CN110186891A - A kind of polypeptide fluorescent probe specifically binding copper ion and cysteine - Google Patents
A kind of polypeptide fluorescent probe specifically binding copper ion and cysteine Download PDFInfo
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Abstract
The invention discloses the polypeptide fluorescent probes of specific binding copper ion and cysteine.The amino acid sequence of the polypeptide is as shown in SEQ ID NO:1；Polypeptide fluorescent probe P is formed with the N-terminal that fluorescence radiation substance modifies the polypeptide, which can target in conjunction with Cu2+, have that selectivity is good, toxicity is low, detection limit is low, and prepare simple, easy to detect, provide a kind of efficient detection method for the biological detection with the copper ion in environmental sample；Probe P and Cu2+In conjunction with P-Cu probe is formed, which is capable of the identification Cys of specificity, and the quick detection for Cys in organism and environmental sample provides a kind of efficient detection method.
Description
Technical field
The invention belongs to technical field of analysis and detection, and in particular to it is a kind of specific binding copper ion and cysteine it is more
Peptide fluorescence probe.
Background technique
Cu2+It is indispensable small molecule in human body with Cys (cysteine), participates in a variety of physiological activities in vivo.
Cu2+For important roles such as gene expression, immunological regulation, hematopoiesis, suitable Cu2+The physiology course of body can be played
Facilitation, but work as intracorporal Cu2+It can assemble in liver when excessive and cause cirrhosis, liver ascites, and Alzheimer's disease, Tang Shi
The diseases such as syndrome are also related with the metabolic imbalance of internal copper.Cys is to biologies such as synthesis, the translation modifications of biological vivo protein
Function plays a crucial role.It will lead to hair bleaching falls off, hypoevolutism, muscle are powerless etc. when internal Cys deficiency
Symptom, but neurotoxic can be caused when internal Cys is excessive.Meanwhile Cu2+With Cys largely making in industry, manufacturing industry
With causing the Cu in environment2+It is polluted with Cys, these pollutants can enter human body with animals and plants and be enriched in vivo, to people's
Life and health brings significant damage.
Therefore simple and quick detection Cu is developed2+Medical diagnosis on disease, environmental monitoring, food and medicine are analyzed with the method for Cys
Etc. there is major and immediate significance.Shen etc. is designed using 1,8- naphthalimide as fluorogen has synthesized a novel small-molecule chemical
Sensor, this small-molecule fluorescent probe are very high to the selectivity of Cys, and the fluorescent quenching of probe after Cys is added, and add into system
Enter Cu2+Afterwards, Cu2+It is complexed with Cys, the fluorescence of probe restores.Liu et al. is developed using phenanthraquinone compound as fluorogen
Cu is detected in pure aquatic system2+With the small-molecule fluorescent probe of Cys, the strong paramagnetic quenching effect based on copper ion identifies Cu2+,
And the sulfydryl of Cys can be coordinated with copper ion, and copper ion is removed from probe molecule, be reached with this to Cys detection
Purpose.But the above-mentioned generally existing sensitivity of probe is low, toxicity is high, it is complicated to prepare, poor biocompatibility and detection limit for height etc.
Disadvantage.
Therefore, researching and developing one kind being capable of specific recognition Cu2+And Cys, and toxicity is low, the sensitive new polypeptide of detection
Fluorescence probe is particularly significant.And a kind of polypeptide fluorescent probe P that the present invention develops is in detection Cu2+When, not by other ion interferences,
Have the characteristics that selective height, good biocompatibility, preparation is simple, detection limit is lower, can be used for specific detection Cu2+, and
The probe and Cu2+The complex P-Cu probe of formation is capable of the detection Cys content of specificity.
Summary of the invention
The present invention provides a kind of polypeptide, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.
The present invention also provides a kind of aforementioned polypeptides to prepare the application in polypeptide fluorescent probe.
The present invention also provides a kind of specific recognition Cu2+Polypeptide fluorescent probe P, the polypeptide fluorescent probe P be by
What the N-terminal of luminescent substance modification to aforementioned polypeptides sequence was prepared.
Preferably, the luminescent substance is fluoresceins, rhodamine, Coumarins, quinolines, naphthalimide, mostly virtue
Any one of base vinyl.
Preferably, the luminescent substance is dansyl Cl.
