CN105259118B - A kind of dual-functional nanometer probe and the preparation method and application thereof based on lanthanide series metal - Google Patents
A kind of dual-functional nanometer probe and the preparation method and application thereof based on lanthanide series metal Download PDFInfo
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Abstract
The invention discloses a kind of dual-functional nanometer probe and the preparation method and application thereof based on lanthanide series metal.Include the following steps: 1) to react with antenna BCTOT first using the polypeptide for containing DEVD amino acid residue as bridging agent the present invention is based on the dual-functional nanometer probe preparation method of lanthanide series metal, obtains the polypeptide of BCTOT modification；2) polypeptide for modifying the BCTOT and lanthanide metal ion chelatropic reaction, the polypeptide solution after being chelated；3) polypeptide solution after the chelating is reacted with nanomaterial solution to get the dual-functional nanometer probe based on lanthanide series metal is arrived.The present invention is based on the dual-functional nanometer probe applications of lanthanide series metal in spectral detection, Mass Spectrometer Method, cell imaging and/or the ICP-MS quantitative detection to caspase-3.The present invention is able to achieve dynamic and visual and the high sensitivity quantitation analysis at biomolecule scene.
Description
Technical field
The present invention relates to a kind of dual-functional nanometer probe and the preparation method and application thereof based on lanthanide series metal, belongs to analysis
Chemical field.
Background technique
Important regulatory molecule is to biology and clinical research in dynamic and visual and highly sensitive quantitative analysis life system
Have great importance.Fluorescence detection method is due to high sensitivity and spatial and temporal resolution, so that fluorescence imaging method exists
Extensive development has been obtained in the detection of the real-time visual of biomolecule and bioprocess.It is given birth to however, being realized using fluorescent method
The quantitative detection of object molecule also faces huge challenge.This is because: firstly, can be with using fluorescence resonance energy transfer method
Designed ratios type fluorescence probe realizes the quantitative detection of protease, but this method needs the emission spectrum and receptor of donor fluorescent
The excitation spectrum of fluorescence has overlapping, application of the limited donor-receptor fluorescent molecule to this method is greatly limited；Secondly, big
Most fluorescent molecules are more sensitive to the variation of physiological environment, such as pH, viscosity, ionic strength, this results in realizing biology
The accurate quantitative analysis in situ detection of molecule has difficulties；Again, the autofluorescence of biological sample, also can be fixed to the fluorescence of biomolecule
Amount detection generates interference.Fluorescent method provides a sensitive analysis method for the real-time monitoring of biomolecule, but more than
Factor limit the accurate quantitative analysis method developed fluorescent method as fluorescent molecule.It is therefore desirable to further study, development
Sensitive qualitative and accurate quantitative analysis the new method of fluorescent molecule.
Using mass spectrometry method carry out protease quantitative study, can to avoid the environmental factor in fluorescence detection influence and
The interference of background fluorescence.In addition, can detecte in entire biological sample for optical detecting method using mass spectrometry method
The activity of biomolecule, and it is not limited only to the activity of cell or tissue surface biomolecule, therefore, mass spectrum can be albumen
The detection of enzyme provides relatively accurate quantitative approach.Inductivity coupled plasma mass spectrometry (ICP-MS), due to low sensitivity and
The wide range of linearity has been successfully used to the quantitative detection of cell, protease and DNA by combining rubidium marking technology
In research.But the detection effect of biomolecule only passes through final mass spectral characteristi and can just learn, cannot achieve biomolecule
Real-time, in situ, visual analysis.
Traditional probe only has single signal response performance, therefore cannot realize the dynamic at biomolecule scene simultaneously
Visualization and highly sensitive quantitative analysis.
Summary of the invention
The object of the present invention is to provide a kind of dual-functional nanometer probe and the preparation method and application thereof based on lanthanide series metal,
The present invention is able to achieve dynamic and visual and the high sensitivity quantitation analysis at biomolecule scene.
Dual-functional nanometer probe provided by the invention based on lanthanide series metal, the nano-probe by polypeptide N-terminal NH2With
SO on BCTOT2Cl generates SO2- NH connection connect on the peptide C end with nano material containing SH, and connects on the polypeptide
BCTOT and the lanthanide metal ion chelating connect forms；
Contain the shearing site of protease on the polypeptide.
