CN106367510A - Preparation method and application of satellite-shaped nanometer assembling body used for duplex detection of intracellular cancer biomarker - Google Patents
Preparation method and application of satellite-shaped nanometer assembling body used for duplex detection of intracellular cancer biomarker Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a satellite-shaped nanometer assembling body used for duplex detection of an intracellular cancer biomarker, and belongs to the technical field of materials chemistry. The preparation method comprises the steps of 1, preparation of the satellite-shaped nanometer assembling body, wherein the preparation of the satellite-shaped nanometer assembling body comprises the steps of synthesis of gold nanorods, preparation of 20 nm upconversion nanoparticles, assembly of gold nanorod dipolymer, coupling of raman beacon molecular DTTC on the gold nanorod dipolymer and assembly of the gold nanorod dipolymer nuclear-upconversion satellite-shaped structure; 2, duplex detection of the intracellular cancer biomarker, wherein the duplex detection comprises the steps of modification of cell-penetrating peptide with the gold nanorod dipolymer nuclear-upconversion satellite-shaped nanometer assembling body, intracellular detection of the gold nanorod dipolymer nuclear-upconversion satellite-shaped nanometer assembling body, representation of the intracellular detection of the gold nanorod dipolymer nuclear-upconversion satellite-shaped nanometer assembling body, establishment of a standard curve and the like. The method can simultaneously detect the intracellular miRNA21content and the telomerase activity through dual signals of raman and fluorescence, and the prepared old nanorod dipolymer nuclear-upconversion satellite-shaped nanometer assembling body is uniform in structure and good in biological compatibility.
Description
Technical field
The present invention relates to a kind of preparation method of the satellite shape Nanoscale assemblies for intracellular cancer markers double check
And application, belong to material chemistry technical field.
Background technology
Microrna is single-stranded endogenouss non-coding rna of a class 18-25 nucleotide, plays important in life sciences
Regulating and controlling effect.Generally believe that the expression change level of mirna is expressed and hereditary and changes in immune function with changing at present
Relevant, mirna is especially in the diagnosis of many entity diversification tumors, and the expression of one or more mirnas is commonly used to
As important diagnostic and prognosis biomarker.Traditional technology, such as gene chip and real-time quantitative pcr, waste time and energy, work
Intensity is big, and expensive, which has limited their application.Therefore, non-invasive and maneuverable living cells situ is quantitative
Detection method has caused substantial amounts of concern.
Telomerase is a kind of ribonucleoprotein complexes, is divided into template to tie up using the ribonucleic acid group that it is had with itself
Hold the length of telomere, telomerase high expression in primary tumor, and immortality in these tumor cells.Therefore, telomerase tool
There iss to be acknowledged as the important biomolecule mark of early clinical diagnosis.Based on conventional polymeric enzyme chain reaction (pcr)
Telomeric repeatamplification protocol (trap), due to the basis of amplification program, cuts out telomerase products distribution, false positive including length
As a result, and primer dimer problem, the telomerase of activity complexity may be can't detect.Recently, in mesoporous silicon dioxide nano
It has been used to the activity of quantization cell telomerase with the fluorescent probe of DNA (deoxyribonucleic acid) preparation in grain duct.However, it is limited
Sensitivity and the suitability remain an important problem in complex matrix.
Therefore build that a kind of structure is homogeneous, the gold rod dimer core of good biocompatibility-above change satellite shape nanostructured,
The detection that the content of intracellular mirna21 and the activity of telomerase are realized by Raman and fluorescent dual signal simultaneously highly significant,
And also do not have been reported that at present.
Content of the invention
It is an object of the invention to provide a kind of satellite shape Nanoscale assemblies are used for the side of intracellular cancer markers double check
Method, the method for the activity of the content of detection intracellular mirna21 and telomerase simultaneously.
