CN106636326A - Method for preparing DNA (deoxyribonucleic acid) nano-probes with electrochemical luminescence activity for detecting micro RNA (ribonucleic acid) - Google Patents
Method for preparing DNA (deoxyribonucleic acid) nano-probes with electrochemical luminescence activity for detecting micro RNA (ribonucleic acid) Download PDFInfo
- Publication number
- CN106636326A CN106636326A CN201610835093.9A CN201610835093A CN106636326A CN 106636326 A CN106636326 A CN 106636326A CN 201610835093 A CN201610835093 A CN 201610835093A CN 106636326 A CN106636326 A CN 106636326A
- Authority
- CN
- China
- Prior art keywords
- dna
- nano
- probes
- micro rna
- electrochemical luminescence
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing DNA (deoxyribonucleic acid) nano-probes with electrochemical luminescence activity for detecting micro RNA (ribonucleic acid). The method includes carrying out self-assembly reaction on DNA probes and silicon dioxide composite nano-particles doped with chitosan and trisruthenium to obtain the DNA nano-probes with the electrochemical luminescence activity. Enrichment effects can be realized by the DNA nano-probes on the surfaces of modified electrodes, and the DNA nano-probes can be used for detecting the micro RNA by means of electrochemical luminescence sensing. Compared with the prior art, the method has the advantages that complicated DNA markers can be omitted, the DNA nano-probes are sensitive, simple and universal, and the micro RNA detection limit can reach 0.1 pmol/L.
Description
Technical field
The invention belongs to nanometer detection technical field, and in particular to a kind of DNA probe by with doping shitosan and join pyrrole
The silica composite nanoparticle self assembly of pyridine ruthenium, prepares the method for detecting the DNA nano-probes of micro RNA.
Background technology
DNA nano-probes, because it has chemically synthesis, stable in properties, identification extensively and with signal reports ability etc.
Advantage, and more and more it is applied to bioanalysis.Existing DNA nano-probes technology of preparing majority is to lead to DNA probe
Cross chemical bonding effect to be fixed on nano-material surface or carry out signaling molecule mark to it, some also need to enzyme and are aided in,
This can make, and experimental procedure is loaded down with trivial details, experimental cost is high；On the other hand, DNA probe is fixed on into a nanometer material by chemical combination bonding action
Material surface can substantially reduce the spatial degrees of freedom of probe so as to due to the shadow of sterically hindered grade during identification target molecule
Ring and reduce molecule distinguishability.Therefore, develop the preparation method of new DNA nano-probes, overcome above-mentioned weak point just
With important science and using value.
The content of the invention
The technical problem to be solved be a kind of preparation process of development it is simple, without the need for the DNA nanometers of DNA marker
The preparation method of probe.
The technical scheme that solution above-mentioned technical problem is adopted is comprised the steps of：
1st, the silica composite nanoparticle of doping shitosan and bipyridyl ruthenium is prepared
By shitosan addition mass fraction be in 0.1% aqueous acetic acid be configured to mass-volume concentration for 0.5～
The chitosan solution of 5mg/mL, is then added to Triton X-100 and n-hexyl alcohol, hexamethylene, ultrapure by gained chitosan solution
In the mixed liquor of water, the bipyridyl ruthenium aqueous solution of 0.01mol/L is added, with 0.1mol/L NaOH aqueous solution regulation system extremely
Neutrality, is stirred at room temperature 30～60 minutes, adds tetraethyl orthosilicate and ammoniacal liquor, continues to stir 20～24 hours, adds acetone breakdown of emulsion,
Centrifugation, gained solid with absolute ethyl alcohol and milli-Q water, obtains the titanium dioxide of doping shitosan and bipyridyl ruthenium successively
Silicon composite nanoparticle.
2nd, DNA nano-probes are prepared
The doping shitosan that step 1 is obtained and the silica composite nanoparticle of bipyridyl ruthenium are scattered in ultra-pure water
In, the DNA probe corresponding with micro RNA to be detected is subsequently adding, adulterate shitosan and bipyridyl in control reaction system
The concentration of the silica composite nanoparticle of ruthenium be 0.10～0.25mg/mL, DNA probe concentration be 5 × 10-10～5 ×
10-8Mol/L, room temperature reaction 30～40 minutes, centrifugation, and with the PB buffer solutions 1～2 of 1.0mmol/L pH=6.0
It is secondary, obtain DNA nano-probes.
