CN106367510B - A kind of preparation method and application of the satellite shape Nanoscale assemblies for cancer markers double check intracellular - Google Patents
A kind of preparation method and application of the satellite shape Nanoscale assemblies for cancer markers double check intracellular Download PDFInfo
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Abstract
A kind of preparation method and application of the satellite shape Nanoscale assemblies for cancer markers double check intracellular, belongs to material chemistry technical field.The present invention includes: the preparation of one, satellite shape Nanoscale assemblies: the synthesis of gold nanorods, the preparation of 20 nm up-conversion nanoparticles, the assembling of golden stick dimer, Raman beacon molecule DTTC is coupled on golden stick dimer, golden stick dimer core-above converts the assembling for defending star topology；Two, cancer markers double check intracellular: golden stick dimer core-above converts satellite shape Nanoscale assemblies modification cell-penetrating peptide, golden stick dimer core-above converts the detection intracellular of satellite shape Nanoscale assemblies, golden stick dimer core-above converts the characterization of satellite shape Nanoscale assemblies detection intracellular, establish standard curve and etc. realization.The present invention provides the content of miRNA21 intracellular and the active method of Telomerase can be detected simultaneously by Raman and fluorescent dual signal, prepare that structure is uniform, the golden stick dimer core-of good biocompatibility above converts satellite shape Nanoscale assemblies.
Description
Technical field
The present invention relates to a kind of preparation methods of satellite shape Nanoscale assemblies for cancer markers double check intracellular
And application, belong to material chemistry technical field.
Background technique
MicroRNA is the single-stranded endogenous non-coding RNA of a kind of 18-25 nucleotide, is played in life science important
Regulating and controlling effect.The expression change level of miRNA is generally believed at present and changes expression and genetic disease and changes in immune function
Related, miRNA is especially commonly used in the diagnosis of many entity diversification tumours, and the expression of one or more miRNAs
As important diagnostic and prognosis biomarker.Traditional technology, time-consuming and laborious such as genetic chip and real-time quantitative PCR, labour
Intensity is big, and expensive, and which has limited their applications.Therefore, in situ quantitation in non-invasive and maneuverable living cells
Detection method has caused a large amount of concern.
Telomerase is a kind of ribonucleoprotein complexes, is divided into template using it with ribonucleic acid group possessed by itself and ties up
Hold the length of telomere, Telomerase high expression in primary tumor, and the immortality in these tumour cells.Therefore, Telomerase has
There is the important biomolecule marker for being acknowledged as early clinical diagnosis.Based on conventional polymeric enzyme chain reaction (PCR)
Telomeric repeatamplification protocol (TRAP) due to amplification program basis, including length cut out telomerase product distribution, false positive
As a result, the Telomerase of active complexity may be can't detect with primer dimer problem.Recently, in mesoporous silicon dioxide nano
The activity of quantization cell Telomerase has been used in grain duct with fluorescence probe prepared by DNA.However, its is limited
Sensibility and applicability be still an important problem in complex matrix.
Therefore constructing that a kind of structure is uniform, the golden stick dimer core-of good biocompatibility above converts satellite shape nanostructure,
The active detection of the content and Telomerase of realizing miRNA21 intracellular simultaneously by Raman and fluorescent dual signal is meaningful,
And it does not have been reported that also at present.
Summary of the invention
The object of the present invention is to provide the sides that a kind of satellite shape Nanoscale assemblies are used for cancer markers double check intracellular
Method, while detecting the content of miRNA21 intracellular and the active method of Telomerase.