Technical scheme, a kind of satellite shape Nanoscale assemblies are used for the side of intracellular cancer markers double check
Method, step is as follows:
First, the preparation of satellite shape Nanoscale assemblies:
(1) synthesis of gold nanorods: horizontal plasmon absorption peaks are synthesized using seeded growth method and controls the gold nanorods in 800nm
gnr；
(2) the excellent dimeric assembling of gold: the gold nanorods gnr that step (1) is obtained modifies mercapto-polyglycol sh-peg-
1000, modify the sulfydryl dna1 containing microrna21 antisense base sequences and partly mutual with dna1 after centrifugation is resuspended more respectively
The sulfydryl dna2 sequence mended, respectively obtains gnr-dna1 and gnr-dna2 complex, then two kinds of complex are mixed, and obtains gold rod
Dimer；
(3) Raman beacon molecule dttc is coupled on gold rod dimer: gold rod dimer prepared by step (2) is divided with Raman beacon
Sub 3.3 '-diethyl thioaldehydes tricarbocyanine iodine dttc mixes, and obtaining surface modification after 8h has the gold rod dimer-dttc of dttc multiple
Fit；
(4) assembling of star topology: the gold rod dimer-dttc centrifugation that step (3) is obtained is defended in gold rod dimer core-upper conversion
After resuspended, modify the primer sequence te primer of telomerase, 20nm upper conversion nano granule is modified and linker dna part
Complementary sulfydryl mismatch dna, is assembled into gold rod dimer core-upper conversion by linker dna and defends star topology assembling
Body, then assembly is passed through gradient centrifugation purification；
2nd, intracellular cancer markers double check:
(5) gold rod dimer core-above conversion satellite shape Nanoscale assemblies modify cell-penetrating peptide: the gold rod dimerization that step (4) is obtained
Body core-upper conversion is defended star topology assembly and is mixed with sh-peg-5000 and cell-penetrating peptide tat, and obtaining surface modification has cell-penetrating peptide
Gold rod dimer core-upper conversion defend star topology assembly；
(6) gold rod dimer core-above changes the detection of satellite shape Nanoscale assemblies intracellular: the gold rod dimerization that step (5) is obtained
The conversion satellite shape Nanoscale assemblies of body core-above are incubated 8h altogether with cell, when there is determinand mirna21 and telomerase simultaneously,
The structure of assembly changes, and gold nanorods and up-conversion nanoparticles dissociate from assembly, causes Raman and glimmering respectively
The change of optical signal, and then detected；
(7) gold rod dimer core-above conversion satellite shape Nanoscale assemblies intracellular detection characterizes: the cell that step (6) is obtained with
Shi Jinhang Raman Characterization and fluorescence imaging, and Criterion curve.
The method that described satellite shape Nanoscale assemblies are used for intracellular cancer markers double check, specifically comprises the following steps that
First, the preparation of satellite shape Nanoscale assemblies:
(1) synthesis of gold nanorods:
A, crystal seed synthesis: under room temperature, by 0.05ml concentration be 10mm three hydration tetra chlorauric acids, be added to 1ml 0.2m ten
In six alkyl trimethyl ammonium bromide solution, solution colour becomes yellowish-brown by colourless, is subsequently adding the new 0.01m preparing of 0.12ml
Sodium borohydride solution, quickly stirs 2min, and solution colour is changed into light brown from yellowish-brown；
B, gold nanorods growth: the three hydration tetra chlorauric acids of the 1mm of 5ml are added to 5ml, 0.2m cetyl trimethylammonium bromide
In solution, add the ultra-pure water of 4ml, mix；Again the 0.01m silver nitrate solution of 0.125ml is added in above-mentioned mixed system,
Mix；Subsequently 70 l, 0.1m ascorbic acid solutions are added, be stirred vigorously, solution becomes colorless, after 2min, add 12 μ l steps
The crystal seed of rapid a preparation, stirs 20s, puts into 30 DEG C of water-baths, 2h；
(2) the excellent dimeric assembling of gold: using the gold nanorods of seeded growth method synthesis, its centrifugal concentrating is resuspended in 5mm
In cetyl trimethylammonium bromide (ctab) solution, the concentration making gold nanorods is 5nm；By gold nanorods and the poly- second of sulfydryl two
Alcohol peg-1000 is mixed with the ratio of molar concentration 1 100, stands 12h, and the gold nanorods taking 100 μ l modified peg afterwards will
It is mixed with the ratio of molar concentration 1 40 with the sulfydryl dna1 containing microrna21 antisense base sequences；Separately take 100 μ l
The sulfydryl dna2 sequence of the gold nanorods of modified peg and dna1 partial complementarity is mixed with the ratio of molar concentration 1 40；Respectively
Add the nacl solution of final concentration of 50mm, after being sufficiently mixed, incubated at room overnight after, centrifugation 3 times remove solution in unreacted