In the present invention, the polypeptide plays the role of connecting BCTOT and nano material, and also acts as with protease
The effect of shearing site.
In above-mentioned nano-probe, the length of the polypeptide is 7~10 amino acid residues；
The nano material is nanogold, nano silver, nano-silicon, graphene, carbon nanotube or upper conversion nano particle；
The lanthanide metal ion is europium ion, terbium ion, erbium ion or ytterbium ion；The symbol of element of europium is Eu, specifically
It can be EuCl3Aqueous solution in the Eu that ionizes out3+；.
In above-mentioned nano-probe, the amino acid sequence of the shearing site of protease is DEVD in the polypeptide；
The nano material is graininess, and the partial size of the nano material is 10nm~100nm.
In above-mentioned nano-probe, the amino acid sequence of the polypeptide is the end N-terminal-GKDEVDAPGC-C.
The preparation method of the present invention also provides the above-mentioned dual-functional nanometer probe based on lanthanide series metal, including walk as follows
It is rapid: 1) to be reacted first with antenna BCTOT using the polypeptide as bridging agent, obtain the polypeptide of BCTOT modification；
2) polypeptide for modifying the BCTOT and the lanthanide metal ion chelatropic reaction, the polypeptide after being chelated are molten
Liquid；
3) polypeptide solution after the chelating is reacted to double based on lanthanide series metal to get arriving with the nanomaterial solution
Function nano probe.
In the present invention, the English of the BCTOT is named as 1,10-bis (5 '-chlorosulfo-thiophene-2 '-
yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone；The DEVD is the bottom Caspase-3
Object.
In above-mentioned method, the molar ratio of the polypeptide and the BCTOT can be 1:0.5~1, concretely 1:1；
In step 1), the temperature of the reaction can be 10~30 DEG C, and the temperature of reaction is room temperature, and general room temperature refers to 10
~30 DEG C；
The time of the reaction can for 1~for 24 hours, concretely 12h；
In step 2), the molar ratio of the lanthanide metal ion and the BCTOT can be 0.1~10:1, concretely 1:
1；
Concentration after the lanthanide metal ion and BCTOT chelating can be 0.1~100 μM, concretely 10 μM；
The temperature of the chelatropic reaction can be 30 DEG C~60 DEG C, and concretely 50 DEG C, the time of the chelatropic reaction can be 1
~8h, concretely 4h；The chelatropic reaction is reacted on shaking table.
In above-mentioned method, the partial size of the nano material can be 10nm~100nm；
The molar ratio of the polypeptide and the nano material can be 100~3000:1, concretely 3000:1；
In step 3), the temperature of the reaction can be 10~30 DEG C, and the temperature of reaction is room temperature, and general room temperature refers to 10
~30 DEG C；
The time of the reaction can be 1~36h, concretely for 24 hours.
In above-mentioned method, in step 1), acid binding agent is additionally added in the system of the reaction, the system of the reaction is being shaken
It is reacted on bed, in the BCTOT modification to the N-terminal of the polypeptide；
The acid binding agent is triethylamine and/or pyridine；
The molar ratio of the polypeptide and the acid binding agent can be 1:0.05~1:0.2.
Dual-functional nanometer probe of the present invention is applied shown in following (1) or (2) or (3):
(1) application of the dual-functional nanometer probe in the qualitative and/or quantitative detection to protease described in；Or it is described
Dual-functional nanometer probe is preparing the application in qualitative and/or quantitative detection protease product (such as kit)；
(2) application of the dual-functional nanometer probe in the intracellular imaging to protease described in；Or it is described difunctional
Nano-probe in preparing the product being imaged into the cell to protease (such as kit) application；
(3) application of the dual-functional nanometer probe in the reinforcing agent or inhibitor of screening protease described in；Or it is described
Application of the dual-functional nanometer probe in the reinforcing agent of preparation screening protease or the model of inhibitor.
For the present invention in application, the qualitative detection uses spectral detection, the quantitative detection uses Mass Spectrometer Method；
The spectral detection is specially cell imaging, and the Mass Spectrometer Method is specially ICP-MS quantitative detection；
And/or described qualitative and/or quantitative detection is to carry out in external and/or living cells.