In above-mentioned steps 1, preferred tetraethyl orthosilicate and chitosan solution, the bipyridyl ruthenium aqueous solution, ammoniacal liquor, the body of ultra-pure water
Product is than being 1:1.5～2.0:0.3～0.8:0.6～0.8:It is 3～3.5, preferred Triton X-100 and n-hexyl alcohol, hexamethylene, super
The volume ratio of pure water is 1:1:4.0～4.3:0.1～0.3.
In above-mentioned steps 2, the silica composite Nano of adulterate in preferred control reaction system shitosan and bipyridyl ruthenium
The concentration of particle be 0.15～0.20mg/mL, DNA probe concentration be 5 × 10-9～4.5 × 10-8Mol/L, wherein described
DNA probe is scattered in the PB buffer solutions of 1.0mmol/LpH=6.0.
The silica composite nanoparticle self assembly that the present invention passes through DNA probe and doping shitosan and bipyridyl ruthenium,
It is prepared into the DNA nano-probes for detecting micro RNA.Compared with prior art, the present invention has following features：
1st, without the need for complicated mark and fixing DNA probe molecule manipulation process, it mainly utilizes DNA probe molecule to the present invention
DNA nano-probes are prepared in the self assembly of nano-material surface, if it is 20 that DNA probe is single stranded DNA and base number～
35, the base sequence of DNA probe is not required, therefore with general applicability.
2nd, because DNA probe is in the special assembling morphology of nanoparticle surface, target micro RNA is made to be more easy to and be assembled in
The DNA probe hybridization of nanoparticle surface, and electrochemical luminescence nano-particle is discharged, thus using electrochemiluminescence analysis skill
Art can fast and efficiently recognize target micro RNA, and the detection limit of target micro RNA is low, up to 0.1pmol/L.
Description of the drawings
Fig. 1 is the transmission electron microscope figure of DNA nano-probes prepared by embodiment 1.
Fig. 2 be embodiment 1 prepare DNA nano-probes (curve a) and DNA nano-probes act on let-7a after (curve
B) electrochemical luminescence comparison diagram.
Fig. 3 is linear relationship chart of the DNA nano-probes of the preparation of embodiment 1 to let-7a.
Fig. 4 is electrochemical luminescence stacking chart of the DNA nano-probes of the preparation of embodiment 1 to let-7a.
Specific embodiment
With reference to the accompanying drawings and examples the present invention is described in more detail, but protection scope of the present invention not only limits this
A little embodiments.
Embodiment 1
1st, the silica composite nanoparticle of doping shitosan and bipyridyl ruthenium is prepared
It is in 0.1% aqueous acetic acid, to be configured to mass-volume concentration by 0.2g shitosans addition 100mL mass fractions
For the chitosan solution of 2mg/mL, then 150 μ L gained chitosan solutions are added into 1.8mL Triton X-100 and 1.8mL
In n-hexyl alcohol, 7.5mL hexamethylenes, the mixed liquor of 300 μ L ultra-pure waters, stir 30 minutes, add 50 μ L 0.01mol/L connection pyrroles
The pyridine ruthenium aqueous solution, and add 0.1mol/L NaOH aqueous solution regulation systems to neutrality, it is stirred at room temperature 60 minutes, sequentially add 90 μ
L tetraethyl orthosilicates and 60 μ L ammoniacal liquor, room temperature continues to stir 24 hours, adds acetone breakdown of emulsion, centrifugation, gained solid to use successively
Absolute ethyl alcohol and milli-Q water, obtain the silica composite nanoparticle of doping shitosan and bipyridyl ruthenium.
2nd, DNA nano-probes are prepared
The doping shitosan that step 1 is obtained and the silica composite nanoparticle of bipyridyl ruthenium are scattered in ultra-pure water
In, (base sequence is AACTATACAACCTACTACCTCA, is scattered in be subsequently adding the DNA probe corresponding with let-7a
In the PB buffer solutions of 1.0mmol/L pH=6.0, concentration is 4 × 10-8Mol/L), adulterate in reaction system shitosan and connection are controlled
The concentration of the silica composite nanoparticle of pyridine ruthenium is 0.15mg/mL, the concentration of DNA probe is 2 × 10-8Mol/L, room temperature
Reaction 30 minutes, centrifugation, and with the PB buffer solutions 1～2 time of 1.0mmol/L pH=6.0, obtain the spy of DNA nanometers
Pin, is dispersed in stored refrigerated in the PB buffer solutions of 1.0mmol/L pH=6.0.Using JEM-2100 type transmission electron microscopies
Mirror (Hitachi, Japan) is characterized respectively to gained DNA nano-probes, as a result sees Fig. 1.As seen from the figure, the DNA nanometers being prepared into
Detecting probe surface can be seen the bubble for significantly being formed outwardly due to hydrophobicity base, illustrate that DNA probe is existed by successful self assembly
Composite nanoparticle surface.