The present invention also provides a kind of specific recognition Cu2+Polypeptide fluorescent probe P detection Cu2+Application in content,
The polypeptide fluorescent probe P and Cu2+It is coordinated and is combined with 1:1, the Cu2+Lowest detection be limited to 52nM, the Cu2+Detection
Method the following steps are included:
(1) to 10mM, 10 the preparation of standard curve: are added in the HEPES buffer solution of pH7.4-5The polypeptide fluorescence of mol/L is visited
Needle P adds the Cu that concentration is 0.0,0.25,0.5,0.75,1.0,1.25 μM2+, and Detection wavelength is the glimmering of solution at 540nm
Luminous intensity, with Cu2+Concentration is abscissa, and fluorescence intensity is ordinate, draws standard curve；
(2) Cu in sample2+The measurement of content: it to 10mM, is added in the HEPES buffer solution of pH7.4 certain density to be measured
Sample, and detect the fluorescence intensity of solution at 540nm；
(3) Cu in sample to be tested is calculated2+Content: by step (2) resulting fluorescence intensity level bring into step (1) preparation
In standard curve, Cu in sample to be tested is calculated2+Content.
The present invention also provides a kind of specific recognition Cu2+Polypeptide fluorescent probe P in preparing cell imaging reagent
Using.
The present invention also provides the polypeptide fluorescent probe P-Cu, the polypeptide fluorescent probe P- of a kind of specific recognition Cys
Cu is by aforementioned polypeptides fluorescence probe P and Cu2+It is coordinated with 1:1 and combines formation.
The present invention provides the polypeptide fluorescent probe P-Cu of specific recognition Cys a kind of to detect the application in Cys content,
The polypeptide fluorescent probe P-Cu is combined with Cys with 2:1 coordination, and the lowest detection of the Cys is limited to 43nM, the detection method
The following steps are included:
(1) to 10mM, 10 the preparation of standard curve: are added in the HEPES buffer solution of pH7.4-5The polypeptide fluorescence of mol/L is visited
Needle P-Cu adds the Cys that concentration is 0.0,0.2,0.4,0.6,0.8,1.0,1.2 μM, and Detection wavelength is solution at 515nm
Fluorescence intensity, using Cys concentration as abscissa, fluorescence intensity is ordinate, draw standard curve；
(2) in sample Cys content measurement: to 10mM, be added in the HEPES buffer solution of pH7.4 certain density to be measured
Sample, and detect the fluorescence intensity of solution at 515nm；
(3) it calculates the content of Cys in sample to be tested: bringing step (2) resulting fluorescence intensity level into step (1) preparation
In standard curve, the content of Cys in sample to be tested is calculated.
The present invention also provides the polypeptide fluorescent probe P-Cu of specific recognition Cys a kind of in preparing cell imaging reagent
Application.
The beneficial effects of the present invention are: 1. a kind of polypeptide provided by the invention, the polypeptide are directly synthesized by solid phase method, make
It is standby simple, it can be used for preparing polypeptide fluorescent probe；2. using the polypeptide fluorescent probe P of polypeptide preparation using polypeptide as primary structure list
Member has the advantages that good biocompatibility, toxicity are low；3. the polypeptide probe is to Cu2+There is preferable response, not by other same main groups
Metal ion disturbance, high specificity；4. the polypeptide probe and Cu2+The P-Cu probe of formation has preferable response to Cys, not by it
Its amino acid and S2-Ion interference, specificity are good.
Detailed description of the invention
The structure chart of Fig. 1 fluorescent polypeptide, wherein Fig. 1 a is the structure chart of polypeptide fluorescent probe P, and Fig. 1 b is P-Cu probe
Structure chart；
The cations recognition spectrogram of Fig. 2 fluorescent polypeptide probe P；
Fig. 3 fluorescent polypeptide probe P is to Cu2+The histogram of other metal ion disturbances when identification；
Fig. 4 fluorescent polypeptide probe P is to Cu2+Fluorescence titration tendency chart；
Fig. 5 fluorescent polypeptide probe P is to Cu2+Detection limit；
The amino acid of Fig. 6 P-Cu probe identifies spectrogram；
The histogram of other amino acid interference when Fig. 7 P-Cu probe identifies Cys；
Fluorescence titration tendency chart of Fig. 8 P-Cu probe to Cys；
Fig. 9 P-Cu probe limits the detection of Cys；
The different pH response diagrams of Figure 10 polypeptide fluorescent probe；
Figure 11 cell imaging figure, wherein a is the fluorescence field figure being added after 10 μM of P incubation 1h into HeLa cell, and b is it
Corresponding light field figure, c are its corresponding fluorescence field and light field stacking chart；D is to add after 10 μM of P is added into HeLa cell
10 μM of Cu2+Fluorescence field figure after being incubated for 1h, e are its corresponding light field figure, and f is its corresponding fluorescence field and light field stacking chart；g
For the fluorescence field figure after the Cys incubation 1h for adding 5 μM is added after 10 μM of P-Cu into HeLa cell, h is that its is corresponding bright
Field figure, j are its corresponding fluorescence field and light field stacking chart.