Dna, be resuspended in respectively in the 5mm ctab solution of 100 μ l, then by bodies such as gnr-dna1 and gnr-dna2 obtaining complexs
Long-pending bulk crossing, adds nacl solution to nacl concentration to carry out for 50mm aging, is incubated 12h under room temperature, obtains gold rod dimer,
Stand-by；
(3) it is coupled Raman beacon molecule dttc on gold rod dimer: by Raman beacon molecule 3.3 '-diethyl thioaldehydes three of 1mm
Carbon cyanines iodine (dttc) is added in gold rod dimerization liquid solution prepared by above-mentioned steps (2), and mix homogeneously keeps dttc final concentration
For 10 μm, incubated at room 8h；
(4) assembling of star topology is defended in gold rod dimer core-upper conversion: step (3) has been modified the gold rod of Raman beacon molecule
Dimer centrifugation is resuspended in 5mm ctab solution, modifies the primer te primer of telomerase with the ratio of molar concentration 1 500
Sequence, after overnight incubation, centrifugation is resuspended in 5mm ctab solution；Change on the amine-modified water solublity of the maleimide of 20 nm
Concentration dilution is 5nm with the tris buffer of 10mm ph 7.4 by nano-particle, is modified and linkerdna with molar concentration 15
The sulfydryl mismatch dna of partial complementarity, ultrafiltration after overnight incubation removes unconjugated mismatch dna, uses 10mm ph
7.4 tris buffer is resuspended stand-by；The gold rod dimer that 50 μ l te primer modify is repaiied with 200 μ l mismatch dna
The upper conversion nano granule mixing of decorations, is assembled into gold rod dimer core-above change satellite shape by 10 μm of linker dna of 5 μ l
Structure assembly, then assembly is passed through gradient centrifugation purification；
2nd, intracellular cancer markers double check:
(5) gold rod dimer core-above changes satellite shape structural modification cell-penetrating peptide: the gold rod dimer core that step (4) is obtained-on
Conversion is defended star topology assembly peg5000 cell-penetrating peptide tat and is mixed with the ratio of molar concentration 1 1,000 100, incubated at room
After 12h, 7500rpm is centrifuged 10min, removes supernatant, and precipitation is resuspended in cell culture fluid；
(6) gold rod dimer core-above changes the detection of satellite shape Nanoscale assemblies intracellular: surface modification has the gold rod two of cell-penetrating peptide
Aggressiveness core-upper conversion is defended star topology assembly and can be directly entered in cell and detected, when there is determinand mirna21,
It is combined with the microrna21 antisense nucleoside acid fragment in sulfydryl dna1 sequence, and the gold rod dimer leading to is dismissed, with
The increase of mirna21 concentration, Raman signal gradually weakens；When there is determinand telomerase and dntp, te primer can be along
Linkerdna transcribes, and replaces the complementary position of mismatchdna and linkerdna, leads to conversion nano granule from assembling
Dissociate on body, with the increase of telomerase activation, fluorescence signal gradually recovers；There is determinand mirna21 and telomere when simultaneously
During enzyme, gold nanorods and up-conversion nanoparticles dissociate from assembly, cause the change of Raman and fluorescence signal respectively, and then
Carry out detection to characterize；
(7) gold rod dimer core-above conversion satellite shape Nanoscale assemblies intracellular detection characterizes: the gold rod two that step (6) is obtained
Aggressiveness core-upper conversion defends star topology assembly and through not commensurability transfection agents Transfected cells and without the cell difference after transfection
Altogether after incubation 8h, use 1ml trypsin digestion and cell, obtain gold rod dimer core-above and change satellite shape structure detection mirna21
Cell suspension after concentration, subsequently carries out Raman Characterization, sets up different mirna21 concentration and Raman signal intensity in cell
Standard curve between the two；
After the golden excellent dimer core that step (6) is obtained-above conversion satellite shape Nanoscale assemblies are suppressed with not commensurability egcg and not
Inhibited cell is respectively altogether after incubation 8h, after obtaining the gold rod conversion satellite shape structure detection telomerase activation of dimer core-above
Cell, subsequently carry out fluorescence imaging, set up in cell different telomerase activations and fluorescence intensity standard curve between the two.
Described dna1 sequence as shown in seq id no. 1, dna2 sequence as shown in seq id no. 2, te primer sequence
Row as shown in seq id no. 3, mismatch dna sequence as shown in seq id no. 4, linker dna sequence such as seq
Shown in id no. 5, tat peptide sequence is as shown in seq id no. 6.Specifically as shown in table 1.