Technical solution of the present invention, a kind of satellite shape Nanoscale assemblies are used for the side of cancer markers double check intracellular
Method, steps are as follows:
One, the preparation of satellite shape Nanoscale assemblies:
(1) lateral plasmon absorption peaks control the synthesis of gold nanorods: is synthesized in the Jenner of 800nm using crystal seed growth method
Rice stick GNR；
(2) assembling of golden stick dimer: the gold nanorods GNR that step (1) is obtained modifies mercapto-polyglycol SH-PEG-
1000, the sulfydryl DNA1 containing microRNA21 antisense base sequences and mutual with the part DNA1 is modified in centrifugation respectively again after being resuspended
The sulfydryl DNA2 sequence of benefit respectively obtains GNR-DNA1 and GNR-DNA2 complex, then two kinds of compounds is mixed, and obtains golden stick
Dimer；
(3) Raman beacon molecule DTTC is coupled on golden stick dimer: the golden stick dimer of step (2) preparation and Raman are believed
It marks molecule 3.3 '-diethyl thioaldehydes tricarbocyanine iodine DTTC to mix, obtains the golden stick dimer-that surface modification has DTTC after 8h
DTTC complex；
(4) assembling of star topology: the golden stick dimer-DTTC that step (3) is obtained is defended in golden stick dimer core-upper conversion
After centrifugation is resuspended, modification and linker DNA on the upper conversion nano particle of primer sequence TE primer, 20nm of Telomerase are modified
The sulfydryl mismatch DNA of partial complementarity, being assembled into golden stick dimer core-by linker DNA, above star topology is defended in conversion
Assembly, then assembly is passed through into gradient centrifugation purification；
Two, cancer markers double check intracellular:
(5) above conversion satellite shape Nanoscale assemblies modify cell-penetrating peptide: the golden stick that step (4) is obtained to golden stick dimer core-
Above star topology assembly and SH-PEG-5000 are defended in conversion to dimer core-and cell-penetrating peptide TAT is mixed, and are obtained surface modification and are worn
Above star topology assembly is defended in conversion to the golden stick dimer core-of film peptide；
(6) golden stick dimer core-above conversion satellite shape Nanoscale assemblies detection intracellular: the golden stick that step (5) is obtained
Dimer core-above converts satellite shape Nanoscale assemblies and cell is incubated for 8h altogether, when existing simultaneously determinand miRNA21 and Telomerase
When, the structure of assembly changes, and gold nanorods and up-conversion nanoparticles are dissociated from assembly, cause respectively Raman and
The variation of fluorescence signal, and then detected；
(7) above the detection intracellular of conversion satellite shape Nanoscale assemblies characterizes golden stick dimer core-: step (6) being obtained thin
Born of the same parents carry out Raman Characterization and fluorescence imaging simultaneously, and establish standard curve.
The method that the satellite shape Nanoscale assemblies are used for cancer markers double check intracellular, the specific steps are as follows:
One, the preparation of satellite shape Nanoscale assemblies:
(1) synthesis of gold nanorods:
A, crystal seed synthesizes: at room temperature, by the three hydration tetra chlorauric acids that 0.05mL concentration is 10mM, being added to the 0.2M of 1mL
Cetyl trimethylammonium bromide solution in, solution colour becomes yellowish-brown by colourless, and 0.12mL is then added and newly prepares
0.01M sodium borohydride solution, quickly stirs 2min, and solution colour becomes light brown from yellowish-brown；
B, gold nanorods are grown: the three hydration tetra chlorauric acids of the 1mM of 5mL are added to 5mL, 0.2M cetyl trimethyl bromine
Change in ammonium salt solution, the ultrapure water of 4mL is added, mixes；The 0.01M silver nitrate solution of 0.125mL is added to above-mentioned mixture again
In system, mix；Then 70 μ L, 0.1M ascorbic acid solution are added, are vigorously stirred, solution becomes colorless, and after 2min, is added 12
The crystal seed of μ L step a preparation, stirs 20s, is put into 30 DEG C of water-baths, 2h；
(2) assembling of golden stick dimer: its centrifugal concentrating is resuspended in using the gold nanorods that crystal seed growth method synthesizes
In 5mM cetyl trimethylammonium bromide (CTAB) solution, make the concentration 5nM of gold nanorods；Gold nanorods and sulfydryl are gathered
Ethylene glycol PEG-1000 is mixed with the ratio of 1 ︰ 100 of molar concentration, is stood 12h, is taken the gold nano of 100 μ L modified PEG later
Stick mixes it with the sulfydryl DNA1 containing microRNA21 antisense base sequences with the ratio of 1 ︰ 40 of molar concentration；Separately take
The gold nanorods of 100 μ L modified PEG and the sulfydryl DNA2 sequence of DNA1 partial complementarity are mixed with the ratio of 1 ︰ 40 of molar concentration；
