CN112410404A

CN112410404A - Open type two-photon nucleic acid probe and application thereof in FISH

Info

Publication number: CN112410404A
Application number: CN202010202893.3A
Authority: CN
Inventors: 饶军; 国立浩; 钭方芳; 郑智
Original assignee: Jiangxi Cancer Hospital (jiangxi Cancer Center)
Current assignee: Jiangxi Cancer Hospital (jiangxi Cancer Center)
Priority date: 2020-05-27
Filing date: 2020-05-27
Publication date: 2021-02-26
Anticipated expiration: 2040-05-27
Also published as: CN112410404B

Abstract

The invention discloses an open type two-photon nucleic acid probe and application thereof in FISH, wherein 8.0ml of 50.0mmol compound 1 and 18.1g of 100mmol compound 2 are selected to be suspended in 20ml of 1, 2-dichlorobenzene to obtain a mixture, the mixture is stirred and reacted for 14-16h at the temperature of 100-120 ℃, and 300ml of dichloromethane is added into the reaction mixture after the reaction solution is cooled to 20-25 ℃ to obtain suspended matters. The open type two-photon nucleic acid probe and the application thereof in FISH have good excitation wavelength permeability and fluorescence signal control performance, have the characteristics of strong color rendering property, good light stability and the like, realize the specific two-photon imaging of DNA in deep tissues or organs, and can be reformed on the premise of not changing the conjugated structure of a dye core, thereby being further developed into a corresponding biological detection probe for physiological and pathological researches.

Description

Open type two-photon nucleic acid probe and application thereof in FISH

Technical Field

The invention relates to the technical field of nucleic acid probes, in particular to an open type two-photon nucleic acid probe and application thereof in FISH.

Background

The spatial structure of DNA, i.e., the dynamic interaction between DNA and protein or RNA, regulates the export of genetic functions, e.g., the sub-nuclear localization of the genome can regulate gene expression, heterochromatin formation and DNA replication. Therefore, exploring the space-time regulation of DNA in cells has important significance for understanding dynamic changes under various physiological environments and verifying the relationship between nucleic acid distribution and physiological performance. Typically, the localization of the DNA of interest in a particular subcellular region is studied by fluorescence in situ hybridization. Although these techniques appear simple and effective, they require specimen fixation and are only suitable for in situ visualization. Fluorescent-labeled DNA binding proteins are often used in order to display specific genomic sites in living cells. Unfortunately, when unbound fluorophores or FPs diffuse freely, there is a significant background of fluorescence and can affect the localization, stability and behavior pattern of DNA. For higher signal-to-noise ratios, tens of motif repeats per target are required. Despite these limitations, these systems have been widely recognized and applied to cellular imaging. To reduce the signal background and provide more vital information, many imaging methods have been devised, such as molecular beacons, GFP recombination and quenching self-ligation. These methods are of great advantage due to the natural physiological environment in which endogenous nucleic acids are observed. However, none of these methods have been applied to study nucleic acid kinetics in vivo or in tissue. Aiming at the problems of low availability, limited sequence design, slow response of reactivity or conformation change, high background fluorescence intensity or low fluorescence reversibility and the like existing in the prior method, a novel fluorescence controllable probe is provided, which is called as an exciton control hybridization sensitive fluorescent oligonucleotide probe. Despite these advantages, due to the limited permeability of visible light, the probe is only suitable for imaging thin tissue sections, not satisfying monitoring of genomic information in deep tissues or organs.

Compared with the traditional single photon excitation technology, the two-photon excitation fluorescence microscope (TPM) has the advantages of small light damage, less photobleaching and high detection sensitivity. In TP, longer wavelength excitation light may also reduce cell damage. This technique may also reduce out-of-focus radiation. Despite the above advantages of two-photon technology in tissue organ imaging, current fluorescent probes do not specifically display target nucleic acids in organs. Therefore, there is still a lack of excellent nucleic acid probes for imaging and detecting target nucleic acids in tissues and organs.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an open type two-photon nucleic acid probe and application thereof in FISH, and solves the problem that the existing fluorescent probe cannot specifically display target nucleic acid in an organ.