In order to prove beneficial effects of the present invention, the DNA nano-probes detection let- that inventor is prepared using embodiment 1
7a, concrete test is as follows：
1st, electrochemical luminescence detection
(1) hybridization reaction of DNA nano-probes and let-7a
The DNA nano-probes that 20 μ L are scattered in the PB buffer solutions of 1.0mmol/L pH=6.0 are taken, after adding 20 μ L annealing
(with the PB buffers of 1.0mmol/L pH=6.0, concentration is 1 × 10 to let-7a-8Mmol/L), after fully mixing, room temperature
Hybridization 40 minutes.
(2) preparation of Nafion/MWNT modified electrodes
By glass-carbon electrode successively with 0.3 μm and 0.05 μm of Al2O3Polishing powder is polished light on polishing cloth, then successively
It is cleaned by ultrasonic respectively 5 minutes with ethanol and distilled water, is dried naturally after taking-up ultrapure water.By 1.5mg MWNT ultrasounds point
Dissipate in the Nafion ethanol solutions that 3.0mL volume fractions are 0.05%, then take the 10 μ L dispersant liquid drops and be coated in what is handled well
Glassy carbon electrode surface, standing under room temperature makes it dry to form Nafion/MWNT modified electrodes naturally.
(3) electrochemical luminescence signals detection
Nafion/MWNT modified electrodes are submerged initially in the DNA nano-probe dispersion liquids that embodiment 1 is obtained, room temperature is quiet
Put 60 minutes, after fully being rinsed and dried with ultra-pure water, conventionally carry out electrochemical luminescence detection, obtain DNA nanometers
Probe is assembled into electrochemical luminescence intensity I corresponding on Nafion/MWNT modified electrodes0；By Nafion/MWNT after having detected
Modified electrode is immersed again in the hybridization reaction solution of step (1) DNA nano-probes and let-7a, after being stored at room temperature 60 minutes, then is adopted
Electrochemical luminescence detection is carried out with RFL-1 types chemiluminescent analyzer, corresponding electrochemical luminescence intensity I is obtainedi, i is 1～n
Between positive integer.As a result Fig. 2 is seen.As seen from the figure, electrochemical luminescence signals after DNA nano-probes and let-7a hybridization reactions
Substantially increase, illustrate that let-7a can make self assembly in the DNA probe of nanoparticle surface by hybridizing from nanoparticle surface
Depart from, so that nanoparticle surface recovers electropositivity and is attracted to modified electrode surface.
2nd, linear relationship
According to the method for above-mentioned steps (3), the DNA nano-probes obtained to embodiment 1 and variable concentrations let-7a standards
Sample (0.1,0.3,0.5,0.7,0.9pmol/L) solution after hybridization reaction carries out electrochemical luminescence signals detection, respectively
To corresponding Ii, draw IiWith the calibration curve of let-7a change in concentration, Fig. 3 and 4 is as a result seen.As seen from the figure, different let-7a
I under concentrationiThe linear relationship good with the presentation of let-7a concentration, its linearly dependent coefficient R=0.985, linear equation is Ii
=-576.85+1500.85c (c is let-7a concentration).
The linear equation of gained is verified using people's urine sample composite sample, the average recovery rate for obtaining is 101%,
Relative standard deviation is 3.4%, shows that the DNA nano-probes detection prepared using the inventive method is realized in actual sample
Micro RNA, with high susceptibility, good reappearance and accuracy.Therefore, the DNA nanometers that prepared by the inventive method are visited
Pin can as micro RNA clinical diagnosis detection material.