Specific embodiment
To be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, below with reference to
Specific embodiment, the present invention is further explained.Following embodiment is only used to illustrate the technical scheme of the present invention and not to limit it, to the greatest extent
Pipe is described the invention in detail referring to preferred embodiment, those skilled in the art should understand that, it can be to this
The technical solution of invention is modified or replaced equivalently, should all without departing from the objective and range of technical solution of the present invention
It is included within the scope of the claims of the present invention.
The preparation of 1 polypeptide of embodiment and polypeptide fluorescent probe P and polypeptide fluorescent probe P-Cu
1.1 preparation method
(1) 100mg Rink ammonia resin is added in the reactor with sieve plate, uses 5mL methylene chloride swellable resins；
By 20% piperidines/n,N-Dimethylformamide (DMF) solution removal N-terminal Fmoc protecting group, it is de- that chromogenic reaction detects protecting group
Except complete；
(2) 4eq N-terminal is dissolved with 4eq HOBt and 4eq HBTU with DMF by fmoc-protected amino acid, low-temperature activation
After 20min, 6eq DIEA is added dropwise into solution, is added in reactor after solution mixing, reacts 3hrs；
(3) after reaction, reaction solution by extracting out in reactor, washed 3 times by 5mL DMF and DCM respectively by resin.Colour developing is anti-
It is complete that amino acid condensation should be detected, resin handles 3 times, respectively 5min, 5min and 15min with 20% piperidines/DMF solution.Tree
Rouge is washed 3 times by 5mL DMF and DCM respectively, and it is complete that chromogenic reaction detects protecting group removing；
(4) step (2) and (3) are repeated, the polypeptide until synthesizing target sequence, the amino acid sequence of the polypeptide such as SEQ
Shown in ID NO:1.
(5) 4eq fluorescence radiation substance dansyl Cl is dissolved with 4eq HOBt and 4eq HBTU with DMF, low-temperature activation
After 20min, 6eq DIEA is added dropwise into solution, is added in reactor after solution mixing, reacts 6hrs.
(6) after reaction, reaction solution by extracting out in reactor, washed 3 times with 5mL DMF and DCM respectively by resin.Colour developing is anti-
Dansyl Cl condensation should be detected completely, resin is washed 3 times with DCM and methanol in turn respectively.Resin is drained, cutting liquid is added
(TFA:TIS: water=95:2.5:2.5) reacts 3hrs；
(7) step (6) resulting reaction solution is added in ice ether, precipitated polypeptide.Precipitating is collected by centrifugation, with a small amount of
TFA dissolution precipitating is added excessive ice ether and precipitates and be collected by centrifugation precipitating again, and precipitating obtains slightly after washing 2 times with ice ether
Peptide, thick peptide are purified to obtain pure peptide by reversed-phase liquid chromatography, then it is freeze-dried obtain polypeptide fluorescent probe P, structure is as shown in Figure 1a；
(8) by the resulting polypeptide fluorescent probe P and Cu of step (7)2+It is mixed with 1:1, forms polypeptide fluorescent probe P-Cu, knot
Structure is as shown in Figure 1 b.
1.2 result
According to the amino acid sequence of the polypeptide of above-mentioned steps preparation as shown in SEQ ID NO:1, the fluorescent polypeptide of preparation is visited
The structure of the P of needle is as shown in Figure 1a, and the structure chart of polypeptide fluorescent probe P-Cu is as shown in 1b.