In order to achieve the purpose, the invention is realized by the following technical scheme: an open type two-photon nucleic acid probe is prepared by the following steps:

s1, preparation of phenethyl cyan pigment:

a1, selecting 8.0ml, namely 50.0mmol of compound 1 and 18.1g, namely 100mmol of compound 2 to suspend in 20ml of 1, 2-dichlorobenzene to obtain a mixture, stirring and reacting the mixture at 100-120 ℃ for 14-16h, adding 300ml of dichloromethane into the reaction mixture when the reaction solution is cooled to 20-25 ℃ to obtain a suspended substance, staying the synthesized suspension at 20-25 ℃ for 2-2.2h, filtering the generated precipitate, washing with dichloromethane, and drying under reduced pressure to obtain a white powdery compound 3, wherein the reaction formula is shown as (1):

a2, selecting 1020mg of the compound 3 prepared in a1, namely 3.00mmol and 521mg of the compound 4 prepared in a1, namely 3.50mmol to be suspended in 30ml of acetic anhydride to obtain a suspension, stirring the obtained suspension at the temperature of 100-120 ℃ for reaction for 30-35min, slowly adding 10ml of distilled water into the reaction mixture, stirring at the temperature of 100-120 ℃ for 26-30min, evaporating the solvent, adding 100ml of acetone into the residue, filtering the precipitate, washing with acetone, and drying under reduced pressure to obtain a purple powdery compound D, wherein the reaction formula is shown as (2):

s2, preparation of the hybridization sensitive fluorescent nucleic acid probe capable of being excited by two photons:

b1, adding 4.6mg of 40 mu mole of N-hydroxysuccinimide and 7.7mg of 40 mu mole of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the DMF solution of 20 mu mole of the synthetic dye D prepared in a2, stirring for 14-16h at 20-25 ℃, and using the reaction mixture and the DNA oligomer for the next reaction without further purification;

b2, synthesizing DNA oligomer by adopting a DNA/RNA synthesizer and a traditional phosphoramidite method, coupling and synthesizing a nucleic acid probe by using commercially available phosphoramides corresponding to dA, dG, dC, dT and diamino modified nucleoside dU, cracking the synthesized DNA by using 28% ammonia water at 50-55 ℃ for 4.6-5H, removing the ammonia water in the reaction mixture by using a vacuum centrifugation method, purifying the corresponding nucleic acid product by using HPLC, wherein the column is a 5-ODA-H reverse column, the elution buffer solution is 0.1M buffer solution with pH7.0TEAA, the elution gradient lasts for 26-30min, the appearance gradient is 5-40%, and the flow rate is 3 ml/min;

b3, mixing DMF solution of dye D succinimide ester with sodium carbonate buffer solution dissolved with active amino nucleic acid, reacting at 23-25 deg.C for 1.5-1.7H, dissolving the reaction mixture in ethanol, centrifuging at-20 deg.C for 18-20min, removing supernatant, dissolving residual solid in small amount of water, filtering with 0.45um filter membrane, purifying corresponding nucleic acid coupled product by HPLC, wherein the column is 5-ODA-H reverse column, the elution buffer solution is 0.1M TEAA buffer solution with pH of 7.0, the elution gradient is continuous for 27-30min, 5-40% of current gradient, and the flow rate is 3 ml/min.

Preferably, the pH of the sodium carbonate buffer in step b3 is 9.

Preferably, the concentration of the fluorescent nucleic acid probe in the step b3 is measured by an ultraviolet spectrophotometer, and the molecular weight of the fluorescent nucleic acid probe is characterized by a MALDI-TOF mass spectrometer.

Preferably, the open two-photon nucleic acid probe is used for carrying out fluorescence imaging on cell line markers, and the cell line is preferably Hela cells.

Preferably, the open type two-photon nucleic acid probe is applied to the fluorescence imaging of organ markers, and the organ is preferably mouse embryonic brain.

Preferably, the open two-photon nucleic acid probe is used in FISH.

Advantageous effects

The invention provides an open type two-photon nucleic acid probe and application thereof in FISH. Compared with the prior art, the method has the following beneficial effects:

1. the open type two-photon nucleic acid probe prepared by the invention has good excitation wavelength permeability and fluorescence signal control performance, has the characteristics of strong color rendering property, good light stability and the like, realizes the specific two-photon imaging of DNA in deep tissues or organs, and can be reformed on the premise of not changing the core conjugated structure of a dye, thereby being further developed into a corresponding biological detection probe for physiological and pathological researches.