Table 1
Beneficial effects of the present invention: the present invention has prepared that structure is homogeneous, the gold rod dimer core of good biocompatibility-upper is changed
Satellite shape nanostructured assembles body, there is provided can detect containing of intracellular mirna21 by Raman and fluorescent dual signal simultaneously
The method of the activity of amount and telomerase, establishes intracellular mirna21 concentration and Raman signal intensity, and cell telomerase is lived
Property and fluorescence intensity between standard curve, have that sensitivity is high, selectivity is good, the advantage that test limit is low, the used time is short, have non-
Often good actual application prospect.
Brief description
Fig. 1 is that gold rod dimer (a) of the present invention, upper conversion nano granule (b), gold rod dimer core-above change satellite
The transmission electron microscope photo of shape Nanoscale assemblies (c).
Fig. 2 is that in the present invention, gold rod dimer core-above conversion satellite shape Nanoscale assemblies enter and turn through not commensurability transfection agents
After dye cell and without transfection cell in, detection intracellular difference mirna21 content Raman spectrogram (a), and Raman letter
Standard curve (b) number with intracellular mirna21 content.
Fig. 3 is that in the present invention, gold rod dimer core-above conversion satellite shape Nanoscale assemblies enter through not commensurability egcg suppression
Afterwards and in not inhibited cell, fluorescence imaging figure (a) of detection intracellular difference telomerase activation, and fluorescence signal and intracellular
The standard curve (b) of telomerase activation.
Specific embodiment
In following examples, biomaterial is purchased from Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 1
All of glass apparatus all soak 24h with chloroazotic acid, and are cleaned with distilled water, dry standby.Used in experiment, water is
The milli-q ultra-pure water of 18.2m ω.
(1) synthesis of gold nanorods:
A, crystal seed synthesis: under room temperature, by 0.05ml concentration be 10mm three hydration tetra chlorauric acids, be added to 1ml 0.2m ten
In six alkyl trimethyl ammonium bromide solution, solution colour becomes yellowish-brown by colourless, is subsequently adding the new 0.01m preparing of 0.12ml
Sodium borohydride solution, quickly stirs 2min, and solution colour is changed into light brown from yellowish-brown；
B, gold nanorods growth: the three hydration tetra chlorauric acids of the 1mm of 5ml are added to 5ml, 0.2m cetyl trimethylammonium bromide
In solution, add the ultra-pure water of 4ml, mix；Again the 0.01m silver nitrate solution of 0.125ml is added in above-mentioned mixed system,
Mix；Subsequently 70 l, 0.1m ascorbic acid solutions are added, be stirred vigorously, solution becomes colorless, after 2min, add 12 μ l's
Crystal seed, stirs 20s, puts into 30 DEG C of water-baths, 2h；
(2) the excellent dimeric assembling of gold: using the gold nanorods of seeded growth method synthesis, its centrifugal concentrating is resuspended in 5 mm
In ctab solution, gold nanorods concentration is made to be 5nm；By gold nanorods with mercapto-polyglycol peg-1000 with molar concentration 1
100 ratio mixes, and stands 12h, and the gold nanorods taking 100 μ l modified peg afterwards are by it and containing microrna21 antisense
The sulfydryl dna1 of nucleotide sequence is mixed with the ratio of molar concentration 1 40；100 μ l are separately taken to modify gold nanorods and the dna1 of peg
The sulfydryl dna2 sequence of partial complementarity is mixed with the ratio of molar concentration 1 40, is separately added into the nacl solution of 50mm, fully mixed
After conjunction, incubated at room overnight after, centrifugation removes unreacted dna in solution 3 times, and the 5 mm ctab being resuspended in 100 μ l respectively are molten
In liquid, then by gnr-dna1 and gnr-dna2 obtaining complex equal-volume bulk crossing, add nacl solution to its final concentration
Carry out for 50mm aging, under room temperature, be incubated 12 h, obtain gold rod dimer, stand-by.
(3) it is coupled Raman beacon molecule dttc on gold rod dimer: by Raman beacon the molecule 3.3 '-diethyl sulfide of 1 mm
Aldehyde tricarbocyanine iodine dttc is added in gold rod dimerization liquid solution prepared by above-mentioned steps (2), and mix homogeneously keeps its final concentration
For 10 μm, incubated at room 8h.