The present invention is in application, the protease is caspase-3；
Or, the method for the external qualitative detection includes the following steps:
It when using spectral detection and/or Mass Spectrometer Method, carries out in accordance with the following steps: 1) by described based on lanthanide series metal
Dual-functional nanometer probe solution is added in external test sample, then the system of the reaction is centrifuged, takes supernatant by reaction
Liquid, spectral detection and/or Mass Spectrometer Method supernatant, if detecting fluorescence and/or lanthanide metal ion signal response,
Show to contain protease in sample to be tested；
Or, the method for the intracellular qualitative detection difunctional receiving of including the following steps: described based on lanthanide series metal
Rice probe solution is added in living cells, reaction, after then the living cells for being incubated for the probe is cracked, centrifugation point
From supernatant is collected, Mass Spectrometer Method detects supernatant, if detecting the lanthanide metal ion signal response, shows to be measured
Into the cell containing protease or show intracellular protease to be measured have proteinase activity；
Or, the method for the Quantitative in vitro detection includes the following steps: first by described based on the difunctional of lanthanide series metal
Nano-probe solution is added in the standard items of the protease, measures the concentration of the lanthanide metal ion, with the albumen
The concentration of the standard items of enzyme is abscissa, and the concentration of the lanthanide metal ion is ordinate, establishes standard curve I；It will be described
Dual-functional nanometer probe solution based on lanthanide series metal is added in external test sample, reaction, then by the reaction
System centrifugation, takes supernatant, Mass Spectrometer Method supernatant, and the concentration for measuring lanthanide metal ion in sample to be tested substitutes into the standard
To get the content of the protease into the sample to be tested in equation I；
Or, the method for the intracellular quantitative detection includes the following steps: double function first by described based on lanthanide series metal
Energy nano-probe solution is added in the standard items of the protease, the concentration of the lanthanide metal ion is measured, with the egg
The concentration of the standard items of white enzyme is abscissa, and the concentration of the lanthanide metal ion is ordinate, establishes standard curve II；By institute
It states the dual-functional nanometer probe solution based on lanthanide series metal to be added in living cells, then reaction will be incubated for the institute of the probe
State after living cells cracked, supernatant is collected in centrifuge separation, Mass Spectrometer Method supernatant, measure in cell to be measured lanthanide series metal from
The concentration of son substitutes into the normal equation II to get the content of the protease into the cell to be measured；
Or, the method for the cell imaging includes the following steps: living cells in culture dish and described based on lanthanide series metal
Dual-functional nanometer probe solution be incubated for, after then the living cells for being incubated for the probe is washed, carry out cell at
As.
The present invention is based on the principle of the preparation method of the dual-functional nanometer probe of lanthanide series metal is as follows:
Utilize the SO on BCTOT under alkaline condition first2The NH of Cl and N-terminal on polypeptide2SO can be generated2- NH, to close
The polypeptide modified at BCTOT；And then Eu is chelated to by the BCTOT modified on polypeptide by the chelating of BCTOT and Eu ion
On, and then using stable Au-S key is formed between the SH and Au on the cysteine residues at peptide C end, by Eu-BCTOT
Chelate modification polypeptide be added in nano-Au solution (chelate modification polypeptide and nanogold according to molar ratio 3000:1 into
Row feeds intake), at room temperature, Eu-BCTOT chelate labels polypeptide is recycled by the way of centrifugation after the arrival design reaction time and is repaired
The nanogold particle (i.e. the dual-functional nanometer probe based on lanthanide series metal) of decorations, using deionized water to obtained nanogold particle
Cleaning centrifugation three times, removes unbonded polypeptide.
The invention has the following advantages that
1) Eu-BCTOT marks the nanogold particle of peptide fragment modification, since Eu ion therein not only has unique light
Property is learned, also there is mass spectrum response property, therefore can respond by the mono-modified dual signal for realizing biomolecule；
2) it responds single label probe due to being provided simultaneously with spectrum and mass signal and has not been reported, with conventional biomolecule
Detection method is compared, which is constructed using Eu ion, so that the probe is not only able to achieve the real-time of biomolecule
Visualized in situ detection, moreover it is possible to realize quantitative detection；
3) good response performance of the Eu ion in ICP-MS, so that Eu ion concentration can be still detected down to ppt level
It measures and, so that the nano-probe can be used for the sensitive quantitative analysis of biomolecule, be therefore particularly suited for intracellular table
Up to the detection of horizontal lower biomolecule；
4) Eu ion there's almost no in the cell or in vivo, therefore realize life using the probe of Eu ion modification
The detection of object analysis can effectively avoid the appearance of false positive signal.