It is separately added into the NaCl solution of final concentration of 50mM, after being sufficiently mixed, after incubation at room temperature overnight, is centrifuged in 3 removing solution not
The DNA of reaction is resuspended in respectively in the 5mM CTAB solution of 100 μ L, then GNR-DNA1 the and GNR-DNA2 complex that will be obtained
Isometric bulk crossing, it is that 50mM carries out aging that NaCl solution to NaCl concentration, which is added, is incubated for 12h at room temperature, obtains golden stick two
Aggressiveness, for use；
(3) Raman beacon molecule DTTC is coupled on golden stick dimer: by Raman beacon 3.3 '-diethyl sulfide of molecule of 1mM
Aldehyde tricarbocyanine iodine (DTTC) is added in the golden stick dimerization liquid solution of above-mentioned steps (2) preparation, is uniformly mixed, and keeps DTTC whole
Concentration is 10 μM, is incubated at room temperature 8h；
(4) assembling of star topology is defended in golden stick dimer core-upper conversion: step (3) has been modified Raman beacon molecule
Golden stick dimer centrifugation is resuspended in 5mM CTAB solution, with the primer TE of the ratio modification Telomerase of 1 ︰ 500 of molar concentration
Primer sequence, after overnight incubation, centrifugation is resuspended in 5mM CTAB solution；The amine-modified water solubility of the maleimide of 20 nm
Concentration dilution is 5nM with the tris buffer of 10mM pH 7.4 by upper conversion nano particle, with 1 ︰ 5 of molar concentration modification with
The sulfydryl mismatch DNA of linkerDNA partial complementarity, the unbonded mismatch DNA of ultrafiltration removal after overnight incubation,
It is resuspended with the tris buffer of 10mM pH 7.4 stand-by；The golden stick dimer and 200 μ L of 50 μ L TE primer modification
The upper conversion nano particle of mismatch DNA modification mixes, and is assembled into golden stick dimer by 5 μ L, 10 μM of linker DNA
Above star topology assembly is defended in conversion to core-, then assembly is passed through gradient centrifugation purification；
Two, cancer markers double check intracellular:
(5) golden stick dimer core-above converts satellite shape structural modification cell-penetrating peptide: the golden stick dimer that step (4) is obtained
Above conversion defends star topology group dress body ︰ PEG5000 ︰ cell-penetrating peptide TAT with the ratio mixing of 1 ︰ of molar concentration, 1000 ︰ 100, room to core-
After temperature is incubated for 12h, 7500rpm is centrifuged 10min, removes supernatant, and precipitating is resuspended in cell culture fluid；
(6) golden stick dimer core-above conversion satellite shape Nanoscale assemblies detection intracellular: surface modification has the gold of cell-penetrating peptide
Stick dimer core-above defend star topology assembly and can be directly entered in cell and detected by conversion, when there are determinand miRNA21
When, in conjunction with the microRNA21 antisense nucleoside acid fragment in sulfydryl DNA1 sequence, caused gold stick dimer is dismissed, with
The increase of miRNA21 concentration, Raman signal gradually weaken；When there are determinand Telomerase and dNTP, TE primer can be along
LinkerDNA transcription, and the position for replacing mismatchDNA complementary with linkerDNA, cause conversion nano particle from assembling
It is dissociated on body, with the increase of telomerase activation, fluorescence signal is gradually recovered；When existing simultaneously determinand miRNA21 and telomere
When enzyme, gold nanorods and up-conversion nanoparticles are dissociated from assembly, cause the variation of Raman and fluorescence signal respectively, in turn
Carry out detection characterization；
(7) above the detection intracellular of conversion satellite shape Nanoscale assemblies characterizes golden stick dimer core-: the gold that step (6) is obtained
Stick dimer core-above defend star topology assembly and through not same amount transfection agents Transfected cells and without the cell after transfection by conversion
After being incubated for 8h altogether respectively, with 1mL trypsin digestion and cell, obtains golden stick dimer core-and above convert satellite shape structure detection
Cell suspension after miRNA21 concentration then carries out Raman Characterization, establishes difference miRNA21 concentration and Raman in cell
The standard curve of signal strength between the two；
After the golden stick dimer core-that step (6) obtains above is converted satellite shape Nanoscale assemblies and not same amount EGCG inhibition
After not inhibited cell is incubated for 8h altogether respectively, obtains golden stick dimer core-and above convert satellite shape structure detection telomere enzyme activity
Property after cell, then carry out fluorescence imaging, establish in cell different telomerase activations and the standard of fluorescence intensity between the two
Curve.
