CN108037100B - A method of detecting two kinds of miRNA while based on FRET effect - Google Patents
A method of detecting two kinds of miRNA while based on FRET effect Download PDFInfo
- Publication number
- CN108037100B CN108037100B CN201711138127.XA CN201711138127A CN108037100B CN 108037100 B CN108037100 B CN 108037100B CN 201711138127 A CN201711138127 A CN 201711138127A CN 108037100 B CN108037100 B CN 108037100B
- Authority
- CN
- China
- Prior art keywords
- dna
- mirna
- stick core
- conversion
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a kind of methods that two kinds of miRNA are detected while effect based on FRET, belong to technical field of material chemistry.The present invention has synthesized conversion on golden stick core one and has defended star topology assembly, establishes standard curve, finally prepare that structure is uniform, converts satellite shape Nanoscale assemblies on golden stick core one of good biocompatibility, the platform that can realize the quantitative of two kinds of miRNA intracellular, super sensitivity detection by FRET effect is provided, is had a good application prospect.
Description
Technical field
The present invention relates to a kind of method for detecting two kinds of miRNA while effect based on FRET, especially one kind is based on
The golden stick-of FRET effect above converts the miRNA super sensitivity detection platform of satellite assembly, belongs to technical field of material chemistry.
Background technique
MicroRNA (miRNA) is the microRNA regulatory factor being widely present in eukaryotic cells, is usually had
The length of 21 bases or so plays a significant role in regulation growth course.The study found that the unconventionality expression of miRNA and more
Kind cancer is related, this also makes miRNA become the new biological marker of cancer diagnosis, provides to the treatment of human diseases a kind of new
Means.But due to the features such as its content in cell is low, size is small and degradable, the intracellular miRNA of quantitative detection
It is very difficult.MiRNA microarray hybridization and realtime quantitative inspection (PCR) are the exceptions for studying miRNA
The conventional method of expression, but equipment is expensive, complex process, sensitivity are limited, constrains its development.
In recent years, controllably independently dress technology has been widely used in bio-sensing to the nanoparticle based on DNA, has
The geometry of rule, stable structure, very strong optical activity.In addition to this, many inspections based on FRET effect have been developed
Survey method detects metallic, cancer markers etc., has become a hot topic and causes extensive concern, still,
It is concentrated mainly on and detects a kind of miRNA, and the disadvantages of organic dyestuff optical signalling is unstable, the service life is short, background signal is strong all limits
The technology has been made intracellular while detecting the application in a variety of miRNA.
Summary of the invention
The present invention establishes a kind of method for detecting two kinds of miRNA while effect based on FRET, is based on FRET effect
Golden stick-above convert satellite assembly two kinds of miRNA of detection method, be a kind of hypersensitive, the original intracellular of high specific
Position detecting method can detect miR-21 and miR-200b content intracellular simultaneously.
Present invention firstly provides satellite shape Nanoscale assemblies are converted on a kind of golden stick core one, preparation method includes following step
It is rapid:
(1) gold nanorods are synthesized, and are coupled DNA2, DNA5；
(2) by up-conversion nanoparticles respectively with DNA1、DNA4Coupling, wherein DNA2With DNA1Partial complementarity, DNA5With
DNA4Partial complementarity；Luminescent dye molecule TAMRA is coupled DNA3, luminescent dye molecule Cy5.5 coupling DNA6；Wherein DNA3With DNA1
Partial complementarity, DNA6With DNA4Partial complementarity；
(3) DNA will be coupled2、DNA5The obtained fluorescent dye modification of gold nanorods and step (2) after upper conversion receive
Rice corpuscles assembling.
Wherein, DNA2With DNA1Partial complementarity, DNA3With DNA1Partial complementarity, DNA1With the first miRNA complete complementary to be measured,
When encountering the first miRNA to be measured, the first miRNA and DNA to be measured2Competitive binding DNA1.Equally, DNA5With DNA4Partial complementarity,
DNA6With DNA4Partial complementarity, DNA4With the second miRNA complete complementary to be measured, when encountering the second miRNA to be measured, second is to be measured
MiRNA and DNA5Competitive binding DNA4.In one embodiment of the invention, the described first miRNA to be measured is miR-21, institute
Stating the second miRNA to be measured is miR-200b.
