-1.28×10-7mol/L、5.8×10-9During mol/L, four-(4- pyridine radicals) zinc protoporphyrin nanosphere with
The fluorescence intensity of CdTe quantum is into good linear relationship.
Further preferably：Reversible nanometer porphyrin fluorescence sensor of the invention is by quantum dot and nanometer porphyrin specificity
With reference to the compound for obtaining.Absorbing wavelength is down to 365 by 641nm red shifts to 649nm, fluorescence intensity by 851.
The D types and L-type amino acid of reversible nanometer porphyrin fluorescence sensor quantitative judge of the invention are respectively D/L- dried meat ammonia
Acid, D/L- lysines, D/L- serines.
Reversible nanometer porphyrin fluorescence transducer sensitivity of the invention is high.The fluorescence intensity of CdTe quantum fluorescence probe with
The increase for four-(4- pyridine radicals) zinc protoporphyrin self assembly solution gradually weakens, it might even be possible to be quenched on earth, as long as the present invention is carried out
Part is quenched or quenching (preferably in linear relation part range), can realize the qualitative and quantitative detection of step (4) completely；
Four-(4- pyridine radicals) zinc protoporphyrin nanosphere concentration (1.46 × 10-9-1.28×10-7Mol/L) with CdTe quantum (2.9 × 10- 8Mol/L fluorescence intensity) is into good linear relationship；The D types and the fluorescence of L-type amino acid recognized in certain scope are strong
Degree has linear relationship.
Reversible nanometer porphyrin fluorescence sensor quantitative judge D-/L- proline of the invention, D-/L- lysines and D-/L-
Serine ability is strong.Concentration is from 1.0 × 10-10Mol/L to 5.0 × 10-6The L-PROLINE of mol/L, 1B and D-Ser
Being combined rear fluorescence intensity with reversible nanometer porphyrin sensors does not have linear relationship.D-PROLINE (1.0 × 10-9-1.5×10- 7Mol/L), D-Lys (1.0 × 10-9-1.5×10-8) and Serine (1.0 × 10 mol/L-9-5.0×10-9Mol/L) with
Fluorescence intensity after reversible nanometer porphyrin fluorescence sensor is combined strengthens as the concentration of amino acid increases, and into good line
Sexual intercourse.Linearly dependent coefficient can be respectively 0.9984,0.9952,0.9931.Due to its binding ability be better than nanometer porphyrin with
Weak electrostatic interaction between quantum dot, adds CdTe quantum reaction a period of time, nanometer porphyrin and CdTe quantum
With reference to dying down, fluorescence recovers.D types and L-type amino acid cause the phenomenon that quantum dot fluorescence recovers to produce obvious difference, realize
Reversible nanometer porphyrin fluorescence sensing modes assistant's acidic amino acid identification with it is quantitative.So as to obtain reversible (On-Off-On) nanometer
Porphyrin fluorescence sensor；So step (3) and (4) merge and can separately obtain identical effect.
Reversible nanometer porphyrin sensors good stability of the invention.The reversible nanometer porphyrin fluorescence sensor is 1.0 × 10- 6Mol/L ions (KCl, Na2SO4、CaCl2、ZnCl2), 1 μ g/mL bio-matrixes it is (human serum albumins, bovine serum albumin(BSA), thin
Born of the same parents' nutrient solution, calf thymus DNA) and 0.1 μ g/mL mixing interference in the case of, with proline, lysine and serine act on it is glimmering
The intensity that light recovers is almost unchanged.
Reversible nanometer porphyrin fluorescence sensor of the invention is to proline, lysine and serine fast response time.It is reversible
After adding proline, lysine and serine in nanometer porphin fluorescent optical sensor, the fast quick-recovery of fluorescence reaches most stationary value in 5 minutes.
Method of the present invention, has many excellent compared to tradition in the method that chromatogram hair method separates identification amino acid chiral
Gesture, including prepare simple, reaction condition gently, the chiral Recognition and quantitation capabilities to proline, lysine and serine are strong, clever
Sensitivity is high, strong antijamming capability, fast response time, and this nanometer of porphyrin fluorescence sensor has in biochemistry, medicine and other fields
Actual application value.