The DNA1 sequence is as shown in SEQ ID NO. 1, and DNA2 sequence is as shown in SEQ ID NO. 2, TE primer sequence
Column are as shown in SEQ ID NO. 3, and mismatch DNA sequence dna is as shown in SEQ ID NO. 4, linker DNA sequence dna such as SEQ
Shown in ID NO. 5, TAT polypeptide sequence is as shown in SEQ ID NO. 6.It is specific as shown in table 1.
Table 1
Beneficial effects of the present invention: the present invention has prepared that structure is uniform, on the golden stick dimer core-of good biocompatibility
It converts satellite shape nanostructure and assembles body, providing by Raman and fluorescent dual signal while can detect miRNA21 intracellular
Content and Telomerase active method, establish telomere in intracellular miRNA21 concentration and Raman signal intensity and cell
Standard curve between enzymatic activity and fluorescence intensity has high sensitivity, selectivity good, the advantage that detection limit is low, the used time is short, tool
There is extraordinary actual application prospect.
Detailed description of the invention
Fig. 1 is golden stick dimer (a) of the invention, upper conversion nano particle (b), gold stick dimer core-above convert satellite
The transmission electron microscope photo of shape Nanoscale assemblies (c).
Fig. 2 is that above conversion satellite shape Nanoscale assemblies enter through not same amount transfection agents turn golden stick dimer core-in the present invention
After dye in cell and cell without transfection, the Raman spectrogram (a) and Raman for detecting difference miRNA21 content intracellular are believed
Standard curve (b) number with miRNA21 content intracellular.
Fig. 3 is that above conversion satellite shape Nanoscale assemblies enter through not same amount EGCG inhibition golden stick dimer core-in the present invention
Afterwards and in not inhibited cell, detect different telomerase activations intracellular fluorescence imaging figure (a) and fluorescence signal with it is intracellular
The standard curve (b) of telomerase activation.
Specific embodiment
Biomaterial is purchased from Sangon Biotech (Shanghai) Co., Ltd. in following embodiment.
Embodiment 1
All glass apparatus are all impregnated for 24 hours with chloroazotic acid, and are cleaned with distilled water, are dried spare.Water used in experiment
It is the Milli-Q ultrapure water of 18.2M Ω.
(1) synthesis of gold nanorods:
A, crystal seed synthesizes: at room temperature, by the three hydration tetra chlorauric acids that 0.05mL concentration is 10mM, being added to the 0.2M of 1mL
Cetyl trimethylammonium bromide solution in, solution colour becomes yellowish-brown by colourless, and 0.12mL is then added and newly prepares
0.01M sodium borohydride solution, quickly stirs 2min, and solution colour becomes light brown from yellowish-brown；
B, gold nanorods are grown: the three hydration tetra chlorauric acids of the 1mM of 5mL are added to 5mL, 0.2M cetyl trimethyl bromine
Change in ammonium salt solution, the ultrapure water of 4mL is added, mixes；The 0.01M silver nitrate solution of 0.125mL is added to above-mentioned mixture again
In system, mix；Then 70 μ L, 0.1M ascorbic acid solution are added, are vigorously stirred, solution becomes colorless, and after 2min, is added 12
The crystal seed of μ L stirs 20s, is put into 30 DEG C of water-baths, 2h；
(2) its centrifugal concentrating the assembling of golden stick dimer: is resuspended in 5 using the gold nanorods that crystal seed growth method synthesizes
In mM CTAB solution, make gold nanorods concentration 5nM；By gold nanorods and mercapto-polyglycol PEG-1000 with molar concentration
The ratio of 1 ︰ 100 mixes, and stands 12h, take later the gold nanorods of 100 μ L modified PEG by its with it is anti-containing microRNA21
The sulfydryl DNA1 of sense nucleotide sequence is mixed with the ratio of 1 ︰ 40 of molar concentration；Separately take 100 μ L modification PEG gold nanorods with
The sulfydryl DNA2 sequence of DNA1 partial complementarity is mixed with the ratio of 1 ︰ 40 of molar concentration, is separately added into the NaCl solution of 50mM, is filled
After dividing mixing, after incubation at room temperature overnight, it is centrifuged unreacted DNA in 3 removing solution, is resuspended in 5 mM of 100 μ L respectively
In CTAB solution, then the isometric bulk crossing of GNR-DNA1 and GNR-DNA2 complex that will be obtained, be added NaCl solution to its
Final concentration of 50mM carries out aging, is incubated for 12 h at room temperature, obtains golden stick dimer, for use.