(4) assembling of star topology is defended in gold rod dimer core-upper conversion: step (3) has been modified Raman beacon molecule
Gold rod dimer centrifugation is resuspended in 5 mm ctab solution, modifies the primer te of upper telomerase with the ratio of molar concentration 1 500
Primer sequence, after overnight incubation, centrifugation is resuspended in 5 mm ctab solution；The amine-modified water solublity of the maleimide of 20 nm
Upper conversion nano granule is diluted to 5nm with the tris buffer of 10 mm ph 7.4, modifies upper and linker with molar concentration 15
The sulfydryl mismatch dna of dna partial complementarity, ultrafiltration after overnight incubation removes unconjugated mismatch dna, uses 10 mm
The tris buffer of ph 7.4 is resuspended stand-by；Gold rod dimer and 200 μ l mismatch dna that 50 μ l te primer modify
The upper conversion nano granule mixing modified, is assembled into gold rod dimer core-above change satellite by 10 μm of linker dna of 5 μ l
Shape structure assembly, then assembly is passed through gradient centrifugation purification.Obtain gold rod dimer, upper conversion nano granule, gold rod
The transmission electron microscope photo of the conversion satellite shape Nanoscale assemblies of dimer core-above is as shown in Figure 1.
(5) gold rod dimer core-above changes satellite shape structural modification cell-penetrating peptide: the gold rod dimer that step (4) is obtained
Core-upper conversion is defended star topology assembly and is mixed with 1 1,000 100 molar concentration with peg5000 and cell-penetrating peptide tat, room temperature
After incubation 12h, 7500 rpm are centrifuged 10 min, remove supernatant, and precipitation is resuspended in cell culture fluid.
(6) gold rod dimer core-above changes the detection of satellite shape Nanoscale assemblies intracellular: surface modification has the gold of cell-penetrating peptide
Excellent dimer core-upper conversion is defended star topology assembly and can be directly entered in cell and detected, when there is determinand mirna21
When, it is combined with the microrna21 antisense nucleoside acid fragment in sulfydryl dna1 sequence, and the gold rod dimer leading to is dismissed, with
The increase of mirna21 concentration, Raman signal gradually weakens；When there is determinand telomerase and dntp, te primer can be along
Linkerdna transcribes, and replaces the complementary position of mismatchdna and linkerdna, leads to conversion nano granule from assembling
Dissociate on body, with the increase of telomerase activation, fluorescence signal gradually recovers；There is determinand mirna21 and telomere when simultaneously
During enzyme, gold nanorods and up-conversion nanoparticles dissociate from assembly, cause the change of Raman and fluorescence signal respectively, and then
Carry out detection to characterize.
(7) gold rod dimer core-above conversion satellite shape Nanoscale assemblies intracellular detection characterizes: will turn through not commensurability transfection agents
Cell and the quantitation carrying out intracellular mirna21 concentration without the cell after transfection with real-time fluorescence pcr after dye；By not commensurability egcg
After suppression and not inhibited cell pyrolysis liquid carries out the quantitation of intracellular telomerase activation with elisa standard curve.By step (6)
The gold rod dimer core obtaining-above change satellite shape Nanoscale assemblies and through not commensurability transfection agents Transfected cells and without transfection
After cell afterwards is incubated 8h respectively altogether, use 1ml trypsin digestion and cell, obtain gold rod dimer core-upper conversion and defend star knot
Structure detects the cell suspension after mirna21 concentration, subsequently carries out Raman Characterization, sets up different mirna21 concentration in cell
As shown in Figure 2 with Raman signal intensity standard curve between the two；Gold rod dimer core-upper conversion that step (6) is obtained
Defend after star topology assembly is suppressed with not commensurability egcg and not inhibited cell is incubated after 8h respectively altogether, obtain gold rod dimerization
Body core-above change the cell after satellite shape structure detection telomerase activation, subsequently carries out fluorescence imaging, sets up different ends in cell
Telomerase activity and fluorescence intensity standard curve between the two is 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 (3)
1. a kind of satellite shape Nanoscale assemblies be used for intracellular cancer markers double check method it is characterised in that step such as
Under:
(1) synthesis of gold nanorods: horizontal plasmon absorption peaks are synthesized using seeded growth method and controls the gold nanorods in 800nm