Detailed description of the invention
Fig. 1 is that the present invention is based on the preparation flow figures of the dual-functional nanometer probe of lanthanide series metal.
Fig. 2 a1) it is the transmission electron microscope photo of nanogold particle modified without peptide fragment；Fig. 2 a2) it is mentioned for the present invention
The transmission electron microscope photo of the nanogold particle of the Eu-BCOT label peptide fragment modification of confession；Fig. 2 b1) it is to be modified without peptide fragment
Nanogold particle partial size statistical chart；Fig. 2 b2) it is the nanogold particle that Eu-BCOT provided by the invention marks peptide fragment modification
Partial size statistical chart.
Fig. 3 is the fluorescence spectra of peptide fragment front and back in conjunction with nanogold particle of Eu-BCOT label, wherein Fig. 3 a is Eu-
BCTOT label peptide fragment, Fig. 3 b be Eu-BCOT label peptide fragment in conjunction with nanogold particle after nano-probe.
Fig. 4 is the response of fluorescence spectrum after the reacting of nano-probe and the caspase-3 of various concentration.
Fig. 5 is that the Eu ion after nano-probe is reacted with the caspase-3 of various concentration, in supernatant carries out ICPMS detection,
Detect linear relationship between obtained Eu signal and caspase-3 concentration.
Fig. 6 be nano-probe (core peptide section sequence is DEVD) and control group probe (core peptide section sequence be DEVG) and
Cell imaging figure after HeLa cell incubation, after adding staurosporine (STS) or Ac-DEVD-CHO incubation.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
In following embodiments, peptide fragment is purchased from Beijing SBS Genetech gene technology Co., Ltd.
Embodiment 1,
Experiment flow such as Fig. 1 institute of nano-probe is obtained using the peptide fragment modified nano gold particle preparation that Eu-BCTOT is marked
Show.
1) optimization of Eu and BCTOT chelating condition: by a certain amount of EuCl3After being dissolved into deionized water, it is added to
In BCTOT solution, after being incubated for a period of time at 50 DEG C, the ultraviolet and fluorescence spectrum of measurement system.By to Eu and BCTOT chela
The solution that obtains after conjunction carries out spectrum analysis, the maximum excitation and launch wavelength of Eu-BCTOT chelate be respectively 350nm and
614nm, this is the characteristic emission spectrum of Eu, illustrates that Eu and BCTOT is successfully chelated, and BCTOT can play the luminous of Eu really
To a good sensibilization.
Then, the best chelating ratio of Eu and BCTOT is investigated, the results showed that when the molar ratio of Eu and BCTOT exist
When within the scope of 0.1~10:1, the fluorescence of Eu-BCTOT chelate increases with the increase of the two ratio, when the molar ratio of the two
When for 1:1, the fluorescence of Eu-BCTOT chelate has reached a platform.
Meanwhile the concentration of Eu-BCTOT chelate is optimized, the results showed that chelate is glimmering when concentration is 10 μM
Light value reaches maximum, when chelate concentration relatively high (100 μM) or it is relatively low when (1 μM) when, can all lead to Eu-BCTOT
Fluorescence is relatively weak.The possible reason is when Eu-BCTOT concentration is lower, since concentration effect is so as to cause the fluorescence of system
It is lower；And when Eu-BCTOT concentration is higher, due to the self-quenching effect between molecule, so that the fluorescence intensity in system becomes
It is weak.Therefore, in the case where no other specified otherwises, in reaction system, the concentration of Eu-BCTOT is selected as 10 μM.
In addition, the fluorescence of Eu-BCTOT chelate increases with the increase of chelating time, and when chelating the time is 1h,
The fluorescence intensity of chelate reaches a platform, and this fluorescent value is not changed significantly interior for 24 hours, illustrates Eu-BCTOT
Chelate is highly stable.