(3) Raman beacon molecule DTTC is coupled on golden stick dimer: by Raman beacon 3.3 '-diethyl sulfide of molecule of 1 mM
Aldehyde tricarbocyanine iodine DTTC is added in the golden stick dimerization liquid solution of above-mentioned steps (2) preparation, is uniformly mixed, is kept its final concentration
It is 10 μM, is incubated at room temperature 8h.
(4) assembling of star topology is defended in golden stick dimer core-upper conversion: step (3) has been modified Raman beacon molecule
Golden stick dimer centrifugation is resuspended in 5 mM CTAB solution, and the primer TE of upper Telomerase is modified with the ratio of 1 ︰ 500 of molar concentration
Primer sequence, after overnight incubation, centrifugation is resuspended in 5 mM CTAB solution；The amine-modified water solubility of the maleimide of 20 nm
Upper conversion nano particle is diluted to 5nM with the tris buffer of 10 mM pH 7.4, modifies upper and linker with 1 ︰ 5 of molar concentration
The sulfydryl mismatch DNA of DNA partial complementarity, the unbonded mismatch DNA of ultrafiltration removal after overnight incubation, with 10 mM
The tris buffer of pH 7.4 is resuspended stand-by；The golden stick dimer and 200 μ L mismatch DNA of 50 μ L TE primer modification
The upper conversion nano particle mixing of modification, is assembled into golden stick dimer core-by 5 μ L, 10 μM of linker DNA and above converts satellite
Shape structure assembly, then assembly is passed through into gradient centrifugation purification.Obtained golden stick dimer, upper conversion nano particle, golden stick
The transmission electron microscope photo that dimer core-above converts satellite shape Nanoscale assemblies is as shown in Figure 1.
(5) golden stick dimer core-above converts satellite shape structural modification cell-penetrating peptide: the golden stick dimer that step (4) is obtained
Above conversion defends star topology assembly and PEG5000 and cell-penetrating peptide TAT with the molar concentration mixing of 1 ︰, 1000 ︰ 100, room temperature to core-
After being incubated for 12h, 7500 rpm are centrifuged 10 min, remove supernatant, and precipitating is resuspended in cell culture fluid.
(6) golden stick dimer core-above conversion satellite shape Nanoscale assemblies detection intracellular: surface modification has the gold of cell-penetrating peptide
Stick dimer core-above defend star topology assembly and can be directly entered in cell and detected by conversion, when there are determinand miRNA21
When, in conjunction with the microRNA21 antisense nucleoside acid fragment in sulfydryl DNA1 sequence, caused gold stick dimer is dismissed, with
The increase of miRNA21 concentration, Raman signal gradually weaken；When there are determinand Telomerase and dNTP, TE primer can be along
LinkerDNA transcription, and the position for replacing mismatchDNA complementary with linkerDNA, cause conversion nano particle from assembling
It is dissociated on body, with the increase of telomerase activation, fluorescence signal is gradually recovered；When existing simultaneously determinand miRNA21 and telomere
When enzyme, gold nanorods and up-conversion nanoparticles are dissociated from assembly, cause the variation of Raman and fluorescence signal respectively, in turn
Carry out detection characterization.