In one embodiment of the invention, the DNA1Sequence is as shown in SEQ ID NO.1, DNA2Sequence such as SEQ
Shown in ID NO.2, DNA3Sequence is as shown in SEQ ID NO.3, DNA4Sequence is as shown in SEQ ID NO.4, DNA5Sequence such as SEQ
Shown in ID NO.5, DNA6Sequence is as shown in SEQ ID NO.6.
In one embodiment of the invention, gold nanorods are resuspended in the CTAB solution of 0.005M by step (1),
The suspension of final concentration of 10nM is obtained, with molar concentration AuNR:DNA2Ratio be 1:80-100 be added DNA2, react at room temperature
Centrifugation removes extra DNA after 12h2, it is resuspended in the CTAB solution of 0.001M, obtains the suspension of final concentration of 10nM；Again with
DNA is added in molar concentration 1:450-5004DNA5, centrifugation removes extra DNA after reacting 12h at room temperature4DNA5, it is resuspended in
In the CTAB solution of 0.001M, the suspension of final concentration of 10nM is obtained.
In one embodiment of the invention, in step (2), up-conversion nanoparticles is diluted to 10nM, respectively take 100
μ L, is coupled DNA respectively1And DNA4, the DNA of end modified fluorescent molecule TAMRA is then added3With end modified fluorescent molecule
The DNA of Cy5.56, hybridized to obtain the up-conversion nanoparticles for being coupled different fluorescent molecules.
In one embodiment of the invention, step (3), to being coupled DNA2、DNA52 μ L of gold nanorods in, be added
It has modified the 20 μ L of up-conversion nanoparticles of fluorescent molecule TAMRA and has modified the up-conversion nanoparticles of fluorescent molecule Cy5.5
100 μ L, the unbonded particle of gradient centrifugation removal after overnight incubation obtain the assembling that star topology is defended in conversion on golden stick core one
Body.
The method for detecting two kinds of miRNA simultaneously using satellite shape Nanoscale assemblies are converted on golden stick core one, including walk as follows
It is rapid:
(1) it prepares and converts satellite shape Nanoscale assemblies on golden stick core one；
(2) it makes the standard curve in cell between the miRNA and fluorescence intensity of various concentration: will be converted on golden stick core one
Star topology assembly is defended with the target cell to be detected after the transfection of not same amount transfection agents and without the mesh to be detected after transfection
Cell is marked, a period of time is incubated for altogether respectively, then carries out fluorescence imaging, establish between the miRNA of various concentration and fluorescence intensity
Standard curve；
(3) cell-penetrating peptide is modified: star topology assembly: PEG5000 is defended in conversion on golden stick core one: cell-penetrating peptide TAT is to rub
The ratio of your concentration 1:1000:100 mixes, and after being incubated at room temperature 12h, centrifugation removal supernatant, gained precipitating is that cell-penetrating peptide is repaired
Satellite shape Nanoscale assemblies are converted on the golden stick core one of decorations；TAT polypeptide sequence is as shown in SEQ ID NO.7；
(4) star topology assembly is defended into conversion on the golden stick core one after the modification modification of film peptide and is transferred to cell to be measured, culture 8
After hour, cell is washed three times with PBS, after removal does not enter the assembly of cell, is carried out up-conversion fluorescence and is copolymerized burnt imaging,
Under 980nm excitation, fluorescent image is obtained, fluorescence intensity is substituted into standard curve, the content of miRNA intracellular is calculated.
Beneficial effects of the present invention: the present invention has prepared that structure is uniform, converts on the golden stick core one of good biocompatibility
Satellite shape nanostructure assembles body, and the fluorescence signal of FRET effect can be passed through while detect two kinds of miRNA intracellular by providing
The method of content establishes the standard curve between intracellular miRNA concentration and fluorescence signal intensity, has high sensitivity, choosing
Selecting property is good, and the advantage that detection limit is low, the used time is short has extraordinary actual application prospect.For sensitivity, as shown in figure 4,
The detection line of miR-21 is 3.2zmol/ngRNA, and the detection line of miR-200b is 10.3zmol/ngRNA.For selectivity, such as
Satellite shape Nanoscale assemblies are converted shown in Fig. 5, on gold nanorods core one, and mismatch, intracellular interference matter and assembly phase is added
When than not apparent change in fluorescence, only addition miR-21 and miR-200b, FRET can just occur, generate change in fluorescence.It says
The bright present invention has good selectivity and sensitivity.