2. The two-photon fluorescence image display after the open type two-photon nucleic acid probe is marked is utilized, the nucleic acid probe can specifically show bright green fluorescence at a target nucleic acid position after entering cells through two-photon excitation, and the open type two-photon nucleic acid probe disclosed by the invention is proved to have excellent hybridization sensitive color rendering performance and two-photon fluorescence imaging characteristic.

3. The open type two-photon nucleic acid probe prepared by the invention has high signal-to-noise ratio, the signal of a target point can be identified without washing after the probe is hybridized, and the preparation method is particularly simple, convenient and quick.

Drawings

FIG. 1 is a structural diagram of a nucleic acid probe of the present invention;

FIG. 2 shows the absorption spectra before and after hybridization of the fluorescent nucleic acid probe 5 '-d (TTTTTTDTTTTTT) -3';

FIG. 3 shows excitation and emission spectra of a fluorescent nucleic acid probe 5 '-d (TTTTTTDTTTTTT) -3' before and after hybridization with complementary DNA;

FIG. 4 shows excitation and emission spectra before and after hybridization of the fluorescent nucleic acid probe 5 '-d (TACCAGDCCAT) -3' with complementary DNA;

FIG. 5 shows excitation and emission spectra of a fluorescent nucleic acid probe 5 '-d (TTTTTTDTTTTTT) -3' before and after hybridization with complementary RNA;

FIG. 6 shows excitation and emission spectra before and after hybridization of fluorescent nucleic acid probe 5 '-d (TACCAGDCCACAT) -3' with complementary RNA;

FIG. 7 shows excitation and emission spectra before and after hybridization of a fluorescent nucleic acid probe 5 '-mUmUmUmUmUmUmUmUmUmU-3' with complementary DNA;

FIG. 8 shows excitation and emission spectra before and after hybridization of a fluorescent nucleic acid probe 5 '-mUmUmUmUmUmUmUmUmUmU-3' with complementary RNA;

FIG. 9 is a circular dichroism spectrum of the fluorescent nucleic acid probe 5 '-d (TTTTTTDTTTT) -3' after hybridization with complementary DNA;

FIG. 10 is a photograph of telomere imaging based on wash-free two-photon fluorescence in situ hybridization;

FIG. 11 is a co-localized image of wash-free two-photon fluorescence in situ hybridization and conventional fluorescence in situ hybridization;

FIG. 12 is a photograph of telomeres in ex vivo mouse embryonic brain tissue based on wash-free two-photon fluorescence in situ hybridization.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-10, the present invention provides three technical solutions: a preparation method of an open type two-photon nucleic acid probe specifically comprises the following embodiments:

example 1

S1, preparation of phenethyl cyan pigment:

a1, selecting 8.0ml, namely 50.0mmol of compound 1 and 18.1g, namely 100mmol of compound 2, to suspend in 20ml of 1, 2-dichlorobenzene to obtain a mixture, stirring the mixture at 100 ℃ for reaction for 14h, adding 300ml of dichloromethane to the reaction mixture after the reaction solution is cooled to 20 ℃ to obtain a suspension, standing the synthesized suspension at 20 ℃ for 2h, filtering the generated precipitate, washing with dichloromethane, and drying under reduced pressure to obtain a white powdery compound 3, wherein the reaction formula is shown as (1):

a2, selecting 1020mg, namely 3.00mmol of compound 3 prepared in a1 and 521mg, namely 3.50mmol of compound 4 to suspend in 30ml of acetic anhydride to obtain a suspension, stirring the obtained suspension at 100 ℃ for reaction for 30min, slowly adding 10ml of distilled water into the reaction mixture, stirring at 100 ℃ for 26min, evaporating the solvent, adding 100ml of acetone into the residue, filtering the precipitate, washing with acetone, and drying under reduced pressure to obtain purple powdery compound D, wherein the reaction formula is shown as (2):

b1, adding 4.6mg of 40 mu mole N-hydroxysuccinimide and 7.7mg of 40 mu mole 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the DMF solution of 20 mu mole synthetic dye D prepared in a2, stirring for 14h at 20 ℃, and using the reaction mixture and DNA oligomer for the next reaction without further purification;