Brief description of the drawings
Fig. 1 is the controllable method for preparing and its Gao Ling of reversible of the present invention (On-Off-On) nanometer porphyrin fluorescence sensor
The method schematic diagram of quick detection chiral amino acid.
Fig. 2 is the ultraviolet of four-(4- pyridine radicals) zinc protoporphyrin self assembly solution in reversible nanometer porphyrin sensors of the invention
Visible light, abscissa is wavelength, and ordinate is absorbance.
Fig. 3 is the transmission of four-(4- pyridine radicals) zinc protoporphyrin self assembly solution in reversible nanometer porphyrin sensors of the invention
Formula electron microscope picture, it is nanosphere.
Fig. 4 is CdTe quantum and four-(4- pyridine radicals) zinc protoporphyrin self assemblies in reversible nanometer porphyrin sensors of the invention
Solution specifically binds forward and backward fluorescence spectra, and abscissa is wavelength, and ordinate is fluorescence intensity.
Fig. 5 is the sensitivity of reversible nanometer porphyrin sensors of the invention.Four-(4- pyridine radicals) zinc protoporphyrin self assembly solution
(1.46×10-9-1.28×10-7Mol/L it is) glimmering after Tris-HCl cushioning liquid (PH=4.02) effect with CdTe quantum
Light spectrogram, abscissa is wavelength, and ordinate is fluorescence intensity.
Fig. 6 is reversible nanometer porphyrin sensors of the invention and various concentrations D-PROLINE (1.0 × 10-9-1.5×10- 7Mol/L the fluorescence after) acting on recovers spectrum, and abscissa is wavelength, and ordinate is fluorescence intensity.
Fig. 7 is reversible nanometer porphyrin sensors of the invention and various concentrations L-PROLINE (1.0 × 10-10-5.0×10- 6Mol/L the fluorescence after) acting on recovers spectrum, and abscissa is wavelength, and ordinate is fluorescence intensity.
Fig. 8 is reversible nanometer porphyrin sensors of the invention and various concentrations D-Lys (1.0 × 10-9-1.5×10- 8Mol/L the fluorescence after) acting on recovers spectrum, and abscissa is wavelength, and ordinate is fluorescence intensity.
Fig. 9 is reversible nanometer porphyrin sensors of the invention and various concentrations 1B (1.0 × 10-10-5.0×10- 6Mol/L the fluorescence after) acting on recovers spectrum, and abscissa is wavelength, and ordinate is fluorescence intensity.
Figure 10 is reversible nanometer porphyrin sensors of the invention and various concentrations Serine (1.0 × 10-9-5.0×10- 9Mol/L the fluorescence after) acting on recovers spectrum, and abscissa is wavelength, and ordinate is fluorescence intensity.
Figure 11 is reversible nanometer porphyrin sensors of the invention and various concentrations D-Ser (1.0 × 10-10-5.0×10- 6Mol/L the fluorescence after) acting on recovers spectrum, and abscissa is wavelength, and ordinate is fluorescence intensity.
Figure 12 is linear correlation figure, horizontal seat after reversible nanometer porphyrin sensors of the invention are acted on various concentrations D-PROLINE
The concentration of D-PROLINE is designated as, ordinate is fluorescence recovery strength (F2) and CdTe quantum raw florescent intensity (F0) ratio.
Figure 13 is linear correlation figure, horizontal seat after reversible nanometer porphyrin sensors of the invention are acted on various concentrations D-Lys
The concentration of D-Lys is designated as, ordinate is the ratio of fluorescence recovery strength and CdTe quantum raw florescent intensity.
Figure 14 is linear correlation figure, horizontal seat after reversible nanometer porphyrin sensors of the invention are acted on various concentrations Serine
The concentration of Serine is designated as, ordinate is the ratio of fluorescence recovery strength and CdTe quantum raw florescent intensity.