2 polypeptide fluorescent probe P of embodiment is to Cu2+The special Journal of Sex Research of detection
The cation selective evaluation experimental of 2.1 polypeptide fluorescent probe P
In HEPES buffer solution (10mM, pH7.4), it is separately added into 10-5The polypeptide fluorescent probe P of mol/L and 10-5mol/
Metal cation (the Ag of L+, Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+),
Detect the fluorescence emission spectrum variation of solution.As a result as shown in Fig. 2, only Cu2+In the presence of, the fluorescence of probe P is real
Existing obvious quenching, other 15 metal ion species cannot make the fluorescence of polypeptide fluorescent probe P that quenching phenomenon occur, illustrate polypeptide
Answer fluorescence probe P that can identify Cu2+。
2.2 polypeptide fluorescent probe P are to Cu2+Specific detection research
In HEPES buffer solution (10mM, pH7.4), it is separately added into 10-5The polypeptide fluorescent probe P of mol/L and 10-5mol/
Various metal cation (the Ag of L+, Al3+, Ca2+, Cd2+, Co2+, Cr3+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+),
The fluorescence emission spectrum variation for detecting solution, is then separately added into 10 into the above solution containing cation again-5Mol/L's
Cu2+, the fluorescence emission spectrum of solution is detected, value corresponding to maximum emission wavelength is taken to map.As a result as shown in figure 3, although depositing
In other cations, Cu2+The fluorescent quenching that can still result in fluorescent polypeptide probe illustrates that polypeptide fluorescent probe P can be special
Property detection Cu2+, not by the interference of other cations.
2.3 polypeptide fluorescent probe P and Cu2+Combination than measurement
To addition 10 in HEPES buffer solution (10mM, pH7.4)-5The polypeptide fluorescent probe P of mol/L, then be added thereto not
With the Cu of concentration2+(0.0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,
1.6,1.7,1.8,1.9,2.0eq) the fluorescence emission spectrum variation of solution, is detected, taking launch wavelength is that the place 540nm is corresponding
Value makees fluorescence titration tendency chart.As a result as shown in figure 4, with Cu2+The increase of concentration, the fluorescence intensity at 540nm constantly drop
It is low, as the Cu that 1.0eq is added2+Afterwards, the fluorescence intensity at 540nm is no longer changed, and illustrates Cu be added2+It has reached
Saturation, i.e. polypeptide fluorescent probe P and Cu2+It is combined with the stoichiometric ratio of 1:1.
2.4 polypeptide fluorescent probe P are to Cu2+Detect the measurement of limit
Firstly, fluorescence data when measuring only polypeptide fluorescent probe P by 10 repeated experiments determines blank
Standard deviation under part；
Secondly, by being gradually incremented by Cu2+Concentration is to measure line fluorescent data at 540nm in launch wavelength, and mapping obtains
The straight line that one linearly dependent coefficient is 0.9908, as a result as shown in Figure 5；
Finally, calculating polypeptide fluorescent probe P to Cu using detection limit LOD=3 σ/k formula2+Minimum detection limit, wherein σ
It is the quasi- deviation of blank condition subscript, k is the slope of curve.By the way that polypeptide fluorescent probe P is calculated to Cu2+Lowest detection be limited to
52nM。
The special Journal of Sex Research that 3 polypeptide fluorescent probe P-Cu of embodiment detects cysteine (Cys)
3.1 polypeptide fluorescent probe P-Cu test the selective evaluation that amino acid detects
10 are separately added into in HEPES buffer solution (10mM, pH7.4)-5The polypeptide fluorescent probe P-Cu of mol/L and 10- 5The cysteine (Cys) of mol/L, glutamic acid (Glu), aspartic acid (Asp), asparagine (Asn), phenylalanine (Phe),
Alanine (Ala), glutamine (Gln), threonine (Thr), serine (Ser), tyrosine (Tyr), lysine (Lys), color
Propylhomoserin (Trp), proline (Pro), glycine (Gly), valine (Val), methionine (Met), leucine (Leu), different bright ammonia
Sour (Ile), arginine (Arg), histidine (His), glutathione (GSH) and S2-, detect the fluorescence emission spectrum change of solution
Change.As a result just cause the significant changes of fluorescence intensity when as shown in fig. 6, Cys is only added, and other substances will not all cause
Fluorescence intensity significant changes.