Claims (5)
1. it is a kind of for micro RNA detection electrochemical luminescence active dna nano-probe preparation method, it is characterised in that it
It is made up of following step：
(1) the silica composite nanoparticle of doping shitosan and bipyridyl ruthenium is prepared
It is in 0.1% aqueous acetic acid, to be configured to mass-volume concentration for 0.5～5mg/mL by shitosan addition mass fraction
Chitosan solution, then by gained chitosan solution be added to Triton X-100 and n-hexyl alcohol, hexamethylene, ultra-pure water it is mixed
In closing liquid, the bipyridyl ruthenium aqueous solution of 0.01mol/L is added, with 0.1mol/L NaOH aqueous solution regulation systems to neutrality, room
Temperature stirring 30～60 minutes, adds tetraethyl orthosilicate and ammoniacal liquor, continues to stir 20～24 hours, adds acetone breakdown of emulsion, centrifugation point
From gained solid uses successively absolute ethyl alcohol and milli-Q water, and the silica for obtaining doping shitosan and bipyridyl ruthenium is combined
Nano-particle；
(2) DNA nano-probes are prepared
The doping shitosan that step (1) is obtained and the silica composite nanoparticle of bipyridyl ruthenium are scattered in ultra-pure water,
It is subsequently adding the DNA probe corresponding with micro RNA to be detected, control to be adulterated in reaction system shitosan and bipyridyl ruthenium
The concentration of silica composite nanoparticle be 0.10～0.25mg/mL, DNA probe concentration be 5 × 10-10～5 × 10- 8Mol/L, room temperature reaction 30～40 minutes, centrifugation, and with the PB buffer solutions 1～2 time of 1.0mmol/LpH=6.0,
Obtain DNA nano-probes.
2. it is according to claim 1 for micro RNA detection electrochemical luminescence active dna nano-probe preparation side
Method, it is characterised in that：It is the tetraethyl orthosilicate and chitosan solution, the bipyridyl ruthenium aqueous solution, ammoniacal liquor, ultrapure in step (1)
The volume ratio of water is 1:1.5～2.0:0.3～0.8:0.6～0.8:3～3.5.
3. it is according to claim 1 and 2 for micro RNA detection electrochemical luminescence active dna nano-probe system
Preparation Method, it is characterised in that：In step (1), the Triton X-100 and n-hexyl alcohol, hexamethylene, the volume ratio of ultra-pure water
For 1:1:4.0～4.3:0.1～0.3.
4. it is according to claim 1 for micro RNA detection electrochemical luminescence active dna nano-probe preparation side
Method, it is characterised in that：In step (2), the silica of doping shitosan and bipyridyl ruthenium is compound in control reaction system receives
The concentration of rice corpuscles be 0.15～0.20mg/mL, DNA probe concentration be 5 × 10-9～4.5 × 10-8mol/L。
5. according to claim 1 or 4 for micro RNA detection electrochemical luminescence active dna nano-probe system
Preparation Method, it is characterised in that：In step (2), described DNA probe is scattered in the PB buffer solutions of 1.0mmol/LpH=6.0
In.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610835093.9A CN106636326A (en) | 2016-09-20 | 2016-09-20 | Method for preparing DNA (deoxyribonucleic acid) nano-probes with electrochemical luminescence activity for detecting micro RNA (ribonucleic acid) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610835093.9A CN106636326A (en) | 2016-09-20 | 2016-09-20 | Method for preparing DNA (deoxyribonucleic acid) nano-probes with electrochemical luminescence activity for detecting micro RNA (ribonucleic acid) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106636326A true CN106636326A (en) | 2017-05-10 |
Family
ID=58852016
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610835093.9A Pending CN106636326A (en) | 2016-09-20 | 2016-09-20 | Method for preparing DNA (deoxyribonucleic acid) nano-probes with electrochemical luminescence activity for detecting micro RNA (ribonucleic acid) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106636326A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522253A (en) * | 2020-12-22 | 2021-03-19 | 中山大学 | Nanometer probe with subcellular targeting function and application thereof |
| CN112557365A (en) * | 2020-12-15 | 2021-03-26 | 黄冈师范学院 | Fluorescent probe and application thereof in DNA detection |
| CN113484383A (en) * | 2021-07-05 | 2021-10-08 | 中国科学院长春应用化学研究所 | Nano particle film and preparation method and application thereof |
| CN114736953A (en) * | 2022-02-24 | 2022-07-12 | 北京组学生物科技有限公司 | ATRX and KDM5A mutation detection kit based on digital PCR technology, device and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104155357A (en) * | 2014-05-23 | 2014-11-19 | 济南大学 | Preparation method and application of three-dimensional cubic duct based mesoporous silica sensor |
-
2016
- 2016-09-20 CN CN201610835093.9A patent/CN106636326A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104155357A (en) * | 2014-05-23 | 2014-11-19 | 济南大学 | Preparation method and application of three-dimensional cubic duct based mesoporous silica sensor |
Non-Patent Citations (2)
| Title |
|---|
| 刘文娜: "Ru(bpy)32+-SiO2纳米粒子电化学发光分析特性研究", 《陕西师范大学硕士学位论文》 * |
| 樊雪梅等: "基于壳聚糖/Ru(bpy)32+/SiO2纳米粒子电化学发光传感器用于尿酸的检测", 《分析化学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112557365A (en) * | 2020-12-15 | 2021-03-26 | 黄冈师范学院 | Fluorescent probe and application thereof in DNA detection |
| CN112522253A (en) * | 2020-12-22 | 2021-03-19 | 中山大学 | Nanometer probe with subcellular targeting function and application thereof |
| CN113484383A (en) * | 2021-07-05 | 2021-10-08 | 中国科学院长春应用化学研究所 | Nano particle film and preparation method and application thereof |
| CN114736953A (en) * | 2022-02-24 | 2022-07-12 | 北京组学生物科技有限公司 | ATRX and KDM5A mutation detection kit based on digital PCR technology, device and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | A SiO2@ MIP electrochemical sensor based on MWCNTs and AuNPs for highly sensitive and selective recognition and detection of dibutyl phthalate | |
| Bagwe et al. | Surface modification of silica nanoparticles to reduce aggregation and nonspecific binding | |
| Li et al. | Immobilization of trypsin on superparamagnetic nanoparticles for rapid and effective proteolysis | |
| CN106636326A (en) | Method for preparing DNA (deoxyribonucleic acid) nano-probes with electrochemical luminescence activity for detecting micro RNA (ribonucleic acid) | |
| CN1215902C (en) | Magnetic fluorescent double functional microballoon with core-shell structure and preparation method thereof | |
| Li et al. | Ionic liquid-functionalized fluorescent carbon nanodots and their applications in electrocatalysis, biosensing, and cell imaging | |
| Liu et al. | Electrochemical immunosensor based on mesoporous nanocomposites and HRP-functionalized nanoparticles bioconjugates for sensitivity enhanced detection of diethylstilbestrol | |
| CN101019019A (en) | Surface enhanced spectrometry-active composite nanoparticles | |
| CN102866139B (en) | Establishment method based on surface plasma reinforcing energy transferring biosensor | |
| CN103937486B (en) | A kind of fluorescent nano probe and its preparation method and application | |
| Marfà et al. | Magnetic-molecularly imprinted polymers in electrochemical sensors and biosensors | |
| Xu et al. | Fabrication of magnetic porous pseudo-carbon paste electrode electrochemical biosensor and its application in detection of schistosoma egg antigen | |
| Kovaleva et al. | Acid–base properties of nanoconfined volumes of anodic aluminum oxide pores by EPR of pH-sensitive spin probes | |
| CN101907625A (en) | Method for preparing quantum dot immune fluorescent probe | |
| Li et al. | A 3D graphene oxide microchip and a Au-enwrapped silica nanocomposite-based supersandwich cytosensor toward capture and analysis of circulating tumor cells | |
| Pourmadadi et al. | Development of polyvinylpyrrolidone-based nanomaterials for biosensors applications: a review | |
| CN107543851B (en) | A kind of preparation method and application of the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex | |
| CN109540991A (en) | Functional metal organic framework material, FKN sensor of its building and preparation method thereof | |
| CN109596668A (en) | The gas sensitive for enhancing gas sensing and its preparation and application are modified based on copper ion | |
| Borbora et al. | Dually reactive multilayer coatings enable orthogonal manipulation of underwater superoleophobicity and oil adhesion via post-functionalization | |
| CN110736724B (en) | Detection method of reduced glutathione | |
| Liang et al. | Conductometric immunoassay of alpha-fetoprotein in sera of liver cancer patients using bienzyme-functionalized nanometer-sized silica beads | |
| CN106771254B (en) | Amination mesoporous silicon oxide-glucose-manganese dioxide nano-composite material and its preparation method and application | |
| JP2008273790A (en) | Method for producing silica nanoparticles using reverse micelle disperse system, silica nanoparticles obtained by the method and labelling reagent using the nanoparticles | |
| CN102608189A (en) | Method for manufacturing nanometer magnetic ferroferric oxide modified immunosensor |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170510 |