gnr；
(2) the excellent dimeric assembling of gold: the gold nanorods gnr that step (1) is obtained modifies mercapto-polyglycol sh-peg-
1000, modify the sulfydryl dna1 containing microrna21 antisense base sequences and partly mutual with dna1 after centrifugation is resuspended more respectively
The sulfydryl dna2 sequence mended, respectively obtains gnr-dna1 and gnr-dna2 complex, then two kinds of complex are mixed, and obtains gold rod
Dimer；
(3) Raman beacon molecule dttc is coupled on gold rod dimer: gold rod dimer prepared by step (2) is divided with Raman beacon
Sub 3.3 '-diethyl thioaldehydes tricarbocyanine iodine dttc mixes, and obtaining surface modification after 8h has the gold rod dimer-dttc of dttc multiple
Fit；
(4) assembling of star topology: the gold rod dimer-dttc centrifugation that step (3) is obtained is defended in gold rod dimer core-upper conversion
After resuspended, modify the primer te primer sequence of telomerase, 20nm upper conversion nano granule is modified and linker dna part
Complementary sulfydryl mismatch dna, is assembled into gold rod dimer core-upper conversion by linker dna and defends star topology assembling
Body, then assembly is passed through gradient centrifugation purification；
(5) gold rod dimer core-above conversion satellite shape Nanoscale assemblies modify cell-penetrating peptide: the gold rod dimerization that step (4) is obtained
Body core-upper conversion is defended star topology assembly and is mixed with sh-peg-5000 and cell-penetrating peptide tat, and obtaining surface modification has cell-penetrating peptide
Gold rod dimer core-upper conversion defend star topology assembly；
(6) gold rod dimer core-above changes the detection of satellite shape Nanoscale assemblies intracellular: the gold rod dimerization that step (5) is obtained
The conversion satellite shape Nanoscale assemblies of body core-above are incubated 8h altogether with cell, when there is determinand mirna21 and telomerase simultaneously,
The structure of assembly changes, and gold nanorods and up-conversion nanoparticles dissociate from assembly, causes Raman and glimmering respectively
The change of optical signal, and then detected；
(7) gold rod dimer core-above conversion satellite shape Nanoscale assemblies intracellular detection characterizes: the cell that step (6) is obtained with
Shi Jinhang Raman Characterization and fluorescence imaging, and Criterion curve.
2. the method that satellite shape Nanoscale assemblies are used for intracellular cancer markers double check according to claim 1, it is special
Levy and be to specifically comprise the following steps that
(1) synthesis of gold nanorods:
A, crystal seed synthesis: under room temperature, by 0.05ml concentration be 10mm three hydration tetra chlorauric acids, be added to 1ml 0.2m ten
In six alkyl trimethyl ammonium bromide solution, solution colour becomes yellowish-brown by colourless, is subsequently adding the new 0.01m preparing of 0.12ml
Sodium borohydride solution, quickly stirs 2min, and solution colour is changed into light brown from yellowish-brown；
B, gold nanorods growth: the three hydration tetra chlorauric acids of the 1mm of 5ml are added to 5ml, 0.2m cetyl trimethylammonium bromide
In solution, add the ultra-pure water of 4ml, mix；Again the 0.01m silver nitrate solution of 0.125ml is added in above-mentioned mixed system,
Mix；Subsequently 70 l, 0.1m ascorbic acid solutions are added, be stirred vigorously, solution becomes colorless, after 2min, add 12 μ l steps
The crystal seed of rapid a preparation, stirs 20s, puts into 30 DEG C of water-baths, 2h；
(2) the excellent dimeric assembling of gold: using the gold nanorods of seeded growth method synthesis, its centrifugal concentrating is resuspended in 5mm
In cetyl trimethylammonium bromide ctab solution, the concentration making gold nanorods is 5nm；By gold nanorods and mercapto-polyglycol
Peg-1000 is mixed with the ratio of molar concentration 1 100, stands 12h, take afterwards 100 μ l modify the gold nanorods of peg by its with
Sulfydryl dna1 containing microrna21 antisense base sequences is mixed with the ratio of molar concentration 1 40；100 μ l are separately taken to modify
The sulfydryl dna2 sequence of the gold nanorods of peg and dna1 partial complementarity is mixed with the ratio of molar concentration 1 40；It is separately added into end
Concentration be 50mm nacl solution, after being sufficiently mixed, incubated at room overnight after, centrifugation 3 times remove solution in unreacted dna,
It is resuspended in respectively in the 5mm ctab solution of 100 μ l, then gnr-dna1 and gnr-dna2 obtaining complex equal-volume is mixed