3.2 polypeptide fluorescent probe P-Cu analyze the specificity of the detection of Cys
10 are separately added into in HEPES buffer solution (10mM, pH7.4)-5The P-Cu of mol/L and 10-5Mol/L (Glu,
Asp, Asn, Phe, Ala, Gln, Thr, Ser, Tyr, Lys, Trp, Pro, Gly, Val, Met, Leu, Ile, Arg, His, GSH with
And S2-), the fluorescence emission spectrum variation of solution is detected, is then separately added into 10 into the solution above containing other substances again- 5The Cys of mol/L detects the fluorescence emission spectrum of solution, and value corresponding to maximum emission wavelength is taken to map.As a result such as Fig. 7 institute
Show, despite the presence of other amino acid and anion, Cys can still result in the recovery of fluorescent polypeptide probe, illustrate polypeptide fluorescence
Probe P-Cu is to the detection of Cys not by other amino acid and S2-Interference, have specificity.
The measurement of the combination ratio of 3.3 polypeptide fluorescent probe P-Cu and Cys
To addition 10 in HEPES buffer solution (10mM, pH7.4)-5The probe of the P-Cu of mol/L, then be added thereto different
Concentration Cys (0.0,0.05,0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.55,0.6,0.65,0.7,
0.75,0.8eq) the fluorescence emission spectrum variation for, detecting solution, taking launch wavelength is that the corresponding value in the place 515nm makes fluorescence
Titrate tendency chart.As a result as shown in figure 8, with Cys concentration increase, the fluorescence intensity at 515nm constantly enhances, and works as addition
0.5 equivalent Cys after, the fluorescence intensity at 515nm is no longer changed, and illustrates that Cys be added has reached saturation, i.e.,
Polypeptide fluorescent probe P-Cu and Cys with the stoichiometric ratio of 2:1 in conjunction with.
Measurement of the 3.4 polypeptide fluorescent probe P-Cu to Cys detection limit
Firstly, fluorescence data when measuring only P-Cu by 10 repeated experiments, determines the standard under the conditions of blank
Deviations；
Secondly, being to measure line fluorescent data at 515nm in launch wavelength, mapping considerable by being gradually incremented by Cys concentration
It observes to obtain the straight line that a linearly dependent coefficient is 0.9967, as a result as shown in Figure 9；
Finally, limiting minimum detection limit when LOD=3 σ/k formula calculates P-Cu probe in detecting Cys using detection, wherein σ is
The quasi- deviation of blank condition subscript, k are the slopes of curve.Polypeptide fluorescent probe P-Cu is calculated to be limited to the lowest detection of Cys
43nM。
The pH responsiveness of 4 polypeptide probe of embodiment measures
In the HEPES buffer solution of different pH (2,4,6,8,10,12), fluorescent polypeptide probe P, polypeptide fluorescent probe are surveyed
The fluorescence emission spectrum of P-Cu and polypeptide fluorescent probe P-Cu+Cys take fluorescence intensity maximum value to do figure.As a result such as Figure 10 institute
Show, the pH scope of application of the system is very wide, can be used not only for the Cu in monitoring environment2+, Cys can also be used to Cu in cell2+、
The detection of Cys.
The cell imaging of 5 polypeptide probe of embodiment
(1) by HeLa cell culture in DMEM medium, at 37 DEG C, 10 μM of probe P is added into the cell and is incubated for
1h, is washed out 3 times, and fluorescence microscope is just being set by Zeissz and is being observed, dark-field imaging；
(2) by HeLa cell culture in DMEM medium, at 37 DEG C, the probe P of 10 μM of addition into the cell, then plus
Enter 10 μM of Cu2+, it is incubated for 1h, is washed out 3 times, fluorescence microscope is just being set by Zeiss and is being observed, dark-field imaging；
(3) by HeLa cell culture in DMEM medium, at 37 DEG C, 10 μM of probe P-Cu is added into the cell,
5 μM of Cys is added, 1h is incubated for, is washed out 3 times, fluorescence microscope is just being set by Zeiss and is being observed, dark-field imaging.
Cell imaging result is as shown in figure 11, and cell has stronger fluorescence after probe P is added, and Cu is then added2+Cell is glimmering
Light is carefully quenched, and finally adds the recovery of Cys cell fluorescence.This illustrates that probe P can permeate HeLa cell living, and energy
Enough detect the Cu in cell2+And Cys.Wherein a is the fluorescence field figure being added after 10 μM of P incubation 1h into HeLa cell, and b is it
Corresponding light field figure, c are its corresponding fluorescence field and light field stacking chart；D is to add after 10 μM of P is added into HeLa cell
10 μM of Cu2+Fluorescence field figure after being incubated for 1h, e are its corresponding light field figure, and f is its corresponding fluorescence field and light field stacking chart；g
For the fluorescence field figure after the Cys incubation 1h for adding 5 μM is added after 10 μM of P-Cu into HeLa cell, h is that its is corresponding bright
Field figure, j are its corresponding fluorescence field and light field stacking chart.