Detailed description of the invention
Fig. 1 transmission electron microscope photo, (a): gold nanorods prepared by embodiment 1, (b): upper conversion nano particle, (c): implementing
Satellite shape Nanoscale assemblies are converted on gold nanorods core one prepared by example 1.
Fig. 2 is to convert satellite shape Nanoscale assemblies on gold nanorods core one in the present invention miR-21 (a) and miR- is added
The transmission electron microscope photo of 200b (b).
Fig. 3 is the evaluation of extracellular detectability in the present invention, (a): adds the miR-21 and miR- of a series of various concentrations
The fluorescence signal figure that 200b is obtained, each curve successively respectively represent addition 1000pM miR-21,500pM miR- from top to bottom
200b、500pM miR-21,200pM miR-200b、100pM miR-21,100pM miR-200b、50pM miR-21,50pM
The fluorescence intensity of miR-200b, 10pM miR-21,20pM miR-200b and 5pM miR-21,10pM miR-200b；(b): glimmering
The standard curve of optical signal and miR-21 content；(c): the standard curve of fluorescence signal and miR-200b content.
Fig. 4 is to convert satellite shape Nanoscale assemblies in the present invention on gold nanorods core one to enter through the transfection of not same amount transfection agents
Afterwards and in the cell without transfection, fluorescence imaging figure when (a) difference miR-21 and miR-200b content intracellular, from left to right 1
Indicate that the content of miR-21 and miR-200b gradually increases to 5 column, corresponding fluorescence intensity also enhances therewith；(b): fluorescence signal
With the standard curve of miR-21 content intracellular；(c): the standard curve of fluorescence signal and miR-200b content intracellular.
Fig. 5 is to convert satellite shape Nanoscale assemblies in the present invention on gold nanorods core one mismatch is added, does into the cell
Disturb matter and the fluorescence signal figure of target miRs.
Specific embodiment
The preparation of satellite shape Nanoscale assemblies is converted on 1 gold medal stick core one of embodiment
All glass apparatus are all impregnated with chloroazotic acid, and are cleaned with distilled water, are dried spare.Water used in experiment is
The Milli-Q ultrapure water of 18.2M Ω.
(1) lateral plasmon absorption peaks control the synthesis of gold nanorods: is synthesized in the Jenner of 780nm using crystal seed growth method
Rice stick；
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 trimethyl smelling ammonium salt solution in, solution colour becomes yellowish-brown by colourless, and 0.12mL is then added and newly prepares
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 and smell
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 the ascorbic acid solution of 70 μ L, 0.1M are added, are vigorously stirred, solution becomes colorless, and after 2min, is added
The crystal seed of 12 μ L step a preparation, stirs 20s, is put into 30 DEG C of water-baths, 2h；
(2) it the preparation of up-conversion nanoparticles: is bought by Beijing Oneder Hightech Co., Ltd.；Upper conversion nano grain
Sub- basic composition unit is NaGdF4, adulterate Yb3+,Er3+, size is 20 ± 3nm, has good light resistance and chemical stabilization
Property, while small toxicity and signal-to-noise ratio are high, are suitable for biological detection.
(3) luminescent dye molecule is coupled on up-conversion nanoparticles: the up-conversion nanoparticles of purchase are diluted to 10nM,
It respectively takes 100 μ L in PCR, is coupled DNA respectively1And DNA4, it is then respectively adding the DNA of end modified fluorescent molecule TAMRA3(with
DNA1Partial complementarity) and end modified fluorescent molecule Cy5.5. DNA6(with DNA4Partial complementarity), hybridized and is coupled
The up-conversion nanoparticles of different fluorescent molecules.