Figure 15 is the stability of reversible nanometer porphyrin sensors of the invention.Reversible nanometer porphyrin sensors exist with D-PROLINE
Ca2+、Zn2+、SO4 2-, human serum albumins (HSA), bovine serum albumin(BSA) (BSA), cell culture fluid (CCF), calf thymus DNA
With the stability after effect in the case of mixing interference (Mixture).Abscissa is added interfering material, and ordinate is vertical
Coordinate is fluorescence recovery strength (F2) and CdTe quantum raw florescent intensity (F0) ratio.
Figure 16 is the stability of reversible nanometer porphyrin sensors of the invention.Reversible nanometer porphyrin sensors exist with D-Lys
Ca2+、Zn2+、SO4 2-, human serum albumins (HSA), bovine serum albumin(BSA) (BSA), cell culture fluid (CCF), calf thymus DNA
With the stability after effect in the case of mixing interference (Mixture).Abscissa is added interfering material, and ordinate is vertical
Coordinate is fluorescence recovery strength (F2) and CdTe quantum raw florescent intensity (F0) ratio.
Figure 17 is the stability of reversible nanometer porphyrin sensors of the invention.Reversible nanometer porphyrin sensors exist with Serine
Ca2+、Zn2+、SO4 2-, human serum albumins (HSA), bovine serum albumin(BSA) (BSA), cell culture fluid (CCF), calf thymus DNA
With the stability after effect in the case of mixing interference (Mixture).Abscissa is added interfering material, and ordinate is glimmering
Light recovery strength (F2) and CdTe quantum raw florescent intensity (F0) ratio.
Specific embodiment
Applicant will the present invention is described in further detail in conjunction with specific embodiments below, so that the skill of this area
Art personnel are more clearly understood from the present invention.But herein below should not be understood as that claims of the present invention is claimed
The limitation of scope.
Embodiment：
It is pure that chemical reagent and solvent used in embodiment are analysis.The stirring uses magnetic stirrer side
Formula.Described fluorescence spectrometry condition is launch wavelength 540-720nm, and excitation wavelength is 380nm, and slit width is 10-
15nm。
Embodiment 1：Identification and quantitative analysis of the reversible nanometer porphyrin fluorescence sensor to Proline, methods described are shown
It is intended to such as 1, step is as follows：
(1) synthesis of CdTe quantum fluorescence probe
Dichloride cadmium (0.1142g, 12.5mM) and N-acetyl-L-cysteine (0.0979g, 15mM) are dissolved in into 40mL to surpass
In pure water, solution PH is adjusted to 8.00 with sodium hydroxide solution after being stirred 15 minutes under normal temperature, normal pressure, then inflated with nitrogen ice bath
Stirring 20 minutes.Sodium tellurite (0.0216g, 2.5mM) is added, is stirred 15 minutes；Add sodium borohydride (0.0113g,
7.5mM), stir 15 minutes.Finally this solution is put into reactor, is reacted 50 minutes in 200 DEG C of baking oven.It is cooled to room
Temperature, obtains 2.9 × 10-7Mol/LCdTe quantum dot fluorescence probes.
The synthesis of (2) four-(4- pyridine radicals) zinc protoporphyrin self assembly solution
Appropriate four-(4- pyridine radicals) zinc protoporphyrins are dissolved in tetrahydrofuran solution, it is 1.46 × 10 to obtain concentration-3mol/L
Four-(4- pyridine radicals) zinc protoporphyrin tetrahydrofuran solutions, its ultraviolet spectrogram such as Fig. 2.By cetyl trimethyl bromination
(0.0183g) is dissolved in the 10mL aqueous solution, adds the zinc protoporphyrin tetrahydrofuran solutions of 240 μ L tetra--(4- pyridine radicals), normal temperature and pressure to stir
Mix 10 minutes, solution is become by muddiness to be clarified, and reaction stops.Obtain 3.42 × 10-5Mol/L tetra--(4- pyridine radicals) zinc protoporphyrin is from group
Dress solution, its ultraviolet spectrogram such as Fig. 2.Its transmission electron microscope characterizes the nanosphere for being shown as particle diameter 40nm or so, such as
Fig. 3.