2) synthesis and characterization of nanogold particle: gold nano grain is obtained by citric acid reduction gold chloride, specific experiment step
Rapid is to be heated to flowing back with vigorous stirring by the chlorauric acid solution of 50mL 0.25mM first, then again by 1.3mL1% (W/V)
Citric acid three sodium solution rapidly join wherein, when solution color from it is light yellow become claret when, stop heating, it is molten
Liquid is continuously stirred at room temperature until solution, is finally transferred in 4 DEG C of refrigerators and saves for use by cooling.Existed according to Gold nanoparticle
Absorbance and its molar absorption coefficient (2.7 × 10 at maximum absorption band at 520nm8M-1cm-1) calculate to obtain gold nano
The concentration of particle is 3nM.
The size and dispersibility of gold nano grain are characterized by transmission electron microscope.By 20 μ L gold nano grain solution
It is added drop-wise on the copper mesh of carbon modification, is then dried at room temperature for be measured overnight.
3) Eu-BCTOT modifies the synthesis of peptide fragment: firstly, utilizing the SO on BCTOT2Cl is easy to the NH on peptide fragment2Reaction life
At covalent SO2The characteristics of-NH, to modifying BCTOT onto the peptide fragment of synthesis first.Concrete operations are: weighing 2.1mg
BCTOT is dissolved in the carbon acid solution of 0.1M pH 9.0, then is weighed the peptide fragment of 0.72mg and be dissolved in 100 μ L deionized waters, will be obtained
Peptide fragment solution be added in BCTOT solution, be incubated for 1h at room temperature.
Then, the EuCl of 0.7mg is weighed3After being dissolved in deionized water, it is then added in the peptide fragment solution of BCTOT modification,
It is incubated for 2 hours at 50 DEG C, obtains the peptide fragment of Eu-BCTOT modification.
4) Eu-BCTOT modifies the surface that peptide fragment is integrated to nanogold: the half Guang ammonia of peptide fragment C-terminal modified using Eu-BCTOT
Strong Au-S key can be formed between SH on acid and the aU of nanometer gold surface, received so that the peptide fragment of Eu-BCTOT modification is fixed to
The surface of meter Jin.Concrete operations are that 0.588mL Eu-BCTOT modification obtained above is added into 9.412mL 3nM Au NPs
Peptide fragment solution (peptide fragment/nanogold molar ratio be 3000/1), after being slowly stirred at a room temperature 48h, excessive Eu-BCTOT
The peptide fragment of modification is finally obtained red heavy by being washed with deionized, being centrifuged removal (12,000rpm × 10min, 3 times)
Shallow lake is then resuspended in a certain amount of PBS buffer solution (pH 7.4), makes the ultimate density 0.5mM of probe (with the peptide fragment amount of starting
It calculates).
The nanogold particle and unmodified nanogold particle for taking peptide fragment modification obtained by the above method are transmitted respectively
Electron-microscopic analysis analyzes result such as Fig. 2.
As shown in Figure 2, modification and unmodified nanogold particle all show good dispersity, show spy of the present invention
Needle can be used for biological detection.In addition, the average grain diameter of nanogold is from Fig. 2 b1) after in conjunction with the peptide fragment that Eu-BCTOT is modified
13.6nm increases to Fig. 2 b2) in 16.2nm, the increase of partial size show Eu-BCTOT modification peptide fragment be successfully incorporated
The surface of nanogold particle.
Meanwhile also to the peptide fragment of Eu-BCTOT modification, the variation of front and back spectrum is investigated in conjunction with nanogold particle,
As a result as shown in Figure 3.
Since nanogold is a good fluorescence quenching, as seen from Figure 3, in conjunction with nanogold particle before, Eu-
Stronger fluorescence is presented in the peptide fragment of BCTOT modification, and after the peptide fragment is integrated on nanogold particle, fluorescence is significantly quenched
It goes out.This is because effective fluorescence resonance energy transfer causes between Eu-BCTOT and nanogold, to show that nanogold can be with
The fluorescence of Eu-BCTOT is effectively quenched, while also from showing peptide fragment that Eu-BCTOT is modified on the other hand successfully
It has been integrated to the surface of nanogold particle.
Embodiment 2, using the nano-probe exogenous being prepared in embodiment 1 caspase-3 carry out spectrum and
Mass Spectrometer Method.
1) by exogenous caspase-3 and 10 μM of the probe of various concentration in PBS (pH 7.4) buffer at 37 DEG C
Lower incubation 2h.To after reaction, solution is centrifuged 10min at 120,000rpm, collects supernatant and carry out fluorescence detection.Detection
Excitation wavelength and launch wavelength be respectively 350nm and 614nm.Acquired results are as shown in Figure 4.