Table 1
(4) assembling for defending star topology is converted on Jin Banghe mono-:
The gold nanorods 7500rpm synthesized in step (1) is centrifuged 15 minutes, Vc, AgNO are removed3Equal small-molecule substances,
10 times of concentration, which is resuspended in the CTAB solution of 0.005M, obtains the suspension of final concentration of 10nM, takes 50 μ L in PCR tubule, to rub
You are concentration AuNR:DNA2DNA is added in 1:80-1002, centrifugation removes extra DNA after reacting 12h at room temperature2, it is resuspended in 0.001M
CTAB solution in make AuNR concentration 10nM；Again with molar concentration AuNR:DNA5DNA is added in 1:450-5005、DNA4, room temperature
Centrifugation removes extra DNA after lower reaction 12h4、DNA5, being resuspended in the CTAB solution of 0.001M makes AuNR concentration 10nM.So
Afterwards, the 2 μ L of gold nanorods of above-mentioned coupling DNA is taken, the up-conversion nanoparticles of modification fluorescent molecule TAMRA in step (3) are added
The 100 μ L of up-conversion nanoparticles of 20 μ L and modification fluorescent molecule Cy5.5, the unbonded grain of gradient centrifugation removal after overnight incubation
Son obtains the assembly that star topology is defended in conversion on golden stick core one.
Obtained gold nanorods, upper conversion nano particle, the transmission electricity that satellite shape Nanoscale assemblies are converted on Jin Banghe mono-
Mirror photo as shown in Figure 1, Fig. 1 a, Fig. 1 b illustrate uniform structure, favorable dispersibility gold nanorods and upper conversion nano particle,
Fig. 1 c indicates that the Jin Banghe-being coupled by DNA above converts satellite shape Nanoscale assemblies.
During golden stick is coupled DNA, since different DNA sequence dnas is modified in end face and side respectively, so control CTAB
Concentration and the concentration of DNA be crucial.When golden stick is resuspended in the CTAB of high concentration, DNA2Preferentially in conjunction with end face, so wanting
Control DNA2Concentration, concentration is too high to lead to excessive DNA2In conjunction with side；At this moment, it in the CTAB of low concentration, is added
Suitable DNA4In conjunction with side.In addition to this, the ratio for controlling golden stick and up-conversion nanoparticles (golden stick: is coupled TAMRA's
Up-conversion nanoparticles: the up-conversion nanoparticles for being coupled Cy5.5 are about 1:10:50) also very crucial, upper conversion content mistake
It is low, it does not become and converts satellite structure on complete golden stick core one；Too high levels, foreign particle is too many, and yield is caused to reduce.
It is above converted to golden stick core-in satellite shape Nanoscale assemblies difference and miR-21 and miR-200b, assembly structure is added
As transmission electron microscope photo is as shown in Figure 2.As shown in Figure 2 a, when miR-21 is added, the complementary series of competitive binding end face, upper conversion
Nanoparticle is dissociated from end face；As shown in Figure 2 b, when miR-200b is added, the complementary series of competitive binding side, so that upper turn
Nanoparticle is changed to dissociate from side.
The detection of the cancer markers intracellular of embodiment 2
(1) satellite Nanoscale assemblies are converted on Jin Banghe mono- modify cell-penetrating peptide: the golden stick that 1 step of embodiment (4) is obtained
Star topology assembly: PEG5000 is defended in conversion on core one: cell-penetrating peptide TAT is mixed with the ratio of molar concentration 1:1000:100, room
After temperature is incubated for 12h, 7000rpm is centrifuged 10min, removes supernatant, and precipitating is resuspended in cell culture fluid；
(2) two kinds of miRNA of satellite Nanoscale assemblies detection intracellular are converted on Jin Banghe mono-: surface modification has the gold of cell-penetrating peptide
Conversion, which is defended star topology assembly and is directly entered in cell, on stick core one is detected, and there are determinand miR-21 when intracellular
When, with sequence DNA1In complementary fragment combine, it is caused gold plane rod end up-conversion nanoparticles dismiss, 980nm's
Under excitation, FRET effect is generated, with the increase of miR-21 concentration, fluorescence signal of the up-conversion nanoparticles in 540nm weakens,
Fluorescent molecule TAMRA signal gradually increases；When into the cell there are when determinand miR-200b, with sequence DNA4In complementary fragment
In conjunction with, cause the up-conversion nanoparticles of golden stick side to be dismissed, under the excitation of 980nm, generation FRET effect, with miR-
The increase of 200b concentration, fluorescence signal of the up-conversion nanoparticles in 660nm weaken, and fluorescent molecule Cy5.5 signal gradually increases；
When existing simultaneously determinand miR-21 and miR-200b into the cell, the up-conversion nanoparticles of gold nanorods end face and side are same
When dissociated from assembly, cause the fluorescence signal at 540nm and 660nm to change respectively, and then carry out detection characterization.