Hybridization, adds nacl solution to nacl concentration to carry out for 50mm aging, is incubated 12h under room temperature, obtains gold rod dimer, stand-by；
(3) it is coupled Raman beacon molecule dttc on gold rod dimer: by Raman beacon molecule 3.3 '-diethyl thioaldehydes three of 1mm
Carbon cyanines iodine dttc is added in gold rod dimerization liquid solution prepared by above-mentioned steps (2), and mix homogeneously keeps dttc final concentration of
10 μm, incubated at room 8h；
(4) assembling of star topology is defended in gold rod dimer core-upper conversion: step (3) is modified the gold rod two of Raman beacon molecule
Aggressiveness centrifugation is resuspended in 5mm ctab solution, modifies the primer te primer sequence of telomerase with the ratio of molar concentration 1 500
Row, after overnight incubation, centrifugation is resuspended in 5mm ctab solution；Change on the amine-modified water solublity of the maleimide of 20 nm and receive
Concentration dilution is 5nm with the tris buffer of 10mm ph 7.4 by rice grain, is modified and linkerdna with molar concentration 15
The sulfydryl mismatch dna of partial complementarity, ultrafiltration after overnight incubation removes unconjugated mismatch dna, uses 10mm ph
7.4 tris buffer is resuspended stand-by；The gold rod dimer that 50 μ l te primer modify is repaiied with 200 μ l mismatch dna
The upper conversion nano granule mixing of decorations, is assembled into gold rod dimer core-above change satellite shape by 10 μm of linker dna of 5 μ l
Structure assembly, then assembly is passed through gradient centrifugation purification；
(5) gold rod dimer core-above changes satellite shape structural modification cell-penetrating peptide: the gold rod dimer core that step (4) is obtained-on
Conversion is defended star topology assembly peg5000 cell-penetrating peptide tat and is mixed with the ratio of molar concentration 1 1,000 100, incubated at room
After 12h, 7500rpm is centrifuged 10min, removes supernatant, and precipitation is resuspended in cell culture fluid；
(6) gold rod dimer core-above changes the detection of satellite shape Nanoscale assemblies intracellular: surface modification has the gold rod two of cell-penetrating peptide
Aggressiveness core-upper conversion is defended star topology assembly and is directly entered in cell and detected, when there is determinand mirna21, its
Be combined with the microrna21 antisense nucleoside acid fragment in sulfydryl dna1 sequence, the gold rod dimer leading to is dismissed, with
The increase of mirna21 concentration, Raman signal gradually weakens；When there is determinand telomerase and dntp, te primer can be along
Linkerdna transcribes, and replaces the complementary position of mismatchdna and linkerdna, leads to conversion nano granule from assembling
Dissociate on body, with the increase of telomerase activation, fluorescence signal gradually recovers；There is determinand mirna21 and telomere when simultaneously
During enzyme, gold nanorods and up-conversion nanoparticles dissociate from assembly, cause the change of Raman and fluorescence signal respectively, and then
Carry out detection to characterize；
(7) gold rod dimer core-above conversion satellite shape Nanoscale assemblies intracellular detection characterizes: the gold rod two that step (6) is obtained
Aggressiveness core-upper conversion defends star topology assembly and through not commensurability transfection agents Transfected cells and without the cell difference after transfection
Altogether after incubation 8h, use 1ml trypsin digestion and cell, obtain gold rod dimer core-above and change satellite shape structure detection mirna21
Cell suspension after concentration, subsequently carries out Raman Characterization, sets up different mirna21 concentration and Raman signal intensity in cell
Standard curve between the two；
After the golden excellent dimer core that step (6) is obtained-above conversion satellite shape Nanoscale assemblies are suppressed with not commensurability egcg and not
Inhibited cell is respectively altogether after incubation 8h, after obtaining the gold rod conversion satellite shape structure detection telomerase activation of dimer core-above
Cell, subsequently carry out fluorescence imaging, set up in cell different telomerase activations and fluorescence intensity standard curve between the two.
3. the method that satellite shape Nanoscale assemblies are used for intracellular cancer markers double check according to claim 1, it is special
Levy and be: described dna1 sequence as shown in seq id no.1, dna2 sequence as shown in seq id no.2, te primer sequence
As shown in seq id no.3, mismatch dna sequence as shown in seq id no.4, linker dna sequence such as seq id
Shown in no.5, tat peptide sequence is as shown in seq id no.6.
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