b2, synthesizing DNA oligomer by adopting a DNA/RNA synthesizer and a traditional phosphoramidite method, coupling and synthesizing a nucleic acid probe by using commercially available phosphoramides corresponding to dA, dG, dC, dT and diamino modified nucleoside dU, cracking the synthesized DNA by using 28% ammonia water at 50 ℃ for 4.6H, removing the ammonia water in the reaction mixture by using a vacuum centrifugation method, purifying the corresponding nucleic acid product by using HPLC, wherein the column is a 5-ODA-H reverse column, the elution buffer solution is 0.1M pH7.0TEAA buffer solution, the elution gradient lasts for 26min, the current gradient is 5-40%, and the flow rate is 3 ml/min;

b3, mixing DMF solution of dye D succinimide ester with sodium carbonate buffer solution dissolved with active amino nucleic acid, reacting for 1.5H at 23 ℃, wherein the pH value of the sodium carbonate buffer solution is 9, dissolving the reaction mixture in ethanol, centrifuging at high speed for 18min at-20 ℃, removing supernatant, dissolving residual solid in a small amount of water, filtering with a 0.45um filter membrane, purifying corresponding nucleic acid coupling products by HPLC, wherein the column is a 5-ODA-H reverse column, the elution buffer solution is 0.1M TEAA buffer solution with pH7.0, and the elution gradient is continuous for 27min, 5-40% of current gradient and 3ml/min of flow rate.

Example 2

S1, preparation of phenethyl cyan pigment:

a1, selecting 8.0ml, namely 50.0mmol of compound 1 and 18.1g, namely 100mmol of compound 2 to suspend in 20ml of 1, 2-dichlorobenzene to obtain a mixture, stirring the mixture at 110 ℃ for reaction for 15h, adding 300ml of dichloromethane to the reaction mixture when the reaction solution is cooled to 23 ℃ to obtain a suspended substance, staying the synthesized suspension at 23 ℃ for 2.1h, filtering the generated precipitate, washing with dichloromethane, and drying under reduced pressure to obtain a white powdery compound 3, wherein the reaction formula is shown as (1):

a2, selecting 1020mg, namely 3.00mmol of compound 3 prepared in a1 and 521mg, namely 3.50mmol of compound 4 to suspend in 30ml of acetic anhydride to obtain a suspension, stirring the obtained suspension at 110 ℃ for reaction for 33min, slowly adding 10ml of distilled water into the reaction mixture, stirring at 110 ℃ for 28min, evaporating the solvent, adding 100ml of acetone into the residue, filtering the precipitate, washing with acetone, and drying under reduced pressure to obtain purple powdery compound D, wherein the reaction formula is shown as (2):

b1, adding 4.6mg of 40 mu mole N-hydroxysuccinimide and 7.7mg of 40 mu mole 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the DMF solution of 20 mu mole synthetic dye D prepared in a2, stirring for 15h at 23 ℃, and using the reaction mixture and DNA oligomer for the next reaction without further purification;

b2, synthesizing DNA oligomer by using a DNA/RNA synthesizer and a traditional phosphoramidite method, coupling and synthesizing a nucleic acid probe by using commercially available phosphoramides corresponding to dA, dG, dC, dT and diamino modified nucleoside dU, cracking the synthesized DNA by using 28% ammonia water at 52 ℃ for 4.8H, removing the ammonia water in the reaction mixture by using a vacuum centrifugation method, purifying the corresponding nucleic acid product by using HPLC, wherein the column is a 5-ODA-H reverse column, the elution buffer solution is 0.1M pH7.0TEAA buffer solution, the elution gradient lasts for 28min, the current gradient is 5-40%, and the flow rate is 3 ml/min;

b3, mixing DMF solution of dye D succinimide ester with sodium carbonate buffer solution dissolved with active amino nucleic acid, reacting for 1.6H at 24 ℃, wherein the pH value of the sodium carbonate buffer solution is 9, dissolving the reaction mixture in ethanol, centrifuging at high speed for 19min at-20 ℃, removing supernatant, dissolving residual solid in a small amount of water, filtering with a 0.45um filter membrane, purifying corresponding nucleic acid coupling products by HPLC, wherein the column is a 5-ODA-H reverse column, the elution buffer solution is 0.1M TEAA buffer solution with pH of 7.0, and the elution gradient lasts for 29min, is 5-40% of current gradient, and has a flow rate of 3 ml/min.