(3) characterization that the detection intracellular of satellite Nanoscale assemblies is converted on Jin Banghe mono-, establishes standard curve: by embodiment 1
Star topology assembly and the Hela cell and not after the transfection of not same amount transfection agents are defended in conversion on the golden stick core one being prepared
After cell after transfecting is incubated for 8h altogether respectively, the cell that satellite shape structure detection miRNA is converted on golden stick core one is obtained, then
Fluorescence imaging is carried out, the miRNA of various concentration and the standard curve of fluorescence intensity between the two in cell are established.
As shown in figure 4, with the increase of the intracellular miR-21 and miR-200b content of Hela, TAMRA (green) and Cy5.5
The fluorescence intensity of (red) increases therewith, and good linear relationship is presented.So the assembly is suitable for intracellular miRNA
Quantitative detection.
The detection of the extracellular cancer markers of embodiment 3
In vitro, the miR-21 and miR- that various concentration is added in satellite Nanoscale assemblies are converted on golden stick core one
200b.As a result as shown in figure 3, with miR-21 and miR-200b concentration increase, the upper conversion nanoparticle of end face and side
Dissociation degree increases, and under 980nm excitation, generates FRET, energy is transferred to fluorescent molecule TAMRA and Cy5.5 respectively, is caused
Fluorescence increases.Meanwhile the concentration of miR-21, miR-200b are good linear with the presentation of the fluorescence intensity of TAMRA and Cy5.5 respectively
Relationship.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
Wuxi Di Tengmin Biotechnology Co., Ltd
<120>method of two kinds of miRNA is detected while a kind of effect based on FRET
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 41
<212> DNA
<213>artificial sequence
<400> 1
aaaaatcatc tatcaacatc agtctgataa gctatagaag c 41
<210> 2
<211> 11
<212> DNA
<213>artificial sequence
<400> 2
aaaaagcttg a 11
<210> 3
<211> 8
<212> DNA
<213>artificial sequence
<400> 3
aaagcttc 8
<210> 4
<211> 41
<212> DNA
<213>artificial sequence
<400> 4
aaaaatcatc tatcatcatt accaggcagt attaaacgtc g 41
<210> 5
<211> 11
<212> DNA
<213>artificial sequence
<400> 5
aaaaaatatg a 11
<210> 6
<211> 8
<212> DNA
<213>artificial sequence
<400> 6
aaacgacg 8
<210> 7
<211> 12
<212> PRT
<213>artificial sequence
<400> 7
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Cys
1 5 10
Claims (9)
1. converting satellite shape Nanoscale assemblies on a kind of gold stick core one, which is characterized in that preparation method includes the following steps:
(1) gold nanorods are synthesized, and are coupled DNA2、DNA5；
(2) by up-conversion nanoparticles respectively with DNA1、DNA4Coupling, wherein DNA2With DNA1Partial complementarity, DNA5With DNA4Portion
Divide complementation；Luminescent dye molecule TAMRA is coupled DNA3, luminescent dye molecule Cy5.5 coupling DNA6；Wherein, DNA3With DNA1Part
Complementation, DNA6With DNA4Partial complementarity；
(3) DNA will be coupled2、DNA5Gold nanorods and step (2) obtain fluorescent dye modification after upper conversion nano grain
Subgroup dress；
Wherein, DNA1With the first miRNA complete complementary to be measured, when encountering the first miRNA to be measured, the first miRNA and DNA to be measured2
Competitive binding DNA1；DNA4With the second miRNA complete complementary to be measured, when encountering the second miRNA to be measured, the second miRNA to be measured with
DNA5Competitive binding DNA4；
When into the cell there are when the first miRNA to be measured, with sequence DNA1In complementary fragment combine, lead to gold nanorods end face
Up-conversion nanoparticles dismiss；When into the cell there are when the second miRNA to be measured, with sequence DNA4In complementary fragment knot
It closes, the up-conversion nanoparticles of gold nanorods side is caused to be dismissed.