Sequence table
<110>Lanzhou University
<120>a kind of polypeptide fluorescent probe for specifically binding copper ion and cysteine
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213>artificial sequence ()
<400> 1
Asp Glu Lys Glu Asp
1 5
Claims (10)
1. a kind of polypeptide, which is characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.
2. a kind of polypeptide as described in claim 1 is preparing the application in polypeptide fluorescent probe.
3. a kind of specific recognition Cu2+Polypeptide fluorescent probe P, which is characterized in that the polypeptide fluorescent probe P is sent out by fluorescence
Stimulative substance and polypeptide as described in claim 1 form, N-terminal of the fluorescence radiation substance modification in the polypeptide.
4. polypeptide fluorescent probe P as claimed in claim 3, which is characterized in that the fluorescence radiation substance be fluoresceins,
Any one of rhodamine, Coumarins, quinolines, naphthalimide, polyaryl vinyl.
5. polypeptide fluorescent probe P as claimed in claim 4, which is characterized in that the fluorescence radiation substance is dansyl Cl.
6. a kind of polypeptide fluorescent probe P as claimed in claim 3 is in detection Cu2+Application in content, which is characterized in that described
Polypeptide fluorescent probe P and Cu2+It is coordinated and is combined with 1:1, the Cu2+Lowest detection be limited to 52nM, the detection method includes
Following steps:
(1) to 10mM, 10 the preparation of standard curve: are added in the HEPES buffer solution of pH7.4-5The polypeptide fluorescent probe P of mol/L,
Add the Cu that concentration is 0.0,0.25,0.5,0.75,1.0,1.25 μM2+, and Detection wavelength is strong for the fluorescence of solution at 540nm
Degree, with Cu2+Concentration is abscissa, and fluorescence intensity is ordinate, draws standard curve；
(2) Cu in sample2+The measurement of content: to 10mM, being added certain density sample to be tested in the HEPES buffer solution of pH7.4,
And detect the fluorescence intensity of solution at 540nm；
(3) Cu in sample to be tested is calculated2+Content: by step (2) resulting fluorescence intensity level bring into step (1) preparation standard
In curve, Cu in sample to be tested is calculated2+Content.
7. a kind of polypeptide fluorescent probe P as claimed in claim 3 is preparing the application in cell imaging reagent.
8. a kind of polypeptide fluorescent probe P-Cu of specific recognition Cys, which is characterized in that the polypeptide fluorescent probe P-Cu be by
Polypeptide fluorescent probe P and Cu as claimed in claim 32+The complex for combining and being formed is coordinated with 1:1.
9. a kind of application of polypeptide fluorescent probe P-Cu as claimed in claim 8 in detection Cys content, which is characterized in that
The polypeptide fluorescent probe P-Cu is combined with Cys with 2:1 coordination, and the lowest detection of the Cys is limited to 43nM, the detection method
The following steps are included:
(1) to 10mM, 10 the preparation of standard curve: are added in the HEPES buffer solution of pH7.4-5The polypeptide fluorescent probe P- of mol/L
Cu adds the Cys that concentration is 0.0,0.2,0.4,0.6,0.8,1.0,1.2 μM, and Detection wavelength is the glimmering of solution at 515nm
Luminous intensity, using Cys concentration as abscissa, fluorescence intensity is ordinate, draws standard curve；
(2) in sample Cys content measurement: to 10mM, certain density sample to be tested is added in the HEPES buffer solution of pH7.4,
And detect the fluorescence intensity of solution at 515nm；
(3) it calculates the content of Cys in sample to be tested: step (2) resulting fluorescence intensity level is brought into the standard of step (1) preparation
In curve, the content of Cys in sample to be tested is calculated.
10. a kind of polypeptide fluorescent probe P-Cu as claimed in claim 8 is preparing the application in cell imaging reagent.
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| CN110746411A (en) * | 2019-11-15 | 2020-02-04 | 辽宁科技大学 | Fluorescent probe and preparation method thereof, fluorescent probe test paper and application thereof |
| CN113336856A (en) * | 2020-02-18 | 2021-09-03 | 华东理工大学 | Tryptophan optical probe and preparation method and application thereof |
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