As shown in Figure 4, increase to 70ng/mL from 5ng/mL with the concentration of caspase-3, the fluorescence in reaction system is strong
Degree also enhances therewith.Show that probe of the present invention can be used for the detection of caspase-3；Meanwhile showing based on FRET principle, successfully
The nano-probe of an open type is constructed, the detection for caspase-3.
Meanwhile in order to verify the enhancing of system fluorescence strictly since shear action of the caspase-3 to substrate peptide fragment is drawn
It rises, by the way that the inhibitor Ac-DEVD-CHO of caspase-3 is added into system, to change the activity of caspase-3, to examine
Examine its influence to system fluorescence.The experimental results showed that after Ac-DEVD-CHO is added, the reactant of probe and caspase-3
Weaker fluorescence is presented in system, to show that the recovery of fluorescence in system is by caspase-3 to the substrate peptide fragment in nano-probe
Cutting cause, also, the recovery of fluorescence and the activity of caspase-3 have and directly contact.
Peptide fragment in nano-probe can be selectively sheared in order to further investigate caspase-3, has synthesized a control
Probe, it is DEVG peptide fragment (GKDEVGAPGC) that it, which contains sequence, instead of the substrate peptide fragment DEVD in former probe.When to control group
In probe be added caspase-3 when, due in peptide section sequence single amino acids by D to G variation so that the fluorescence of control probe
It has almost no change, this shows that control group probe cannot be cut by caspase-3.To that is to say, nano-probe of the present invention is illustrated
There is very high selectivity to caspse-3, therefore the selective enumeration method of caspase-3 may be implemented.
2) and in quantitative analysis, other operations are consistent, ICP-MS detection is carried out after only finally collecting supernatant.Then
Linear fit is carried out with the concentration of corresponding caspase-3 to the signal value of Eu in ICP-MS detection, as a result as shown in Figure 5.
As shown in Figure 5, experimental result is shown, after the caspase-3 of various concentration is added into probe solution, Eu exists
Response in ICP-MS is consistent with trend of the probe to the fluorescence response of caspase-3.Peptide fragment after being sheared of Eu label
ICP-MS signal increases with the increase of caspase-3 concentration.And even if the concentration range in caspase-3 is two quantity
Grade when (0.5~70ng/mL), good linear relationship is presented between the two, obtain linear equation [Eu] (μM)=0.0072 ×
[caspase-3] (ng/mL) -0.024 (r=0.99), this shows that the probe can be used for the sensitive quantitative detection of caspase-3.
Real-time example 3, using the nano-probe being prepared in embodiment 1 to intracellular caspase-3 carry out imaging and
Quantitative analysis.
1) in cell imaging research, probe or control group probe of the cell first with 10 μM in culture dish exist
12h is incubated in 1mL culture medium at 37 DEG C, then, outwells culture medium, after cleaning 3 times with PBS, is added new culture medium, and to
The STS or Ac-DEVD-CHO that various concentration is added in culture dish use 0.1M after then cell is incubated for 4h at 37 DEG C again
PBS carries out after repeatedly washing cell, carries out cell imaging detection.As a result as shown in Figure 6.
It will be appreciated from fig. 6 that the experimental results showed that being incubated for has the HeLa cell of probe with weaker when no any processing
Fluorescence, this shows under normal cell state, and the concentration level of intracellular caspase-3 is lower.However, into cell
After 2 μM of STS are added, apparent fluorescence enhancement can see in cell, and after the STS of higher concentration (4 μM) is added, carefully
Stronger fluorescence is presented in born of the same parents.The enhancing of fluorescence can be attributed to the fact that activation of the STS to caspase-3 in cell.It was furthermore observed that
Fluorescence is primarily present in cytosolic domain, shows the subcellular localization for the caspase-3 that STS induction generates.On the other hand, when
Incubation has the HeLa cell of probe while after 4 μM of STS and Ac-DEVD-CHO processing, intracellular to generate weaker fluorescence,
This is because the activity of caspase-3 restrained effectively by Ac-DEVD-CHO.Meanwhile control group probe (DEVG) is incubated for
HeLa cell, even if being also nearly free from apparent fluorescence under the processing of 4 μM of STS, this is also just demonstrated, glimmering in cell
Caused by the generation of light is the specificity cutting due to caspase-3 to DEVD peptide fragment in probe.Above the result shows that being based on
The open type fluorescence probe of FRET principle building, has high selectivity, can be used for the inspection of endogenous caspase-3 in living cells
It surveys.
2) in the operation for carrying out quantitative analysis to the caspase-3 in HeLa cell, other operations are as before, with 0.1M PBS
Cell is carried out after repeatedly washing, cell is digested, culture medium is added and terminates digestion, by it under the piping and druming of culture dish bottom
Come, after piping and druming sufficiently, calculates cell density with cell counting board.Then 0.1M is used on 4 DEG C of centrifuges with by obtained cell
PBS is centrifuged 3 times under the conditions of 100,000rpm × 10min removes culture medium, then again carries out cell on Ultrasonic Cell Disruptor
Cell cracking.Product after cracking is centrifuged under 4 DEG C, 120,000rpm × 10min, collect supernatant, then by obtained supernatant into
Row ICP-MS detection is to obtain the quantitative information of Eu.The results are shown in Table 1.
Quantitative analysis results of the probe of the present invention of table 1 to the caspase-3 in the HeLa cell under different condition processing
By the result shows that under conditions of no any processing, cell density is 2.00 × 10 in table 16Cell/mL situation
Under, the content using caspase-3 intracellular detected by probe of the present invention is 1.0ng/mL, shows probe tool of the present invention
There is high sensitivity, detectable concentration can be detected down to ppb level when the concentration of the STS of processing cell gradually increases
The content of active caspase-3 is also to increase with being positively correlated, this is consistent with phenomenon what is observed in cell imaging.If
When carrying out STS and Ac-DEVD-CHO processing to the cell for being incubated for probe simultaneously, the reduction of Eu signal will lead to, this shows
The activity of caspase-3 is inhibited.Similarly, after the cell for being incubated for control group probe (DEVG) is handled with STS,
A lower Eu signal can be obtained, this shows that caspase-3 has high selectivity to substrate, it cannot cut off DEVG.On
State content also further illustrate be incorporated on nanogold particle be not cut off Eu-BCTOT modification peptide fragment, by from
After the heart, it can be separated well with the Eu-BCTOT peptide fragment segment of cutting, and do not influence probe and active caspase-3 is determined
Quantity research.Experiment above provides the absolute quantification method of caspase-3 in HeLa cell a kind of for the first time, it can contribute to
Research to caspase-3 function.In addition, STS and Ac-DEVD-CHO can be by corresponding on the active influence of caspase-3
The variation of Eu signal realize and delicately monitor that this is just the reinforcing agent and inhibitor of caspase family active accurate
Screening provides an effective approach, and these reinforcing agents or inhibitor be certain cancers, autoimmune disorder disease with
And the drug therapy target of heart disease.
Claims (6)
1. a kind of dual-functional nanometer probe based on lanthanide series metal, it is characterised in that: the nano-probe by polypeptide N-terminal NH2With
SO on BCTOT2Cl generates SO2- NH connection connect on the peptide C end with nano material containing SH, and connects on the polypeptide
BCTOT and the lanthanide metal ion chelating connect forms；
Contain the shearing site of protease on the polypeptide；
The nano material is nanogold, nano silver, nano-silicon, graphene, carbon nanotube or upper conversion nano particle；
The lanthanide metal ion is europium ion；
The amino acid sequence of the shearing site of protease is DEVD in the polypeptide；
The nano material is graininess, and the partial size of the nano material is 10nm~100nm；
The amino acid sequence of the polypeptide is the end N-terminal-GKDEVDAPGC-C.
2. a kind of preparation method of the dual-functional nanometer probe described in claim 1 based on lanthanide series metal, includes the following steps:
1) it is reacted first with BCTOT described in antenna using the polypeptide as bridging agent, obtains the polypeptide of BCTOT modification；
2) polypeptide for modifying the BCTOT and the lanthanide metal ion chelatropic reaction, the polypeptide solution after being chelated；
3) polypeptide solution after the chelating is reacted with the nanomaterial solution to get to based on the difunctional of lanthanide series metal
Nano-probe；
The molar ratio of the polypeptide and the BCTOT are 1:0.5~1；
In step 1), the temperature of the reaction is 10~30 DEG C；
The time of the reaction be 1~for 24 hours；
In step 2), the molar ratio of the lanthanide metal ion and the BCTOT are 0.1~10:1；
Concentration after the lanthanide metal ion and BCTOT chelating is 0.1~100 μM；
The temperature of the chelatropic reaction is 30 DEG C~60 DEG C, and the time of the chelatropic reaction is 1~8h；
The partial size of the nano material is 10nm~100nm；
The molar ratio of the polypeptide and the nano material is 100~3000:1；
In step 3), the temperature of the reaction is 10~30 DEG C；
The time of the reaction is 1~36h.
3. according to the method described in claim 2, it is characterized by: being additionally added in the system of the reaction in step 1) and tiing up acid
Agent；
The acid binding agent is triethylamine and/or pyridine；
The molar ratio of the polypeptide and the acid binding agent is 1:0.05~1:0.2.
The application during 4. dual-functional nanometer probe described in claim 1 is one of any in following (1)-(6):
(1) application in the qualitative and/or quantitative detection to protease；
(2) application in qualitative and/or quantitative detection protease product is being prepared；
(3) application in the intracellular imaging to protease；
(4) application in the product being imaged into the cell to protease is being prepared；
(5) application in the reinforcing agent or inhibitor of screening protease；
(6) application in the reinforcing agent of preparation screening protease or the model of inhibitor；
The protease is caspase-3.
5. application according to claim 4, it is characterised in that: the qualitative detection uses spectral detection, the quantitative inspection
It surveys and uses Mass Spectrometer Method；
The spectral detection is specially cell imaging, and the Mass Spectrometer Method is specially ICP-MS quantitative detection；
And/or described qualitative and/or quantitative detection is to carry out in external and/or living cells.
6. application according to claim 5, it is characterised in that: the method for the external qualitative detection includes the following steps:
When being examined using spectral detection and/or mass spectrum, carry out in accordance with the following steps: 1) by described based on the difunctional of lanthanide series metal
Nano-probe solution is added in external test sample, then the system of the reaction is centrifuged, takes supernatant, light by reaction
Spectrum detection and/or Mass Spectrometer Method supernatant show if detecting fluorescence and/or lanthanide metal ion signal response
Contain protease in sample to be tested；
Or, the method for intracellular qualitative detection includes the following steps: that the dual-functional nanometer probe by described based on lanthanide series metal is molten
Liquid is added in living cells, reaction, after then being cracked the living cells for being incubated for the probe, in centrifuge separation collection
Clear liquid, Mass Spectrometer Method detect supernatant, if detecting the lanthanide metal ion signal response, show that cell to be measured includes
There is protease or shows that intracellular protease to be measured has proteinase activity；
Or, the method for Quantitative in vitro detection includes the following steps: the dual-functional nanometer probe first by described based on lanthanide series metal
Solution is added in the standard items of the protease, measures the concentration of the lanthanide metal ion, with the standard of the protease
The concentration of product is abscissa, and the concentration of the lanthanide metal ion is ordinate, establishes standard curve I；Described it will be based on group of the lanthanides
The dual-functional nanometer probe solution of metal is added in external test sample, then the system of the reaction is centrifuged by reaction,
Supernatant, Mass Spectrometer Method supernatant are taken, the concentration for measuring lanthanide metal ion in sample to be tested substitutes into the normal equation I,
Obtain the content of protease in the sample to be tested；
Or, the method for intracellular quantitative detection includes the following steps: that the dual-functional nanometer first by described based on lanthanide series metal is visited
Needle solution is added in the standard items of the protease, measures the concentration of the lanthanide metal ion, with the mark of the protease
The concentration of quasi- product is abscissa, and the concentration of the lanthanide metal ion is ordinate, establishes standard curve II；Described it will be based on lanthanum
It is in the dual-functional nanometer probe solution addition living cells of metal, then reaction will be incubated for the living cells of the probe
After being cracked, supernatant is collected in centrifuge separation, and Mass Spectrometer Method supernatant measures the concentration of lanthanide metal ion in cell to be measured
It substitutes into the normal equation II to get the content of the protease into the cell to be measured；
Or, the method for cell imaging includes the following steps: that living cells in culture dish is received with difunctional based on lanthanide series metal
Rice probe solution is incubated for, and after then being washed the living cells for being incubated for the probe, carries out cell imaging.
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