2. converting satellite shape Nanoscale assemblies on a kind of golden stick core one according to claim 1, which is characterized in that described the
One miRNA to be measured is miR-21, and second miRNA to be measured is miR-200b.
3. converting satellite shape Nanoscale assemblies on a kind of golden stick core one according to claim 1, which is characterized in that described
DNA1Sequence is as shown in SEQ ID NO.1, DNA2Sequence is as shown in SEQ ID NO.2, DNA3Sequence as shown in SEQ ID NO.3,
DNA4Sequence is as shown in SEQ ID NO.4, DNA5Sequence is as shown in SEQ ID NO.5, DNA6Sequence is as shown in SEQ ID NO.6.
4. converting satellite shape Nanoscale assemblies on a kind of golden stick core one according to claim 1, which is characterized in that step
(1), gold nanorods are resuspended in the CTAB solution of 0.005M, obtain the suspension of final concentration of 10nM, with molar concentration AuNR:
DNA2Ratio be 1:80-100 be added DNA2, centrifugation removes extra DNA after reacting 12h at room temperature2, it is resuspended in 0.001M's
In CTAB solution, the suspension of final concentration of 10nM is obtained；DNA is added with molar concentration 1:450-500 again4DNA5, react at room temperature
Centrifugation removes extra DNA after 12h4 DNA5, it is resuspended in the CTAB solution of 0.001M, obtains the suspension of final concentration of 10nM.
5. converting satellite shape Nanoscale assemblies on a kind of golden stick core one according to claim 1, which is characterized in that step
(2) in, up-conversion nanoparticles are diluted to 10nM, 100 μ L is respectively taken, is coupled DNA respectively1And DNA4, then it is added end modified
The DNA of fluorescent molecule TAMRA3With the DNA of end modified fluorescent molecule Cy5.56, hybridized to obtain and be coupled different fluorescent molecules
Up-conversion nanoparticles.
6. converting satellite shape Nanoscale assemblies on a kind of golden stick core one according to claim 1, which is characterized in that step
(3), to being coupled DNA2、DNA52 μ L of gold nanorods in, be added and modified the up-conversion nanoparticles 20 of fluorescent molecule TAMRA
μ L and the 100 μ L of up-conversion nanoparticles for having modified fluorescent molecule Cy5.5, the unbonded grain of gradient centrifugation removal after overnight incubation
Son obtains the assembly that star topology is defended in conversion on golden stick core one.
7. conversion satellite shape Nanoscale assemblies detect two kinds simultaneously on a kind of any golden stick core one of application claim 1~6
The method of miRNA, which comprises the steps of:
(1) it prepares and converts satellite shape Nanoscale assemblies on golden stick core one；
(2) it makes the standard curve in cell between the miRNA and fluorescence intensity of various concentration: satellite will be converted on golden stick core one
Shape structure assembly is thin with the target cell to be detected after the transfection of not same amount transfection agents and without the target to be detected after transfection
Born of the same parents are incubated for a period of time altogether respectively, then carry out fluorescence imaging, establish the mark between the miRNA of various concentration and fluorescence intensity
Directrix curve；
(3) cell-penetrating peptide is modified: star topology assembly: PEG5000 is defended in conversion on golden stick core one: cell-penetrating peptide TAT is with mole dense
The ratio for spending 1:1000:100 mixes, and after being incubated at room temperature 12h, centrifugation removal supernatant, gained precipitating is cell-penetrating peptide modification
Satellite shape Nanoscale assemblies are converted on golden stick core one；TAT polypeptide sequence is as shown in SEQ ID NO.7；
(4) star topology assembly is defended into conversion on the golden stick core one after the modification modification of film peptide and is transferred to cell to be measured, cultivate 8 hours
Afterwards, cell is washed three times with PBS, after removal does not enter the assembly of cell, is carried out up-conversion fluorescence and is copolymerized burnt imaging, in 980nm
Under excitation, fluorescent image is obtained, fluorescence intensity is substituted into standard curve, the content of miRNA intracellular is calculated.