Example 3

S1, preparation of phenethyl cyan pigment:

a1, selecting 8.0ml, namely 50.0mmol of compound 1 and 18.1g, namely 100mmol of compound 2, to suspend in 20ml of 1, 2-dichlorobenzene to obtain a mixture, stirring the mixture at 120 ℃ for reaction for 16h, adding 300ml of dichloromethane to the reaction mixture after the reaction solution is cooled to 25 ℃ to obtain a suspension, standing the synthesized suspension at 25 ℃ for 2.2h, filtering the generated precipitate, washing with dichloromethane, and drying under reduced pressure to obtain a white powdery compound 3, wherein the reaction formula is shown as (1):

a2, selecting 1020mg, namely 3.00mmol of compound 3 prepared in a1 and 521mg, namely 3.50mmol of compound 4 to suspend in 30ml of acetic anhydride to obtain a suspension, stirring the obtained suspension at 120 ℃ for 35min, slowly adding 10ml of distilled water into the reaction mixture, stirring at 120 ℃ for 30min, evaporating the solvent, adding 100ml of acetone into the residue, filtering the precipitate, washing with acetone, and drying under reduced pressure to obtain purple powdery compound D, wherein the reaction formula is shown as (2):

b1, adding 4.6mg of 40 mu mole of N-hydroxysuccinimide and 7.7mg of 40 mu mole of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the DMF solution of 20 mu mole of the synthetic dye D prepared in a2, stirring for 16h at 25 ℃, and using the reaction mixture and the DNA oligomer for the next reaction without further purification;

b2, synthesizing DNA oligomer by adopting a DNA/RNA synthesizer and a traditional phosphoramidite method, coupling and synthesizing a nucleic acid probe by using commercially available phosphoramides corresponding to dA, dG, dC, dT and diamino modified nucleoside dU, cracking the synthesized DNA by using 28% ammonia water at 55 ℃ for 5H, removing the ammonia water in the reaction mixture by using a vacuum centrifugation method, purifying the corresponding nucleic acid product by using HPLC, wherein the column is a 5-ODA-H reverse column, the elution buffer solution is 0.1M pH7.0TEAA buffer solution, the elution gradient lasts for 30min, the current gradient is 5% -40%, and the flow rate is 3 ml/min;

b3, mixing DMF solution of dye D succinimide ester with sodium carbonate buffer solution dissolved with active amino nucleic acid, reacting for 1.7H at 25 ℃, wherein the pH value of the sodium carbonate buffer solution is 9, dissolving the reaction mixture in ethanol, centrifuging at-20 ℃ for 20min at high speed, removing supernatant, dissolving residual solid in a small amount of water, filtering with a 0.45um filter membrane, purifying corresponding nucleic acid coupling products by HPLC, wherein the column is a 5-ODA-H reverse column, the elution buffer solution is 0.1M TEAA buffer solution with pH value of 7.0, and the elution gradient lasts for 30min, is 5-40% of current gradient, and has a flow rate of 3 ml/min.

Research on probe absorption spectrum, excitation spectrum, emission spectrum and circular dichroism spectrum:

detection of visible ultraviolet absorption, excitation, emission and circular dichroism spectra of the fluorescent nucleic acid probe prepared as described above (FIG. 1), simulated physiological conditions, experiments were performed at pH7.0 (PBS buffer), concentrations of the nucleic acid probe and complementary nucleic acid were performed at 1uM, absorption and emission of the DNA probe labeled with a double dye D were investigated before and after hybridization with the complementary DNA strand (FIGS. 2, 3 and 4), fluorescence emission was observed immediately after addition of the probe to the complementary nucleic acid solution, while emission was suppressed in the non-hybridized state, absorption spectra could be used to confirm the mechanism of fluorescence control, and the absorption band of the non-hybridized probe exhibited a blue shift compared to the hybridized probe, which blue shift indicated that the excited state was split due to H aggregation of dye D and was only allowed to a higher energy level, the excited state will rapidly transition to a lower energy level, but the path from this energy level to the ground state is not emissive, the exciton interaction of the non-hybridized dye suppresses the emission of fluorescence, and after hybridization with the target nucleic acid, the emission intensity will greatly increase with the dissociation of the polymer;

it is also apparent that when the target is RNA, the control of fluorescence emission by exciton interaction ((FIGS. 5 and 6), fluorescence shows shift of absorption band and conversion of fluorescence intensity upon hybridization with complementary RNA strand, and then, absorption, excitation and emission spectra before and after hybridization of fluorescent nucleic acid probe having 2' -OMe RNA as backbone with complementary DNA or RNA strand (FIGS. 7 and 8) are measured, the absorption spectrum of 2' -OM-RNA probe shows red shift of maximum peak after hybridization, the strong emission intensity of 2' -OME RNA probe is similar to that of DNA probe after hybridization with DNA, 2' -OME RNA probe shows better fluorescence ratio upon hybridization with complementary RNA than corresponding DNA probe, which should be attributed to the fact that 2' -OME RNA binds to complementary RNA sequence more closely than similar DNA oligonucleotide, therefore, modification of sugar group from hydrogen atom to methoxy group does not inhibit control of fluorescence by exciton interaction of dye in probe, the fluorescence control effect on RNA imaging is better;

the fluorescence control of the fluorescent nucleic acid probe is due to the dye D on the probe being intercalated into the hybridized nucleic acid double strand, which is further confirmed by the Circular Dichroism (CD) data of the hybridized probe (FIG. 9), and in the CD spectrum, an induced CD peak in the range of 450-550nm, which is a characteristic signal of the dye intercalated into the double strand nucleic acid, can be observed.

Washing-free fluorescent in situ hybridization:

because of the high fluorescent signal-to-noise ratio of the novel fluorescent nucleic acid probe, tedious and time-consuming washing steps can be avoided, parallel experiments (fig. 10) on telomeres were synthesized and applied with Telo-FAM and Telo-D in order to compare the performance of the hybridization sensitive probe with that of the conventional FISH probe, and experiments revealed that, without washing steps, Telo-FAM only showed noise, telomere localization patterns could not be identified, while Telo-D showed scattered fluorescence spots, and when the Telo-D fluorescent probe was used, characteristic fluorescence distribution patterns were observed regardless of whether washing steps were performed or not.

Washing-free co-localization fluorescence in situ hybridization:

to test the specificity of wash-free fluorescent signals, we introduced complementary telomere probes Telo-FAM-L and Telo-D into immobilized MEF7 cells, the Telo-FAM-L in situ hybridization used a traditional FISH protocol with stringent washes, while Telo-D was a simple FISH protocol without washes (FIG. 11), and after applying Telo-D in combination with Telo-FAM-L in the traditional FISH protocol, co-localized FISH signals were detected, indicating that the wash-free fluorescent in situ hybridization Telo-D signals were from telomere hybridization.

Fluorescence in situ hybridization of isolated mouse embryonic brain tissue:

a method for rapidly and reliably detecting a deep tissue genome sequence is helpful for diagnosis, in order to discuss the possibility that a novel two-photon hybridization sensitive fluorescent probe is applied to two-photon excitation FISH imaging in a tissue section, a simple 1030nm two-photon excitation FISH method is introduced into an adult mouse brain section, the change of the positions of fluorescent points at different depths of the brain section is found under a two-photon microscope (figure 12), and a characteristic telomere distribution mode is observed in a defined region, so that the washing-free fluorescence in situ hybridization based on the two-dye D modified probe is also suitable for DNA imaging in the tissue section.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. An open two-photon nucleic acid probe, comprising: the preparation method specifically comprises the following steps:

s1, preparation of phenethyl cyan pigment:

2. The two-photon nucleic acid probe of claim 1, wherein: the pH value of the sodium carbonate buffer solution in the step b3 is 9.

3. The two-photon nucleic acid probe of claim 1, wherein: the concentration of the fluorescent nucleic acid probe in the step b3 is measured by an ultraviolet spectrophotometer, and the molecular weight of the fluorescent nucleic acid probe is characterized by a MALDI-TOF mass spectrometer.

4. The two-photon nucleic acid probe of claim 1, wherein: the use of the open two-photon nucleic acid probe in fluorescence imaging of a cell line marker, preferably Hela cells.

5. The two-photon nucleic acid probe of claim 1, wherein: the open type two-photon nucleic acid probe is applied to the fluorescence imaging of organ markers, and the organ is preferably mouse embryonic brain.

6. The two-photon nucleic acid probe of claim 1, wherein: the open type two-photon nucleic acid probe is applied to FISH.