(3) preparation of nanometer porphyrin fluorescence sensor is switched
200 μ L2.9 × 10 are added in 1.5mL cuvettes-8The CdTe quantum and 800 μ LPH of mol/L steps (1) synthesis
=4.02 Tris-HCl cushioning liquid, carries out fluorescence spectrometry at 540-720nm, fluorescence intensity is obtained at 641nm and is
851 peak, such as Fig. 4.200 μ L2.9 × 10 are added in 1.5mL cuvettes-8Mol/L steps (1) synthesis CdTe quantum and
70μL3.42×10-6Four-(4- pyridine radicals) zinc protoporphyrin self assembly solution of synthesis, add 730 μ LPH in mol/L steps (2)
=4.02 Tris-HCl cushioning liquid, after mixing 5 minutes, carries out fluorescence spectrometry at 540-720nm, at 649nm
To the peak that fluorescence intensity is 365, such as Fig. 4.
(4) identification and quantitative analysis of the reversible nanometer porphyrin fluorescence sensor to D-/L- proline
The 100 μ LD-/L- proline aqueous solution, 70 μ L3.42 × 10 are added in 1.5mL cuvettes-6In mol/L steps (2)
Four-(4- pyridine radicals) zinc protoporphyrin self assembly solution of synthesis and the Tris-HCl cushioning liquid of 630 μ LPH=4.02, stand 5 points
Clock.Add 200 μ L2.9 × 10-8The CdTe quantum of mol/L steps (1) synthesis, carries out fluorescence spectrum at 540-720nm
Determine, measure its spectrum after 5 minutes.D-PROLINE (1.00 × 10-9-1.5×10-7Mol/L) sensed with nanometer porphyrin fluorescence
Fluorescence intensity after device is combined strengthens, such as Fig. 6 as the concentration of amino acid increases, and linearly dependent coefficient is 0.9984, is such as schemed
12.L-PROLINE (1.00 × 10-10-5.0×10-6Mol/L) rear fluorescence intensity is combined with nanometer porphyrin sensors not close linearly
System, such as Fig. 7.100 μ LD- proline, 100 μ L interfering materials, 70 μ L3.42 × 10 are added in 1.5mL cuvettes-6Mol/L is walked
Suddenly four-(4- pyridine radicals) the zinc protoporphyrin self assembly solution and the Tris-HCl cushioning liquid of 530 μ LpH=4.02 for synthesizing in (2),
Stand 5 minutes.Add 200 μ L2.9 × 10-8The CdTe quantum of mol/L steps (1) synthesis, uses fluorescence at 540-720nm
Spectroscopic assay, measures its spectrum after 5 minutes, and fluorescence recovers the influence of almost interference-free factor, shows very strong anti-dry
Disturb ability, such as Figure 15.
Embodiment 2：Reversible nanometer porphyrin fluorescence the sensor identification and quantitative analysis chiral to lysine, methods described are shown
It is intended to such as 1, step is as follows：
(1) synthesis of CdTe quantum fluorescence probe
Method using step (1) in embodiment 1 synthesizes CdTe quantum fluorescence probe.
The synthesis of (2) four-(4- pyridine radicals) zinc protoporphyrin self assembly solution
Method using step (2) in embodiment 1 synthesizes four-(4- pyridine radicals) zinc protoporphyrin self assembly solution.
(3) preparation of nanometer porphyrin fluorescence sensor is switched
Nanometer porphyrin fluorescence sensor is prepared using the method for step (3) in embodiment 1.