8. a kind of for detecting the kit of miRNA intracellular, which is characterized in that contain any golden stick core of claim 1~6
Satellite shape Nanoscale assemblies are converted on one.
9. application of the kit described in claim 8 in detection miRNA.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711138127.XA CN108037100B (en) | 2017-11-16 | 2017-11-16 | A method of detecting two kinds of miRNA while based on FRET effect |
| PCT/CN2018/114795 WO2019096069A1 (en) | 2017-11-16 | 2018-11-09 | Method for simultaneously detecting two types of mirnas on the basis of fret effects |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711138127.XA CN108037100B (en) | 2017-11-16 | 2017-11-16 | A method of detecting two kinds of miRNA while based on FRET effect |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108037100A CN108037100A (en) | 2018-05-15 |
| CN108037100B true CN108037100B (en) | 2019-09-03 |
Family
ID=62092822
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711138127.XA Active CN108037100B (en) | 2017-11-16 | 2017-11-16 | A method of detecting two kinds of miRNA while based on FRET effect |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN108037100B (en) |
| WO (1) | WO2019096069A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108037100B (en) * | 2017-11-16 | 2019-09-03 | 江南大学 | A method of detecting two kinds of miRNA while based on FRET effect |
| CN109540865B (en) * | 2018-11-15 | 2020-08-11 | 江南大学 | Detection method of cytochrome c in living cell based on Raman-fluorescence dual-mode probe |
| CN109781705B (en) * | 2019-01-31 | 2020-09-04 | 江南大学 | High-flux and ultra-sensitive detection lattice array enhanced chip |
| CN111515410B (en) * | 2020-04-23 | 2022-12-02 | 江南大学 | Preparation method based on gold nanoparticle chiral three-dimensional structure conformation transformation |
| CN112961907B (en) * | 2021-03-04 | 2022-11-15 | 中南大学 | Fluorescent biosensor for simultaneously detecting two RNAs and preparation and use methods thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506136A (en) * | 2016-01-21 | 2016-04-20 | 武汉顺可达生物科技有限公司 | Method for detecting microRNA (microribonucleic acid) on basis of rolling circle amplification and upconversion material |
| CN106498047A (en) * | 2016-10-21 | 2017-03-15 | 江南大学 | Method based on tetrahedral dual signal in situ detection intracellular microRNA of golden up-conversion nanoparticles |
| CN106520964A (en) * | 2016-11-18 | 2017-03-22 | 南京中医药大学 | Double-recognition quantitative detection method for microRNA |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102782573A (en) * | 2009-11-22 | 2012-11-14 | 圣诺制药公司 | Rare earth-doped up-conversion nanoparticles for therapeutic and diagnostic applications |
| CN105705938B (en) * | 2013-08-19 | 2020-11-06 | 休斯敦大学*** | Phosphorescent reporters |
| EP3206987B1 (en) * | 2014-10-14 | 2020-07-01 | The University of Chicago | Nanoparticles for photodynamic therapy, x-ray induced photodynamic therapy, radiotherapy, chemotherapy, immunotherapy, and any combination thereof |
| CN106282175B (en) * | 2015-06-07 | 2019-12-20 | 复旦大学 | Hairpin type DNA template of fluorescent nano copper cluster and application thereof |
| CN108037100B (en) * | 2017-11-16 | 2019-09-03 | 江南大学 | A method of detecting two kinds of miRNA while based on FRET effect |
-
2017
- 2017-11-16 CN CN201711138127.XA patent/CN108037100B/en active Active
-
2018
- 2018-11-09 WO PCT/CN2018/114795 patent/WO2019096069A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506136A (en) * | 2016-01-21 | 2016-04-20 | 武汉顺可达生物科技有限公司 | Method for detecting microRNA (microribonucleic acid) on basis of rolling circle amplification and upconversion material |
| CN106498047A (en) * | 2016-10-21 | 2017-03-15 | 江南大学 | Method based on tetrahedral dual signal in situ detection intracellular microRNA of golden up-conversion nanoparticles |
