CN108085369A - A kind of method of-four serobila DNA of quantum dot fluorescence coding microball-rolling circle amplification high throughout screening of G - Google Patents
A kind of method of-four serobila DNA of quantum dot fluorescence coding microball-rolling circle amplification high throughout screening of G Download PDFInfo
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Abstract
The invention discloses the methods of quantum dot fluorescence coding microball-tetra- serobila DNA of rolling circle amplification screening G a kind of, include the following steps：Prepare the quantum dot fluorescence coding microball of label probe；Tetra- serobila DNA sequence dnas of target G carry out rolling circle amplification reaction on quantum dot fluorescence coding microball surface；Amplified production is dyed by tetra- serobila specific recognition molecules of G；Tetra- serobila DNA of G are screened using flow cytometer.The method of quantum dot fluorescence coding microball-tetra- serobila DNA of rolling circle amplification screening G in the present invention, rolling circle amplification and quantum dot fluorescence coding microball are combined for the first time, tetra- serobila DNA of realization G are highly sensitive, specific detection, and accuracy is high, detection range is wide, for tetra- serobila DNA of G are quick, high flux screening provides new method.
Description
Technical field
The invention belongs to biomedical sectors, and in particular to a kind of quantum dot fluorescence coding microball-Rolling Circle Amplification methods are high
Tetra- serobila DNA of flux screening G-.
Background technology
Malignant tumour is to endanger the major disease of human life and health.It is carried out in recent years based on bio-target molecule anti-swollen
The screening of tumor medicine becomes new research direction.Containing can largely form the sequences of tetra- serobilas of G- (about in human genome
376000) chromosome telomere region (telo), gene promoter region (such as k-ras, bcl-2), immune protein, are widely present in
Among switch region, exon region and RNA.The occurrence and development of tetra- serobilas of G- and tumour are closely related, stable tetra- chains of G-
Body can inhibit the activity of cancer cell telomerase or the transcription of some genes is closed as silencer element.Different sequences
Tetra- stranded structures of G- of formation have differences in the screw orientation of chain, polarity, space structure characteristic etc., cause and drug point
The strong and weak difference of son effect.Therefore carrying out screening anticancer medicine as action target spot using tetra- serobilas of G- becomes the hot spot of research.
There are many methods for detecting tetra- serobilas of G- at present, including molecular imprinting method, direct sequencing, real-time quantitative
PCR, planar microarrays, albumen probe and Small-molecule probe etc..But these methods lack high-throughput detectability.
High-throughput detection based on fluorescence-encoded micro-beads incorporates Flow Cytometry and the advantage of fluorescent microsphere technology, will
Fluorescence-encoded micro-beads technology, laser technology, fluidics, high-speed digital signal technology and computer algorithm are combined,
Have many advantages, such as it is high-throughput, at high speed, it is reproducible, easy to operate.However the sensitivity in most cases detected can not also
Meet the requirement of clinical analysis.
New approach is provided to improve detection sensitivity based on the method for amplifying signal of enzymatic amplification.Rolling circle amplification is a kind of
Constant temperature nucleic acid amplification method.Two termini-complementaries of target DNA and padlock probe match, and connecting cyclisation using ligase forms ring-type
DNA, and as template, for target DNA as primer, the template that enzymatically synthesis is repeated comprising hundreds and thousands of is complementary
The single stranded DNA of segment.Signal amplification based on rolling circle amplification has the characteristics that high sensitivity, specificity are good, is commonly applied to monokaryon
The detections such as nucleotide polymorphism, animals and plants virus.
The content of the invention
Goal of the invention：For presently, there are the problem of, the present invention provides a kind of quantum dot fluorescence coding microball-rolling ring and expands
Increase the method for tetra- serobila DNA of screening G-, this method can sensitive, special, quick ,-four serobila DNA of high throughout screening of G.
Technical solution：To achieve these goals, a kind of quantum dot fluorescence coding microball-rolling ring expands as described herein
Increase the method for tetra- serobila DNA of screening G-, include the following steps：
(1) the quantum dot fluorescence coding microball of label probe is prepared；
(2) tetra- serobila DNA sequence dnas of target G- carry out rolling circle amplification reaction on quantum dot fluorescence coding microball surface；
(3) by tetra- serobila specific recognition molecules of G- to step (2) quantum dot fluorescence coding microball surface rolling circle amplification
Product is dyed；
(4) tetra- serobila DNA of G- are screened using flow cytometer.
Wherein, the label probe includes three kinds of capture probes and three kinds of padlock probes.
The capture probe is included for the first capture probe capture of tetra- serobila sequence screenings of G- in k-ras genes
1, for the second capture probe capture 2 of tetra- serobila sequence screenings of G- in bcl-2 genes, for chromosome telomere region end
Hold the 3rd capture probe capture 3 of tetra- serobila sequence screenings of telo G-, the 5' Amino End Groupizations modification of three kinds of capture probes.
The padlock probe includes the first padlock probe padlock 1 hybridized with capture 1；It is miscellaneous with capture 2
The the second padlock probe padlock 2 handed over；The 3rd padlock probe padlock 3 hybridized with capture 3；Three kinds of padlock probes
5 ' end phosphorylation modifications.
Step (1) the quantum dot fluorescence coding microball for preparing label probe is respectively to be coupled to three kinds of capture probes
Three kinds of label probes are made in corresponding quantum dot fluorescence-encoded micro-beads surface, and hybridizing respectively with corresponding three kinds of padlock probes
Quantum dot fluorescence coding microball.
Step (2) the quantum dot fluorescence coding microball surface carries out rolling circle amplification reaction, three kinds of tetra- serobila DNA of target G-
Sequence hybridizes with corresponding padlock probe and is connected cyclization, carries out rolling circle amplification reaction.Three kinds of target G-, the tetra- serobila DNA sequence dnas
The respectively tetra- serobila DNA sequence dna target 1 of G- of k-ras genes are used to hybridize padlock 1, tetra- serobilas of G- of bcl-2 genes
DNA sequence dna target 2 is used to hybridize padlock 2, tetra- serobila DNA sequence dna target of chromosome telomere area distal telo G-
3 are used to hybridize padlock 3.
The time of step (2) the quantum dot fluorescence coding microball surface rolling circle amplification reaction is respectively 20,40,60,80
With 100 minutes, the fluorescence intensity and microballoon number of amplified production are measured by flow cytometer, finally preferably 60 minutes
Time as the reaction of quantum dot fluorescence coding microball surface rolling circle amplification.
By the quantum dot fluorescence coding microball mixed in equal amounts of three kinds of label probes, three kinds of target DNA of different ratio are added in
(molar ratio of three kinds of targets is respectively 0 to mixture:0:0,1:0:0,0:1:0,0:0:1,0:1:1,1:0:1,1:1:0,1:1:
1), rolling circle amplification reacts 60 minutes.
Step (3) the tetra- serobila specific recognition molecules of G- are macrocycle molecule Phthalocyanine Zinc or complex ruthenium chemical combination
Object.Usually Phthalocyanine Zinc (ZnPc) is selected to dye amplified production.
Step (4) is described to screen tetra- serobila DNA of G- using flow cytometer.By flow cytometer to quantum dot
Fluorescence-encoded micro-beads are analyzed, and encoded signal identifies for target, and report signal is demarcated for target content, so as to fulfill G-
Four serobila DNA are screened.
The principle process of the quantum dot fluorescence coding microball of the present invention-tetra- serobila DNA of Rolling Circle Amplification methods screening G- is as schemed
Shown in 1.Polymer microballoon is primarily based on, two kinds of color quantum dots of " swelling-volatilization " method doping different proportion is used, prepares three
The fluorescence-encoded quantum dot polymer microspheres of kind, and pass through improvedThe amount of high stability is made in method coated silica
Son point fluorescence-encoded micro-beads.By carbonyl diimine reaction by three kinds of quantum dot fluorescence coding microballs respectively with corresponding tetra- chains of G-
Body capture probe is coupled, and is hybridized with corresponding padlock probe, and the quantum dot fluorescence coding microball of label probe is made.Add in target
Mark tetra- serobila DNA of G- are attached cyclization with padlock probe, using the capture probe of microsphere surface as primer, to connecting into
The padlock probe of ring carries out rolling circle amplification.By the optimization to proliferation time, 60 minutes are selected as the excellent of rolling circle amplification reaction
Change time, the progress that amplified reaction can quickly, stable, while do not influence quantum dot fluorescence-encoded micro-beads dispersiveness.Use spy
Dyestuff-Phthalocyanine Zinc (ZnPc) of opposite sex identification tetra- serobilas of G- dyes amplified production, by flow cytometer with quantum dot fluorescence
Coding realizes the screening to tetra- serobila DNA of G- as identification signal, ZnPc fluorescence as detection signal.
Advantageous effect：Compared with prior art, the invention has the advantages that：
The method of the tetra- serobila DNA of quantum dot fluorescence coding microball rolling circle amplification screening G- of the present invention, for the first time expands rolling ring
Increase and quantum dot fluorescence coding microball be combined, realize that G- tetra- serobila DNA are sensitive, specific detection, be tetra- serobila DNA of G- it is quick,
High flux screening provides new method.
The method that the present invention is used for-four serobila DNA of high throughout screening of G, accuracy can reach 95%；The range of linearity is
10fM-1nM reaches 5 orders of magnitude；Detection is limited to 2.2fM, and detection limits low, high sensitivity.
Description of the drawings
Fig. 1 screens tetra- serobila DNA flow diagrams of G- for quantum dot fluorescence coding microball-Rolling Circle Amplification methods；
(A) is quantum dot fluorescence coding microball scanning electron microscope (SEM) characterization and particle diameter distribution in Fig. 2；(B) it is three
Kind quantum dot fluorescence coding microball flow cytometer characterization；
Fig. 3 is fluorescent stabilization of the quantum dot fluorescence coding microball under the conditions of conjugated probes, rolling circle amplification and dyeing
Property characterization schematic diagram；
Fig. 4 is the flow cytometer characterization that quantum dot fluorescence coding microball surface rolling circle amplification changes over time；
Fig. 5 is report signal average fluorescent strength (MFI) and corresponding target based on three kinds of quantum dot fluorescence coding microballs
The relation schematic diagram of tetra- serobila DNA concentrations of G-；
Fig. 6 is the accuracy schematic diagram of quantum dot fluorescence coding microball rolling circle amplification detection method；
Flow cytometer of Fig. 7 quantum dot fluorescence coding microballs Rolling Circle Amplification methods for tetra- serobila DNA of G- screenings characterizes
Schematic diagram；The wherein orange tetra- serobila DNA fragmentations of G- for representing k-ras genes, dark yellow represent tetra- serobilas of G- of bcl-2 genes
DNA fragmentation；Green represents tetra- serobila DNA fragmentations of chromosome telomere area distal telo G-；Minus sign represents to deposit without corresponding target
Corresponding target sequence is added in the expression of, plus sige.
Specific embodiment
Below in conjunction with drawings and examples, the invention will be further described.
Embodiment 1
The nucleotide sequence design of tetra- serobila DNA of capture probe, padlock probe and target G-
The tetra- serobila DNA fragmentations of G- of tetra- serobila DNA fragmentations (target 1) of G-, bcl-2 genes for k-ras genes
(target 2), tetra- serobila segments (target 3) of chromosome telomere area distal telo G- devise point for being capable of specificity
Padlock probe (padlock 1, the padlock not hybridized with three target sequences (target 1, target 2, target 3)
2、padlock 3).The probe sequence includes two parts：Target cog region, for hybridizing with target-complementary, in connection enzyme effect
Lower connection cyclization；Padlock probe can be connected to microsphere surface by capture probe cog region by hybridization.All padlock probes are caught
Obtain probe, tetra- serobila DNA of target G- are synthesized by Invitrogen companies.The end of padlock probe 5 ' carries out phosphorylation modification；Capture
5 ' ends of probe carry out amination modification.Each sequence is as shown in table 1.Padlock probe underscore part is known for capture probe in table 1
Underscore partial complementarity in other area, with capture probe (capture 1, capture 2, capture 3)；Padlock probe overstriking
Part is target cog region, is matched with target-complementary and bends cyclization；Italic is mutating alkali yl.
The nucleotide sequence of tetra- serobila DNA of 1 capture probe of table, padlock probe and target G-
Embodiment 2
It is prepared by the quantum dot fluorescence coding microball of label probe
The chloroform soln (2mg/mL) of polystyrene microsphere and the chloroform soln of green, orange two amounts point mix
It closes, stirring at normal temperature to solvent is volatilized completely, and quantum dot fluorescence coding microball is obtained after centrifugal drying.It is added in 20mL ethyl alcohol
When 600mg PVP and 4mg microballoon and small stirring 6,20mL ethyl alcohol is scattered in after centrifuge washing, 400 μ L ammonium hydroxide is added in, is added dropwise
800 μ L TEOS and stir 12 it is small when, centrifuge washing obtains the quantum dot fluorescence coding microball of coated with silica.Pass through scanning
Electron microscope is characterized, and is by the quantum dot fluorescence coding microball grain size that coated with silica can be drawn in Fig. 2 (A)
8.0 μm, smooth surface.By adjusting the ratio of green, orange two amounts point as shown in Fig. 2 (B), it is glimmering green, orange quantum dot has been obtained
Intensity ratio is 0:1、1:1、1:0 three kinds of quantum dot fluorescence coding microballs.
Quantum dot fluorescence coding microball be stirred at room temperature under different chemical environments 6 it is small when, measure green, orange two amounts son point
Fluorescence intensity characterizes the fluorescent stability of quantum dot fluorescence coding microball.Coupling reagent concentration used is：5mM EDC、
5mM NHS；Amplifing reagent concentration is：0.25UμL-1T4ligase, 10 μ L PEG 4000 (50% solution), 7 μM of BSA, 0.04U
μL-1Phi 29,10mM dNTP and 20 μM of ZnPc of dyestuff.From the figure 3, it may be seen that quantum dot fluorescence coding microball is in conjugated probes, rolling
There is higher fluorescent stability under the conditions of circle amplification and dyeing.
Be separately added into 1 milliliter of pH 6.0MES buffer solution the same quantum dot fluorescence coding microballs of 4mg, 100 microlitres 10 μM
Capture and 20 microlitre 50mg/mL EDC corresponding with the coding and stir 2 it is small when, add 20 microlitres of 50mg/mL EDC
And stir 3 it is small when, then add in Tris-HCl buffer solutions (pH 7.4) containing 0.1%Tween-20 and stir 30 minutes, centrifugation
Tris-HCl buffer solutions (1.0 × 10 are scattered in after washing4A quantum dot fluorescence coding microball/100 microlitre).It is micro- to be eventually adding 40
10 μM of corresponding padlock probes are risen, when concussion reaction 1 is small under the conditions of 37 DEG C, milli-Q water obtains the quantum dot of label probe
Fluorescence-encoded micro-beads.(coding 0:1 quantum dot fluorescence microballoon coupling capture1, hybridizes with padlock1；Coding 1:1 amount
Son point fluorescent microsphere coupling capture2, hybridizes with padlock2；Coding 1:0 quantum dot fluorescence microballoon coupling capture3,
Hybridize with padlock3).
Embodiment 3
The rolling circle amplification reaction of quantum dot fluorescence coding microball surface and dyeing
(contain 1.0 × 10 in 120 microlitres of ultra-pure waters4The quantum dot fluorescence coding microball of a same label probe), add
Enter 10 microlitres 10 μM and the corresponding target DNA of the coding, 15 microlitres of 10 × T4ligase buffer solutions and 5 microlitres of T4ligase,
When reaction 1 is small at room temperature.Milli-Q water three times after, be dissolved in 80 microlitres of ultra-pure waters again, and it is slow to add in 10 microlitres of 10 × phi 29
Fliud flushing, 6 microlitres of 10mM dNTPs and 4 microlitres of 29 polymerases of phi choose 20,40,60,80 and 100 points under the conditions of 38 DEG C respectively
Clock is reacted.Finally amplified production is dyed with ZnPc.
The results are shown in Figure 4, flow cytometer the result shows that, rolling circle amplification reaction carry out 60 minutes, report signal reaches
Highest, and microballoon keeps good dispersion.So it is quantum dot fluorescence coding microball surface rolling circle amplification and reacts for 60 minutes
Optimize the time.
Embodiment 4
The sensitivity of quantum dot fluorescence coding microball-rolling circle amplification detection method
Contain 1.0 × 10 in 10 microlitres of amplification buffers3The quantum dot fluorescence coding microball of a same label probe, is adopted
Be incubated with the method for embodiment 3 with the corresponding target of various concentration (concentration be respectively 0,0.1fM, 1fM, 10fM, 50fM,
100fM, 500fM, 1pM, 5pM, 10pM, 50pM, 100pM, 500pM, 1nM, 5nM, 10nM), rolling circle amplification reacts 60 minutes, leads to
Overflow-type cell instrument measures report signal average fluorescent strength, draws standard curve, the results are shown in Figure 5.This is calculated by Fig. 5
Method is limited to 2.2fM, range of linearity 10fM-1nM, related coefficient >=0.990 to the detection of tetra- serobila DNA of G-.
Embodiment 5
The accuracy of quantum dot fluorescence coding microball-rolling circle amplification detection method
Change content (its difference of molar ratio in biased sample of mutant nucleotide sequence in telo and telo mutation biased samples
For 0:100,1:100,1:50,1:20,1:10,1:5), reacted 60 minutes using the method rolling circle amplification of embodiment 3, pass through stream
Formula cell instrument measures report signal average fluorescent strength, calculates accuracy in detection.The results are shown in Figure 6, when telo-mut contents
Reach 1:When 20, mutation can be detected significantly, and the accuracy of tetra- serobila DNA of this method detection G- is 95%.
Embodiment 6
The screening of tetra- serobila DNA of G-
(contain the quantum dot fluorescence coding microball there are three types of label probe, the quantity of each microballoon is equal in 360 microlitres of ultra-pure waters
For 1.0 × 104It is a), target DNA mixtures (mole score of three kinds of targets of different ratio is added in using the method for embodiment 3
It Wei 0:0:0,1:0:0,0:1:0,0:0:1,0:1:1,1:0:1,1:1:0,1:1:1), rolling circle amplification reacts 60 minutes.With stream
Formula cell instrument analyzes quantum dot fluorescence coding microball, and the results are shown in Figure 7.
Shown by Fig. 7 in the presence of corresponding target, corresponding report signal enhances 100 times or more, and only corresponding
Target can start the rolling circle amplification process on corresponding quantum dot fluorescence-encoded micro-beads surface, and report signal is caused to significantly increase.Cause
This quantum dot fluorescence coding microball-Rolling Circle Amplification methods screens suitable for tetra- serobila DNA of G-.
Sequence table
<110>Southeast China University
<120>A kind of method of-four serobila DNA of quantum dot fluorescence coding microball-rolling circle amplification high throughout screening of G
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 51
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tttttttttt ttaacgctta acgcttaacg cccacacttc ccatccatct c 51
<210> 2
<211> 90
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cttcccacac cgcccttatc ctgctcgcca gttaggtttt tttttgagat ggatgggaag 60
tgtggttttt ttttcctccc cctcttccct 90
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
agggcggtgt gggaagaggg aagaggggga gg 32
<210> 4
<211> 51
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tttttttttt ttaacgctta acgcttaacg ccgatagata tacttgtctt a 51
<210> 5
<211> 85
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tcctcccgcg cccgtatcct gctcgccagt taggtttttt ttttaagaca agtatatcta 60
tcgttttttt ttgctcccgc cccct 85
<210> 6
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cgggcgcggg aggaaggggg cgggagc 27
<210> 7
<211> 51
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
tttttttttt ttaacgctta acgcttaacg ctgcgctatc tcgtgtgacc a 51
<210> 8
<211> 82
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
cctaacccta atatcctgct cgccagttag gttttttttt tggtcacacg agatagcgca 60
tttttttttc cctaacccta ac 82
<210> 9
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ttagggttag ggttagggtt aggg 24
<210> 10
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
ttagggttag agttagggtt aggg 24
Claims (7)
1. the method for a kind of quantum dot fluorescence coding microball-tetra- serobila DNA of rolling circle amplification screening G-, which is characterized in that including such as
Lower step：
(1) the quantum dot fluorescence coding microball of label probe is prepared；
(2) tetra- serobila DNA sequence dnas of target G- carry out rolling circle amplification reaction on quantum dot fluorescence coding microball surface；
(3) by tetra- serobila specific recognition molecules of G- to step (2) quantum dot fluorescence coding microball surface rolling circle amplification product
It is dyed；
(4) tetra- serobila DNA of G- are screened using flow cytometer.
2. the method for quantum dot fluorescence coding microball according to claim 1-tetra- serobila DNA of rolling circle amplification screening G-,
It is characterized in that, the label probe includes three kinds of capture probes and three kinds of padlock probes；The capture probe is included for k-ras
First capture probe capture 1 of tetra- serobila sequence screenings of G- in gene, for tetra- serobila sequence screenings of G- in bcl-2 genes
The second capture probe capture 2, for tetra- serobila sequence screenings of chromosome telomere area distal telo G- the 3rd capture
Probe capture 3, the 5' Amino End Groupizations modification of three kinds of capture probes；The padlock probe includes what is hybridized with capture 1
First padlock probe padlock 1；The the second padlock probe padlock 2 hybridized with capture 2；Hybridize with capture 3
The 3rd padlock probe padlock 3；5 ' end phosphorylation modifications of three kinds of padlock probes.
3. the method for quantum dot fluorescence coding microball according to claim 1-tetra- serobila DNA of rolling circle amplification screening G-,
It is characterized in that, step (1) the quantum dot fluorescence coding microball for preparing label probe is respectively by three kinds of capture probes coupling
To quantum dot fluorescence coding microball surface, and hybridize respectively with corresponding three kinds of padlock probes, the amount of three kinds of label probes is made
Son point fluorescence-encoded micro-beads.
4. the method for quantum dot fluorescence coding microball according to claim 1-tetra- serobila DNA of rolling circle amplification screening G-,
It is characterized in that, step (2) the quantum dot fluorescence coding microball surface carries out rolling circle amplification reaction.Three kinds of tetra- serobilas of target G-
DNA sequence dna padlock probe corresponding to quantum dot fluorescence coding microball surface hybridizes and connects cyclization, carries out rolling circle amplification reaction；Institute
The tetra- serobila DNA sequence dna target 1 of G- that three kinds of tetra- serobila DNA sequence dnas of target G- are respectively k-ras genes are stated to be used to hybridize
The tetra- serobila DNA sequence dna target 2 of G- of padlock 1, bcl-2 gene are used to hybridize padlock 2, chromosome telomere region
Telo G- tetra- serobila DNA sequence dna target 3 in end are used to hybridize padlock 3.
5. the method for quantum dot fluorescence coding microball according to claim 1-tetra- serobila DNA of rolling circle amplification screening G-,
It is characterized in that, step (2) the quantum dot fluorescence coding microball surface carry out the time of rolling circle amplification reaction for 20,40,60,
80 or 100 minutes.
6. the method for quantum dot fluorescence coding microball according to claim 1-tetra- serobila DNA of rolling circle amplification screening G-,
It is characterized in that, step (3) the tetra- serobila specific recognition molecules of G- are macrocycle molecule Phthalocyanine Zinc or complex ruthenium
Close object.
7. the method for quantum dot fluorescence coding microball according to claim 1-tetra- serobila DNA of rolling circle amplification screening G-,
It is characterized in that, step (4) is described to screen tetra- serobila DNA of G- using flow cytometer.By flow cytometer to quantum
Point fluorescence-encoded micro-beads are analyzed, and encoded signal identifies for target, and report signal is demarcated for target content, so as to fulfill
Tetra- serobila DNA of G- are screened.
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| CN110004198A (en) * | 2019-04-11 | 2019-07-12 | 中国农业大学 | The preparation method of soft brush Rapid self assembly functional nucleic acid hydrogel |
| CN110295219A (en) * | 2019-07-27 | 2019-10-01 | 福州大学 | A kind of preparation method for the DNA nanowire cluster loading phenanthroline ruthenium |
| CN110305937A (en) * | 2019-07-04 | 2019-10-08 | 东南大学 | Polynary nucleic acid detection method and kit |
| CN110878342A (en) * | 2019-12-25 | 2020-03-13 | 陕西师范大学 | Digital absolute quantitative analysis method for nucleic acid marker based on single fluorescent particle counting of flow cytometer |
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| CN110878342A (en) * | 2019-12-25 | 2020-03-13 | 陕西师范大学 | Digital absolute quantitative analysis method for nucleic acid marker based on single fluorescent particle counting of flow cytometer |
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