(4) identification and quantitative analysis of the reversible nanometer porphyrin fluorescence sensor to D-/L- lysines
100 μ LD-/L- lysine solutions, 70 μ L3.42 × 10 are added in 1.5mL cuvettes-6In mol/L steps (2)
Four-(4- pyridine radicals) zinc protoporphyrin self assembly solution of synthesis and the Tris-HCl cushioning liquid of 630 μ LPH=4.02, stand 5 points
Clock.Add 200 μ L2.9 × 10-8The CdTe quantum of mol/L steps (1) synthesis, carries out fluorescence spectrum at 540-720nm
Determine, measure its spectrum after 5 minutes.D-Lys (1.00 × 10-9-1.50×10-8Mol/L) passed with nanometer porphyrin fluorescence
Fluorescence intensity after sensor is combined strengthens, such as Fig. 8 as the concentration of amino acid increases, and linearly dependent coefficient is 0.9952 as schemed
13.1B (1.00 × 10-10-5.0×10-6Mol/L) rear fluorescence intensity is combined with nanometer porphyrin sensors not close linearly
System, such as Fig. 9.100 μ LD- lysines, 100 μ L interfering materials, 70 μ L3.42 × 10 are added in 1.5mL cuvettes-6Mol/L is walked
Suddenly four-(4- pyridine radicals) the zinc protoporphyrin self assembly solution and the Tris-HCl cushioning liquid of 530 μ LPH=4.02 for synthesizing in (2),
Stand 5 minutes.Add 200 μ L2.9 × 10-8The CdTe quantum of mol/L steps (1) synthesis, uses fluorescence at 540-720nm
Spectroscopic assay, measures its spectrum after 5 minutes, and fluorescence recovers the influence of almost interference-free factor, shows very strong anti-dry
Disturb ability, such as Figure 16.
Embodiment 3：Reversible nanometer porphyrin fluorescence the sensor identification and quantitative analysis chiral to serine, methods described are shown
It is intended to such as 1, step is as follows：
(1) synthesis of CdTe quantum fluorescence probe
Method using step (1) in embodiment 1 synthesizes CdTe quantum fluorescence probe.
The synthesis of (2) four-(4- pyridine radicals) zinc protoporphyrin self assembly solution
Method using step (2) in embodiment 1 synthesizes four-(4- pyridine radicals) zinc protoporphyrin self assembly solution.
(3) preparation of nanometer porphyrin fluorescence sensor is switched
Nanometer porphyrin fluorescence sensor is prepared using the method for step (3) in embodiment 1.
(4) identification and quantitative analysis of the reversible nanometer porphyrin fluorescence sensor to D-/L- serines
The 100 μ LD-/L- serine aqueous solution, 70 μ L3.42 × 10 are added in 1.5mL cuvettes-6In mol/L steps (2)
Four-(4- pyridine radicals) zinc protoporphyrin self assembly solution of synthesis and the Tris-HCl cushioning liquid of 630 μ LPH=4.02, stand 5 points
Clock.Add 200 μ L2.9 × 10-8The CdTe quantum of mol/L steps (1) synthesis, carries out fluorescence spectrum at 540-720nm
Determine, measure its spectrum after 5 minutes.Serine (1.00 × 10-9-5.00×10-9Mol/L) passed with nanometer porphyrin fluorescence
Fluorescence intensity after sensor is combined strengthens, such as Figure 10 as the concentration of amino acid increases, and linearly dependent coefficient is 0.9931, such as
Figure 14.D-Ser (1.00 × 10-10-5.0×10-6Mol/L rear fluorescence intensity) is combined with nanometer porphyrin sensors without linear
Relation, such as Figure 11.100 μ LL- serines, 100 μ L interfering materials, 70 μ L3.42 × 10 are added in 1.5mL cuvettes-6mol/L
The Tris-HCl bufferings of four-(4- pyridine radicals) zinc protoporphyrin self assembly solution of synthesis and 530 μ LPH=4.02 are molten in step (2)
Liquid, stands 5 minutes.Add 200 μ L2.9 × 10-8The CdTe quantum of mol/L steps (1) synthesis, uses at 540-720nm
Fluorescence spectrometry, measures its spectrum after 5 minutes, and fluorescence recovers the influence of almost interference-free factor, shows very strong
Antijamming capability, such as Figure 17.