| CN106520964A (en) * | 2016-11-18 | 2017-03-22 | 南京中医药大学 | Double-recognition quantitative detection method for microRNA |
Non-Patent Citations (1)
| Title |
|---|
| Dual quantification of MicroRNAs and telomerase in living cells;Wei Ma 等;《Journal of the American chemical society》;20170801;第139卷(第34期);第11752-11759页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108037100A (en) | 2018-05-15 |
| WO2019096069A1 (en) | 2019-05-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108037100B (en) | A method of detecting two kinds of miRNA while based on FRET effect | |
| CN106367510B (en) | A kind of preparation method and application of the satellite shape Nanoscale assemblies for cancer markers double check intracellular | |
| Lei et al. | Applications of mesenchymal stem cells labeled with Tat peptide conjugated quantum dots to cell tracking in mouse body | |
| Choi et al. | Developing luminescent silver nanodots for biological applications | |
| Kong et al. | Carbon dot‐based inorganic–organic nanosystem for two‐photon imaging and biosensing of pH variation in living cells and tissues | |
| WO2018072745A1 (en) | Method for double-signal in-situ detection of intracellular microrna based on gold-upconversion nanoparticle tetrahedron | |
| Subramaniam et al. | Generation of a library of non-toxic quantum dots for cellular imaging and siRNA delivery | |
| Li et al. | Standoff optical glucose sensing in photosynthetic organisms by a quantum dot fluorescent probe | |
| Mao et al. | Hydrophobic carbon nanodots with rapid cell penetrability and tunable photoluminescence behavior for in vitro and in vivo imaging | |
| CN106432203B (en) | Gemini type amphiphilic compound and its preparation method and application based on tetraphenyl ethylene base | |
| CN107325814B (en) | A kind of fluorescence silicon nano dots and the preparation method and application thereof | |
| Kostiv et al. | RGDS-and TAT-conjugated upconversion of NaYF4: Yb3+/Er3+ &SiO2 nanoparticles: in vitro human epithelioid cervix carcinoma cellular uptake, imaging, and targeting | |
| CN109307664B (en) | Fluorescent detection probe capable of detecting metal ions in living cells | |
| Jiang et al. | Persistent luminescence nanorod based luminescence resonance energy transfer aptasensor for autofluorescence-free detection of mycotoxin | |
| Zhang et al. | Controlling carbon encapsulation of gold nano-aggregates as highly sensitive and spectrally stable SERS tags for live cell imaging | |
| CN109897093B (en) | Multiple improved orange/red fluorescent proteins | |
| CN110003185B (en) | Macrocyclic polyamine amphiphilic compound based on green fluorescent protein chromophore BI and preparation method and application thereof | |
| CN106884016A (en) | The expression vector of long-chain non-coding RNA LINC00472, tumor suppression reagent and its application | |
| Liu et al. | Triple-emitting dumbbell fluorescent nanoprobe for multicolor detection and imaging applications | |
| Ding et al. | Luminescent silica nanosensors for lifetime based imaging of intracellular oxygen with millisecond time resolution | |
| EP3476402A1 (en) | Capsule for drug delivery systems of target tissue-specific delivery type using carbosilane dendrimer | |
| US8143013B2 (en) | Visible to near-infrared light probe using energy transfer between luciferase and an organic dye via a sugar chain | |
| Shen et al. | A one-step synthesis of novel high pH-sensitive nitrogen-doped yellow fluorescent carbon dots and their detection application in living cells | |
| Tsai et al. | Steroid probes conjugated with protein-protected gold nanocluster: specific and rapid fluorescence imaging of steroid receptors in target cells | |
| Patsula et al. | Polymer-coated hexagonal upconverting nanoparticles: chemical stability and cytotoxicity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |