CN105823770A - Optical-interference-free Raman labeling probe and preparation method and application thereof - Google Patents

Optical-interference-free Raman labeling probe and preparation method and application thereof Download PDF

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CN105823770A
CN105823770A CN201610351941.9A CN201610351941A CN105823770A CN 105823770 A CN105823770 A CN 105823770A CN 201610351941 A CN201610351941 A CN 201610351941A CN 105823770 A CN105823770 A CN 105823770A
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raman
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沈爱国
陈勇
任家强
胡继明
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Wuhan University WHU
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    • 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
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Abstract

The invention designs a series of signal molecules with Raman scattering peaks in the wave number area of 1800-2400 cm<-1> by using a sulfydryl phenylacetylene as a body structure, selects gold and silver nano-particles as reinforcing substrates of Raman signals, enables alkyne signal molecules with sulfydryls to be self-assembled on the surfaces of the reinforcing substrates, uses mercaptoacetic acid to optimize SERS signals, then selects a light-transmitting and hydrophilic package material rich in active reaction groups for probe package and finally grafts specific biological targeting functional molecules to an outer layer of the package material .A probe has strong optical response to Raman scattering, adopts narrow-band unimodal transmission, is free of optical background interferences and has the great significance in the biological imaging field of simultaneous marking of multiple components .The technology can extend to design and development of a no-raster ultra-fast Raman imager using a bandpass filter as a beam splitting device and using a photomultiplier tube (PMT) or an avalanche photodiode (APD) as a detecting device, overcome the technical bottlenecks of Raman imaging point-by-point scanning and slow imaging and fills the blank in the market of optical imaging instruments .

Description

A kind of Raman labels probe without optical interference and its preparation method and application
Technical field
The invention belongs to bio-imaging technical field, relate to a kind of novel alkynes coding, the surface enhanced of biological targeting draws The preparation method of graceful scattering (Surface-enhanced Raman scattering, SERS) active nano label probe.Heavier Want, relate to bandpass filter as light-splitting device, with photomultiplier tube (PMT) or avalanche photodide (APD) for inspection Survey the design and development of the no-raster ultrafast Raman image instrument of device.
Background technology
Bio-imaging (such as living cells imaging) technology makes scientist can observe organism internal structure in real time and dynamically And physiological process, thoroughly reform the mode of biologist postgraduate's object movement mechanism.In numerous bio-imaging technology, Fluorescent labeling imaging applications is the most extensive and ripe.It utilizes the light of absorbable specific wavelength and launches the light that wavelength is longer The signal probe of (fluorescence), it is specific that the least molecule organic dyestuff or fluorescin come in specifically labelling living things system Component, and the distribution situation of target organism material is obtained by the location of its fluorescence signal, greatly improve people and explore Organism internal structure and the ability of process.But, this imaging technique is substantially limited by the inherent shortcoming of fluorescent spectrometry, i.e. Traditional fluorescent dye is launched signal overlap when peak width (about 50nm) wide meeting causes polychrome (or multicomponent) labelling and is difficult to district Point, discrimination.Technology of quantum dots progress in recent years overcomes above-mentioned tradition organic fluorescent dye and the inherent shortcoming of fluorescin, Its emission peak is narrow and symmetrical, and overlapped little and its emission peak wavelength can be fine-tuned etc. consisting of material and particle diameter Good characteristic extremely cell marking and the favor of imaging research, but its potential bio-toxicity makes it be applied to the one of living imaging A little further investigation difficulties have new progress.
Surface enhanced raman spectroscopy (Surface-enhanced Raman scattering, SERS) technology is non-except having Outside destructive, EO-1 hyperion specificity and not advantage by normal Raman spectroscopy such as water interference, also there is lower detection limit and more High sensitivity (possessing the ability of Single Molecule Detection), particularly gets involved the situation of biomedical sector in nanotechnology advantage Under, various gold (silver-colored) nano-particle with surface enhanced Raman scattering effect be widely used in biological sample analysis detection with Bio-imaging is studied.In order to improve the sensitivity of Raman detection, selection and the preparation of SERS base material have been carried out greatly by people Quantifier elimination.But, determining, as another, the key factor that probe is good and bad, selection and the design of signaling molecule the most do not cause The attention that people are enough.The proposition of novel signaling molecule typically requires through reasonably design, careful screening and system Characterize, seem the most loaded down with trivial details the most undoubtedly for the SERS probe compared to selection " ready-made ", thus cause nowadays about letter The work of number MOLECULE DESIGN is few.But in the middle of complicated research system, the random selection of SERS probe molecule but may be used Can become the obstacle of research development further, because disturbing molecule or detection molecules are easily sent out with traditional SERS signal molecule The phenomenon of raw spectra overlapping, this will have a strong impact on the accuracy of result.Along with the proposition of multiple target detection concept simultaneously, this Puzzlement even can extend between all kinds of SERS probe molecule, at this moment classical signal molecule select difficulty will further by Amplify.In the face of such predicament, people do for the anti-of complex system in the urgent need to a large amount of mutual non-overlapping novel signaling molecules Disturb, multiobjective analysis detects.
The raman characteristic peak of alkynyl is at 2120cm-1Near, it is not at the intracellular common interfering material of this SPECTRAL REGION There is Raman response, so people are by the biological metabolism molecule of alkynes labelling in many researchs in recent years, such as DNA, RNA, egg White matter, lipid etc. are for biomarker imaging and achieve good effect；Additionally, the Raman shift of alkynyl and intensity can because The difference of the substituent group being attached thereto and occur substantially to offset, be only even by the carbon atom of alkynyl12C is replaced by its coordination Element13C, Raman shift will produce the skew of 100 wave numbers, and these 2 derivants predictive of alkynes are extremely suitable jamproof Multiobjective analysis detects.But, the extensively application of alkynes class signaling molecule is the most still the most weak by spontaneous Raman scattering signal intensity Problem seriously limit, in order to extremely the faintest alkynyl Raman scattering signal be detected, people have to improve time of exposure or Use high intensity laser beam, but thus may cause the damage of sample；The most also have tried to use stimulated Raman scattering (Stimulated Raman scattering, SRS) technology carries out correlational study, but the instrument price of costliness allow people hope and While raw fear, its having too many difficulties to cope with in multiple target imaging simultaneously also makes it cannot become the general instrument in bio-imaging field Device.In conjunction with the SERS technology that recent researches is more, it provides 106～1012Signal again strengthens, and will be an improvement in theory The desirable route that alkynyl spontaneous Raman scattering signal is more weak.
Summary of the invention
It is an object of the invention to provide a kind of novel alkynes coding, the surface enhanced raman spectroscopy of biological targeting The preparation method of (Surface-enhanced Raman scattering, SERS) active nano label probe, prior It is, development a kind of brightness height, polychrome output, optical stabilization and glitch-free novel SERS imaging technique.This technology (includes probe Product) bio-imaging application widely can be realized on conventional commercialization confocal spectrometer；Meanwhile, also can be used for band Pass filter is light-splitting device, surpasses with the no-raster that photomultiplier tube (PMT) or avalanche photodide (APD) are detection device The design and development of fast Raman image instrument.
The technical scheme that the present invention provides is as follows:
The preparation method of a kind of Raman labels probe without optical interference, comprises the following steps: Raman scattering peak position existed 1800-2400cm-1Wave number district, the unimodal signaling molecule in arrowband with a width of 1-2nm self-assemble to increase by Au-S key or Ag-S key Surface at the bottom of strong basis, the case material then using printing opacity hydrophilic carries out probe encapsulation, i.e. obtains the Raman mark without optical interference Note probe；
Described arrowband unimodal Raman signal molecule, has a structure shown in formula I:
Wherein, R1、R2、R3、R4For-NO2、-CH3Or H, R5For H or-Si (CH3)3、-C2H5、-CH2CH2CH2OH, phenyl or Alkynyl；
Described strengthens the nanoparticle that substrate is particle diameter 30-60nm, and described nanoparticle is (i) nanometer gold, (ii) Nanometer silver or (iii) be the core-shell structure nanometer particle as shell with nanometer gold or nanometer silver；
Described case material is the one in biomolecule, polymer, silicon dioxide.
Further, R5Self-assemble to strengthen the surface of substrate for the unimodal signaling molecule in arrowband of H after, with mercaptan carboxylic acid couple The unimodal signaling molecule in arrowband and the connected mode strengthening substrate are optimized, and the case material then using printing opacity hydrophilic is visited Pin encapsulates.
Yet further, by probe encapsulate after on case material grafting biological targeting molecule.
Described arrowband unimodal Raman signal molecule is
Described mercaptan carboxylic acid is one or more in TGA, mercaptopropionic acid, mercaptobutyric acid；Described biological target It is the one in antibody, part, polypeptide to molecule.
Described antibody is luteinising hormone-releasing hormo, and described part is folic acid, and described polypeptide is cell-penetrating Peptide or nuclear location peptide.
Described biological targeting molecule is grafted onto detecting probe surface by reacting with EDC-NHS or glutaraldehyde cross-linking.
A kind of Raman labels probe without optical interference, by the preparation side of the described Raman labels probe without optical interference Method prepares.
The application in bio-imaging of the described Raman labels probe without optical interference.
A kind of highly sensitive ultrafast Raman image instrument, with the described Raman labels probe without optical interference for detection object, On the basis of conventional optical microscope, using laser as excitation source, configure high-precision quick scanning sample stage, with ultra-narrow The unimodal superpower Raman scattering signal in arrowband of probe is extracted, with overdelicate avalanche photodide for detection with bandpass filter Element, by the photon signal of the two-dimensional scan pointwise acquisition probe of sample stage and make Pseudo Col ored Image.
This quick, sensitive and high-space resolution Raman image instrument is with the Wavelength calibration aberration of bandpass filter, with photon The intensity calibration colourity of signal.
Multiple targets are marked by the described Raman labels probe without optical interference, use the scanning automatically of band two dimension The Raman spectrometer of platform, pointwise gathers spectrum rapidly, and then fakes the superpower Raman scattering signal that probe arrowband is unimodal Color Image Processing, the displacement of its scattering peak is used for demarcating aberration, and the intensity of scattering peak is used for demarcating colourity；Described Quick Acquisition The condition of spectrum is: time of exposure is 1s, and wave-length coverage is Δ 50cm-1；Described target is cell, tissue or organism.
The principle of the present invention is:
1. with to sulfydryl phenylacetylene as agent structure, change alkynyl Raman scattering by replacing alkynyl terminal substituent group The displacement of signal, can carry out one according to Density function theory to the relation of the structure-Raman shift of alkynes class signaling molecule Serial anticipation and theoretical research, filter out the molecular structure that different arrowband is unimodal.
2. synthesis difformity, the gold, silver of size and core-shell structure nanometer particle thereof, filters out the high nanometer strengthening activity Particle strengthens substrate as SERS, by alkynes signaling molecule by Au-S or Ag-S key self-assembled monolayer in strengthening substrate surface.
3. the self assembling process of signaling molecule can use TGA etc. to signaling molecule and the connection side strengthening substrate Formula is optimized, the Interference Peaks caused due to spatial orientation difference with elimination, it is ensured that 1800～2400cm-1In spectrum range The unimodal output mode in arrowband.
4. there is signaling molecule expensive using biomacromolecule, polymer or silicon dioxide material as protection shell, encapsulation self assembly The probe parents such as metal nanoparticle, its outer surface provides transferring rich in reaction active groups beneficially subsequent bio functional molecular Connect.
5. (such as cell-penetrating peptides, appraise and decide with part (such as folic acid), antibody (such as luteinising hormone-releasing hormo), many peptides Position peptide etc.) etc. biologically functional molecule by the cross-linking reaction such as EDC-NHS or glutaraldehyde to detecting probe surface, there is special life The SERS image probe of thing target function.
Relative to existing SERS image probe, the present invention has the following advantages and beneficial effect:
1. this probe is at 1800-2400cm-1The superpower Raman scattering signal that transmitting arrowband, spectral regions (1-2nm) is unimodal, energy Enough it is prevented effectively from cellular endogenous material at lower wave number region (< 1800cm-1) intrinsic Raman signal and the interference of fluorescence signal；Should The alkynyl Raman shift of the used signaling molecule of probe can be by changing alkynyl end in sulfydryl phenylacetylene agent structure Substituted radical regulates, and can be prevented effectively from the problem overlapped each other between marking signal in polychrome imaging applications；This probe is believed Number intensity is big, optical stabilization, uses normal Raman spectrogrph can effectively shorten the bio-imaging time, the output mould that its arrowband is unimodal Formula can be used for the design and development of no-raster ultrafast Raman image instrument.
2. the optic response of this probe Raman scattering is strong, the unimodal transmitting in arrowband, without optics ambient interferences, at multicomponent simultaneously The bio-imaging field of labelling is significant.This technology may extend to bandpass filter as light-splitting device, with photomultiplier tube (PMT) or the design and development of no-raster ultrafast Raman image instrument that avalanche photodide (APD) is detection device, overcome The technical bottleneck that Raman image point by point scanning, imaging are slow, will fill up the market vacancy of optical imagery quasi-instrument.
Accompanying drawing explanation
Fig. 1 is the principle schematic that signaling molecule and the connected mode strengthening substrate are improved by TGA.
Fig. 2 is the Raman shift spectrum comparison diagram of tri-kinds of signaling molecules of B1, B2, B3.
Fig. 3 is the spectral intensity comparison diagram that signaling molecule and the connected mode strengthening substrate are improved by TGA.
Fig. 4 is the SERS probe prepared of B3 signaling molecule and pollen cell endogenous substance cell imaging at different wave numbers Comparison diagram；Wherein, Fig. 4 (A) is 1580cm-1Cell imaging figure, Fig. 4 (B) is 2212cm-1Cell imaging figure.
Fig. 5 is the attached cell image of SERS probe prepared by tri-kinds of signaling molecules of B1, B2, B3.
Fig. 6 is the tri-kinds of SERS probes of B1, B2, B3 spectrograms in same cell.
Fig. 7 is the tri-kinds of SERS probes of B1, B2, B3 cell imaging figures in same cell.
Detailed description of the invention
Below in conjunction with example, the present invention is described in further detail, and prove the advantage of this method with example, but this The embodiment of method is not limited to this.
Embodiment 1: the preparation of arrowband unimodal Raman signal molecule
Alkynes class arrowband unimodal Raman signal MOLECULE DESIGN: the present invention with to sulfydryl phenylacetylene as agent structure, by replacing Change alkynyl terminal substituent group to change the displacement of alkynyl Raman scattering signal, and utilize Density function theory that signal is divided The relation of the structure-Raman shift of son carries out a series of prediction and theoretical research, has obtained the chemical constitution in following table and has drawn Graceful displacement, it was demonstrated that when can effectively regulate the unimodal signaling molecule in arrowband when the alkynyl end of sulfydryl phenylacetylene is changed group The displacement of Raman signal.
Signaling molecule spectrum optimizes: with the silver-colored gold-covered nano particle of certain size for enhancing substrate, use 532nm to mercapto The Raman spectrum of base phenylacetylene molecule is optimized, and finds when golden nanometer particle adds sulfydryl phenylacetylene, can be at 1972cm-1 And 2105cm-1Place produces a Raman signal respectively, confirms that this is due to signaling molecule and increasing through Density function theory Spatial orientation difference at the bottom of strong basis causes, and finding to add TGA through research can be to signaling molecule and enhancing substrate Connected mode improve, result schematic diagram is as shown in Figure 1.
Signaling molecule SERS spectra: according to signaling molecule spectrum optimum results, select three kinds of arrowband unimodal signaling molecule B1, As a example by B2, B3, the actual SERS spectra obtained is as in figure 2 it is shown, the alkynyl Raman shift of three lays respectively at 2105cm-1、 2158cm-1、2212cm-1Place, can be effectively prevented from interference each other.
Embodiment 2
(1) AuNPs-labelling molecule: take 10mL silver gold-covered nano colloidal sol three parts, being added thereto to 10 μ L concentration respectively is The tetrahydrofuran solution of arrowband unimodal signaling molecule B1, B2, B3 of 1mM.
(2) TGA optimization: add the water of the TGA that 100 μ L concentration are 10mmol/L after above-mentioned solution left standstill 3h Solution.(this step only needs when the alkynyl end of signaling molecule does not replace, such as with B1 as signaling molecule).
(3) protection shell: continuously add polycyclic aromatic hydrocarbon (PAH) aqueous solution that 1mL concentration is 0.1wt ‰ after standing 3h again.
(4) biologically functional molecule connects: be simultaneously introduced after 12h 1-(3-dimethylamino-propyl) that 10 μ L concentration are 10mM- 3-ethyl-carbodiimide hydrochloride (EDC HCl) aqueous solution and N-hydroxy-succinamide (NHS) water that 20 μ L concentration are 1mM Solution, stands activated carboxyl, and adding 20 μ L concentration after 1h respectively in three parts of solution is the folic acid (FA) of 1mM, metakentrin Releasing hormone (LHRH), cell-penetrating peptides (CALNNR8) solution.
(5) centrifugal after standing a night collect and resuspended be about 10 to particle concentration12Individual/mL, puts in refrigerator and saves backup. The SERS probe that wherein FA and LHRH modifies has cell membrane targeting ability, CALNNR8The SERS probe modified has endoplasmic reticulum Targeting ability.
With B1 as signaling molecule, different amounts of TGA is used to be optimized: to take the B1-AuNPs nanometer of three parts of 10mL Particle, is separately added into the aqueous solution of the TGA that 0,1,10,100 μ L concentration are 10mmol/L, after standing three hours wherein The SERS spectra recorded, as shown in Figure 3.
Embodiment 3
Take 10mL particle concentration and be about 1012The silver-colored gold-covered nano colloidal sol of individual/mL, being added thereto to 20 μ L concentration is 1mM's B3 signaling molecule, continuously adds polycyclic aromatic hydrocarbon (PAH) aqueous solution that 1mL concentration is 0.1wt ‰ after standing 3h, continue to stand 12h Rear prepared SERS probe.
Take that 10mL is dispersed lily powder aqueous solution, and be added thereto to 1mLSERS probe, shaking table mixes After by pollen solution lyophilizing to remove moisture.
The sucrose solution of compounding high concentration dropping are a small amount of to microscope slide, dry rear top layer and form the sucrose of homogeneous transparent Layer.Take a small amount of pollen particles trickle down on sucrose layer and blow gently to apply pressure, make pollen be firmly adhered to sucrose table Raman image experiment can be carried out behind face.
With the flavonoid class material in pollen at 1580cm-1Raman signal be that Interference Peaks carries out confirmatory experiment.Can send out Existing, alkynyl signaling molecule is at 1580cm-1The Raman signal in lower wave number region is easy to and the Interference Peaks generation spectrum weight in pollen Folding phenomenon (see Fig. 4 (A)), this can produce greatly interference to final result, and uses 2212cm-1Marker peak then can be prevented effectively from Problems (see Fig. 4 (B)).Confirm the alkynyl SERS probe anti-interference energy to cellular endogenous material in cell imaging is studied Power.
Embodiment 4
Take 10mL particle concentration and be about 1012The silver-colored gold-covered nano colloidal sol three parts of individual/mL, is added thereto to 20 μ L concentration respectively Tetrahydrofuran solution for arrowband unimodal signaling molecule B1, B2, B3 of 1mM.Continuously adding 1mL concentration after standing 3h is Mercapto-polyglycol (HS-PEG) aqueous solution of 0.1wt ‰, can be prepared by corresponding SERS probe after standing 6h.
In batch cultur ware (35mm), put into the glass cover-slip (all through high-temperature sterilization) of a diameter of 20mm, take thin After the final step dilution that born of the same parents are passed on, finely dispersed cell suspending liquid (300～500 μ L) uniformly drops to coverslip surface.? After standing 2-4h in incubator, cell is the most adherent, adds 2.5mL fresh cell medium, waits 4h.
Take the attached cell that three parts of said methods prepare, and be separately added into three kinds of SERS probes wherein, in incubator Take out after continuing to co-culture 12h, use PBS buffer solution flush cover slide gently, remove and be adsorbed in surface of cell membrane and do not enter Enter the SERS probe in cell, imaging experiment can be carried out afterwards.
As it is shown in figure 5, three kinds of SERS probes all present good distribution situation intracellular, and each passage it Between entirely without impact, it was demonstrated that three kinds of alkynes SERS probes will not disturb each other.
Embodiment 5
Take 10mL particle concentration and be about 1012The silver-colored gold-covered nano colloidal sol three parts of individual/mL, is added thereto to 20 μ L concentration respectively Tetrahydrofuran solution for arrowband unimodal signaling molecule B1, B2, B3 of 1mM.Continuously adding 1mL concentration after standing 3h is The polypropylene amine aqueous solution of 0.1wt ‰.1-(3-the dimethylamino-propyl)-3-second that 10 μ L concentration are 10mM it is simultaneously introduced after 12h Base carbodiimide hydrochloride (EDC HCl) aqueous solution and N-hydroxy-succinamide (NHS) aqueous solution that 20 μ L concentration are 1mM. Stand that to add 20 μ L concentration after activated carboxyl 1h respectively in three parts of probes be that the folic acid (FA) of 1mM, metakentrin release swash Element (LHRH), cell-penetrating peptides (CALNNR8) solution.After standing a night, centrifugal collection can be prepared by three kinds of corresponding SERS spies Pin.
In batch cultur ware (35mm), put into the glass cover-slip (all through high-temperature sterilization) of a diameter of 20mm, take thin After born of the same parents pass on final step dilution, finely dispersed cell suspending liquid (300～500 μ L) uniformly drops to coverslip surface.In training Supporting after standing 2-4h in case, cell is the most adherent, adds 2.5mL fresh cell medium, waits 4h.
The complete adherent rear addition of cell has the SERS probe (B3-CLANNR of endoplasmic reticulum targeting ability8-NPs) 200 μ L are also Mix gently.Take out after incubator continuing co-culture 12h, use PBS buffer solution flush cover slide gently, remove absorption The endoplasmic reticulum targeted probes being introduced in surface of cell membrane in cell, then adds in 3mL fresh culture extremely little culture dish, And add two kinds of each 200 μ L of cell membrane targeting SERS probe (B1-FA-NPs and OPE1-LHRH-NPs).After continuing to cultivate 2-4h SERS imaging research can be carried out.
Fig. 6 is the more typical SERS spectra recorded in cell, it appeared that the signal of three kinds of labelling molecules can Most clearly distinguish, and will not be disturbed by cellular endogenous material completely.Fig. 7 is three kinds of probes warps in same cell Crossing the imaging results that detection actually obtains, three kinds of SERS probes all achieve selectively targeted according to the effect of biologically functional molecule Result.

Claims (10)

1. the preparation method without the Raman labels probe of optical interference, it is characterised in that comprise the following steps: Raman is dissipated Penetrate peak position at 1800-2400cm-1Wave number district, the unimodal signaling molecule in arrowband with a width of 1-2nm by Au-S key or Ag-S key from Being assembled into the surface strengthening substrate, the case material then using printing opacity hydrophilic carries out probe encapsulation, i.e. obtains without optical interference Raman labels probe；
Described arrowband unimodal Raman signal molecule, has a structure shown in formula I:
Wherein, R1、R2、R3、R4For-NO2、-CH3Or H, R5For H or-Si (CH3)3、-C2H5、-CH2CH2CH2OH, phenyl or alkynyl；
Described strengthens the nanoparticle that substrate is particle diameter 30-60nm, and described nanoparticle is (i) nanometer gold, (ii) nanometer Silver or (iii) be the core-shell structure nanometer particle as shell with nanometer gold or nanometer silver；
Described case material is the one in biomolecule, polymer, silicon dioxide.
The preparation method of the Raman labels probe without optical interference the most according to claim 1, it is characterised in that: R5For H's After the unimodal signaling molecule in arrowband self-assembles to the surface of enhancing substrate, with mercaptan carboxylic acid's signaling molecule unimodal to arrowband and enhancing base The connected mode at the end is optimized, and the case material then using printing opacity hydrophilic carries out probe encapsulation.
The preparation method of the Raman labels probe without optical interference the most according to claim 1 and 2, it is characterised in that: will Probe encapsulation after on case material grafting biological targeting molecule.
The preparation method of the Raman labels probe without optical interference the most according to claim 3, it is characterised in that: described Arrowband unimodal Raman signal molecule is
The preparation method of the Raman labels probe without optical interference the most according to claim 3, it is characterised in that: described Mercaptan carboxylic acid is one or more in TGA, mercaptopropionic acid, mercaptobutyric acid；Described biological targeting molecule be antibody, One in part, polypeptide.
The preparation method of the Raman labels probe without optical interference the most according to claim 5, it is characterised in that: described Antibody is luteinising hormone-releasing hormo, and described part is folic acid, and described polypeptide is cell-penetrating peptides or nuclear location peptide.
The preparation method of the Raman labels probe without optical interference the most according to claim 5, it is characterised in that: described Biological targeting molecule is grafted onto detecting probe surface by reacting with EDC-NHS or glutaraldehyde cross-linking.
8. the Raman labels probe without optical interference, it is characterised in that: by described in any one of claim 1-7 without optics The preparation method of the Raman labels probe of interference prepares.
9. the application in bio-imaging of the Raman labels probe without optical interference described in claim 8.
10. a highly sensitive ultrafast Raman image instrument, it is characterised in that: with the Raman without optical interference described in claim 8 Label probe is detection object, on the basis of conventional optical microscope, using laser as excitation source, configure high-precision soon Speed scanning sample stage, extracts the unimodal superpower Raman scattering signal in arrowband of probe, with overdelicate with super narrow band pass filter Avalanche photodide is detecting element, by the photon signal of the two-dimensional scan pointwise acquisition probe of sample stage and make pseudo-colours Process.
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