(7) above the detection intracellular of conversion satellite shape Nanoscale assemblies characterizes golden stick dimer core-: will turn through not same amount transfection agents
After dye cell and without transfection after cell carry out quantifying for miRNA21 concentration intracellular with real-time fluorescence PCR；To not same amount EGCG
After inhibition and not inhibited cell pyrolysis liquid with ELISA standard curve carries out quantifying for telomerase activation intracellular.By step (6)
Obtained golden stick dimer core-above converts satellite shape Nanoscale assemblies and through not same amount transfection agents Transfected cells and without transfection
After cell afterwards is incubated for 8h altogether respectively, with 1mL trypsin digestion and cell, obtaining golden stick dimer core-, above star knot is defended in conversion
Structure detects the cell suspension after miRNA21 concentration, then carries out Raman Characterization, establishes difference miRNA21 concentration in cell
It is as shown in Figure 2 with the standard curve of Raman signal intensity between the two；The golden stick dimer core-that step (6) is obtained above is converted
It defends after star topology assembly inhibits with not same amount EGCG and after not inhibited cell is incubated for 8h altogether respectively, obtains golden stick dimerization
Body core-above converts the cell after satellite shape structure detection telomerase activation, then carries out fluorescence imaging, establishes different ends in cell
Telomerase activity and the standard curve of fluorescence intensity between the two are as shown in Figure 3.
SEQ ID NO.1:
DNA1:5 '-AAAAATCATC TATCAACATC AGTCTGATAA GCTATAGAAG C-3'；
SEQ ID NO.2:
DNA2:5 '-AAAAAGCTTG A-3 '；
SEQ ID NO.3:
TE primer:5 '-TTTTTTAATC CGTCGAGCAG AGTT-3'；
SEQ ID NO.4:
Mismatch DNA:5 '-ATAGGTATAG GGTTTTTT-3'；
SEQ ID NO.5:
Linker DNA:5 '-CCCTAACCCT AA AACTCTGC TCGACGGATT-3 '；
SEQ ID NO.6:
TAT:5 '-YGRKKRRQRR RC-3 '.
Claims (2)
1. a kind of preparation method of the satellite shape Nanoscale assemblies for cancer markers double check intracellular, it is characterised in that step
It is rapid as follows:
(1) lateral plasmon absorption peaks control the synthesis of gold nanorods: is synthesized in the gold nanorods of 800nm using crystal seed growth method
GNR；
(2) assembling of golden stick dimer: the gold nanorods GNR that step (1) is obtained modifies mercapto-polyglycol SH-PEG-
1000, the sulfydryl DNA1 containing microRNA21 antisense base sequences and mutual with the part DNA1 is modified in centrifugation respectively again after being resuspended
The sulfydryl DNA2 sequence of benefit respectively obtains GNR-DNA1 and GNR-DNA2 complex, then two kinds of compounds is mixed, and obtains golden stick
Dimer；
(3) Raman beacon molecule DTTC is coupled on golden stick dimer: by the golden stick dimer and Raman beacon point of step (2) preparation
3.3 '-diethyl thioaldehydes tricarbocyanine iodine DTTC of son are mixed, and obtaining surface modification after 8h has the golden stick dimer-DTTC of DTTC multiple
It is fit；
(4) assembling of star topology is defended in golden stick dimer core-upper conversion: the golden stick dimer-DTTC that step (3) is obtained is centrifuged
After resuspension, the primer TE primer sequence of Telomerase is modified；Modification and the part linker DNA on the upper conversion nano particle of 20nm
Complementary sulfydryl mismatch DNA；Being assembled into golden stick dimer core-by linker DNA, above star topology assembling is defended in conversion
Body, then assembly is passed through into gradient centrifugation purification；
(5) above conversion satellite shape Nanoscale assemblies modify cell-penetrating peptide: the golden stick dimerization that step (4) is obtained to golden stick dimer core-
Above star topology assembly and SH-PEG-5000 are defended in conversion to body core-and cell-penetrating peptide TAT is mixed, and obtaining surface modification has cell-penetrating peptide
Golden stick dimer core-above conversion defend star topology assembly；
The DNA1 sequence is as shown in SEQ ID NO.1, and DNA2 sequence is as shown in SEQ ID NO.2, and TE primer sequence is such as
Shown in SEQ ID NO.3, mismatch DNA sequence dna is as shown in SEQ ID NO.4, linker DNA sequence dna such as SEQ ID NO.5
Shown, TAT polypeptide sequence is as shown in SEQ ID NO.6.