Claims (6)
1. a kind of reversible nanometer porphyrin fluorescence sensor recognition quantitative chiral amino acid method, it is characterised in that including following step
Suddenly：
(1) dichloride cadmium and N-acetyl-L-cysteine are dissolved in ultra-pure water, are used after being stirred 15 minutes under normal temperature, normal pressure
PH value of solution is adjusted to 8.00 by sodium hydroxide solution, and then inflated with nitrogen ice bath is stirred 20 minutes；Sodium tellurite is added, 15 points are stirred
Clock；Sodium borohydride is added, is stirred 15 minutes；Finally this solution is put into reactor, 50 points are reacted in 200 DEG C of baking oven
Clock, obtains the feux rouges CdTe quantum fluorescence probe that launch wavelength is 641nm；
(2) four-(4- pyridine radicals) zinc protoporphyrins are dissolved in tetrahydrofuran solution, to cetyl trimethylammonium bromide (CTAB) water
Four-(4- pyridine radicals) zinc protoporphyrin tetrahydrofuran solutions, normal temperature, atmospheric agitation 10 minutes, solution are added to become clear by muddiness in solution
Clearly, reaction stops, and obtains four-(4- pyridine radicals) zinc protoporphyrin self-assembly for nanosphere solution；
(3) four-(4- pyridine radicals) zinc protoporphyrin self-assembly for nanosphere solution are added in CdTe quantum fluorescence probe, are added
The Tris-HCl cushioning liquid of pH=4.02, four-(4- pyridine radicals) zinc protoporphyrin self-assembly for nanosphere solution by electro transfer and
FRET is acted on, and is quenched quantum dot fluorescence, the compound obtained by specific binding, there is provided quantum dot one
The state of " Turn-off "；
(4) by answering that the quantum dot fluorescence that the D types of various concentrations scope and L-type amino acid addition step (3) are obtained suitably is quenched
In compound, quantum dot fluorescence recovers, and D types and L-type amino acid cause the phenomenon that quantum dot fluorescence recovers to produce obvious difference, real
Showed identification in reversible nanometer porphyrin fluorescence sensing modes assistant's acidic amino acid with it is quantitative；So as to obtain reversible (On-Off-On)
Nanometer porphyrin fluorescence sensor；
Or directly by step (3) and (4) merging：By synthesis in the D types and L-type amino acid of various concentrations scope, step (2)
The Tris-HCl cushioning liquid mixing of four-(4- pyridine radicals) zinc protoporphyrin self assembly solution, PH=4.02, stands 5 minutes；Add
The CdTe quantum of step (1) synthesis, carries out fluorescence spectrometry at 540-720nm, measures its spectrum after 5 minutes.
2. according to a kind of reversible nanometer porphyrin fluorescence sensor recognition quantitative chiral amino acid method described in claim 1, its
It is characterised by, dichloride cadmium, N-acetyl-L-cysteine, the ratio of the amount of the material of sodium tellurite are：1.0:(1.2~1.5):
0.2。
3. according to a kind of reversible nanometer porphyrin fluorescence sensor recognition quantitative chiral amino acid method described in claim 1, its
It is characterised by, four in step (2)-(4- pyridine radicals) zinc protoporphyrin and cetyl trimethylammonium bromide material in mixed solution
Amount is than being 1:(1.3~1.5).
4. according to a kind of reversible nanometer porphyrin fluorescence sensor recognition quantitative chiral amino acid method described in claim 1, its
It is characterised by, four-(4- pyridine radicals) zinc protoporphyrins are molten with CdTe quantum in step (3) four-(4- pyridine radicals) zinc protoporphyrin nanosphere
The amount ratio of the material of liquid is 40~42:1.
5. according to a kind of reversible nanometer porphyrin fluorescence sensor recognition quantitative chiral amino acid method described in claim 1, its
It is characterised by, four in step (3) mixed solution-concentration of (4- pyridine radicals) zinc protoporphyrin nanosphere, CdTe quantum concentration difference
It is 8.0 × 10-9-1.28×10-7mol/L、5.8×10-9During mol/L, four-(4- pyridine radicals) zinc protoporphyrin nanosphere is measured with CdTe
The fluorescence intensity of son point is into good linear relationship.
6. according to a kind of reversible nanometer porphyrin fluorescence sensor recognition quantitative chiral amino acid method described in claim 1, its
It is characterised by, the D types and L-type amino acid of reversible nanometer porphyrin fluorescence sensor quantitative judge are respectively D/L- proline, D/L-
Lysine, D/L- serines.
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