2. the preparation side for the satellite shape Nanoscale assemblies of cancer markers double check intracellular according to claim 1
Method, it is characterised in that specific step is as follows:
(1) synthesis of gold nanorods:
A, crystal seed synthesizes: at room temperature, by the three hydration tetra chlorauric acids that 0.05mL concentration is 10mM, being added to the ten of the 0.2M of 1mL
In six alkyl trimethyl ammonium bromide solution, solution colour becomes yellowish-brown by colourless, and the 0.01M that 0.12mL is newly prepared then is added
Sodium borohydride solution, quickly stirs 2min, and solution colour becomes light brown from yellowish-brown；
B, gold nanorods are grown: the three hydration tetra chlorauric acids of the 1mM of 5mL are added to 5mL, 0.2M cetyl trimethylammonium bromide
In solution, the ultrapure water of 4mL is added, mixes；The 0.01M silver nitrate solution of 0.125mL is added in above-mentioned mixed system again,
It mixes；Then 70 μ L, 0.1M ascorbic acid solution are added, are vigorously stirred, solution becomes colorless, and after 2min, 12 μ L step is added
The crystal seed of rapid a preparation, stirs 20s, is put into 30 DEG C of water-baths, 2h；
(2) its centrifugal concentrating the assembling of golden stick dimer: is resuspended in 5mM using the gold nanorods that crystal seed growth method synthesizes
In cetyl trimethylammonium bromide CTAB solution, make the concentration 5nM of gold nanorods；By gold nanorods and mercapto-polyglycol
PEG-1000 is mixed with the ratio of 1 ︰ 100 of molar concentration, stands 12h, take later the gold nanorods of 100 μ L modification PEG by its with
Sulfydryl DNA1 containing microRNA21 antisense base sequences is mixed with the ratio of 1 ︰ 40 of molar concentration；100 μ L are separately taken to modify
The gold nanorods of PEG and the sulfydryl DNA2 sequence of DNA1 partial complementarity are mixed with the ratio of 1 ︰ 40 of molar concentration；It is separately added into end
Concentration is the NaCl solution of 50mM, after being sufficiently mixed, after incubation at room temperature overnight, is centrifuged unreacted DNA in 3 removing solution,
It is resuspended in the 5mM CTAB solution of 100 μ L respectively, then obtained GNR-DNA1 and GNR-DNA2 complex is mixed in equal volume
Hybridization, it is that 50mM carries out aging that NaCl solution to NaCl concentration, which is added, is incubated for 12h at room temperature, obtains golden stick dimer, for use；
(3) Raman beacon molecule DTTC is coupled on golden stick dimer: by Raman beacon 3.3 '-diethyl of molecule thioaldehydes three of 1mM
Carbon cyanines iodine DTTC is added in the golden stick dimerization liquid solution of above-mentioned steps (2) preparation, is uniformly mixed, and keeps DTTC final concentration of
10 μM, it is incubated at room temperature 8h；
(4) assembling of star topology is defended in golden stick dimer core-upper conversion: by the golden stick two of step (3) modification Raman beacon molecule
Aggressiveness centrifugation is resuspended in 5mM CTAB solution, with the primer TE primer sequence of the ratio modification Telomerase of 1 ︰ 500 of molar concentration
It arranges, after overnight incubation, centrifugation is resuspended in 5mM CTAB solution；It converts and receives in the amine-modified water solubility of the maleimide of 20 nm
Concentration dilution is 5nM with the tris buffer of 10mM pH 7.4 by rice grain, with 1 ︰ 5 of molar concentration modification and linkerDNA
The sulfydryl mismatch DNA of partial complementarity, the unbonded mismatch DNA of ultrafiltration removal after overnight incubation, with 10mM pH
7.4 tris buffer is resuspended stand-by；The golden stick dimer of 50 μ L TE primer modification is repaired with 200 μ L mismatch DNA
The upper conversion nano particle mixing of decorations, is assembled into golden stick dimer core-by 5 μ L, 10 μM of linker DNA and above converts satellite shape
Structure assembly, then assembly is passed through into gradient centrifugation purification；
(5) golden stick dimer core-above converts satellite shape structural modification cell-penetrating peptide: on the golden stick dimer core-that step (4) is obtained
Conversion is defended star topology group dress body ︰ PEG5000 ︰ cell-penetrating peptide TAT and is mixed with the ratio of 1 ︰ of molar concentration, 1000 ︰ 100, incubation at room temperature
After 12h, 7500rpm is centrifuged 10min, removes supernatant, and precipitating is resuspended in cell culture fluid.
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