CN113960003A - 基于dna银纳米簇和金纳米棒检测四环素的适配体传感器 - Google Patents
基于dna银纳米簇和金纳米棒检测四环素的适配体传感器 Download PDFInfo
- Publication number
- CN113960003A CN113960003A CN202111219184.7A CN202111219184A CN113960003A CN 113960003 A CN113960003 A CN 113960003A CN 202111219184 A CN202111219184 A CN 202111219184A CN 113960003 A CN113960003 A CN 113960003A
- Authority
- CN
- China
- Prior art keywords
- dna
- tetracycline
- solution
- agncs
- gold nanorods
- 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.)
- Granted
Links
- 239000004098 Tetracycline Substances 0.000 title claims abstract description 73
- 229960002180 tetracycline Drugs 0.000 title claims abstract description 70
- 229930101283 tetracycline Natural products 0.000 title claims abstract description 70
- 235000019364 tetracycline Nutrition 0.000 title claims abstract description 70
- 150000003522 tetracyclines Chemical class 0.000 title claims abstract description 70
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 229910052709 silver Inorganic materials 0.000 title claims abstract description 41
- 239000004332 silver Substances 0.000 title claims abstract description 41
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 33
- 108020004414 DNA Proteins 0.000 claims description 44
- 239000000243 solution Substances 0.000 claims description 33
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 18
- 102000053602 DNA Human genes 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 9
- 229960005070 ascorbic acid Drugs 0.000 claims description 9
- 235000010323 ascorbic acid Nutrition 0.000 claims description 9
- 239000011668 ascorbic acid Substances 0.000 claims description 9
- 239000010931 gold Substances 0.000 claims description 8
- 229910052737 gold Inorganic materials 0.000 claims description 8
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 8
- 239000005457 ice water Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000012279 sodium borohydride Substances 0.000 claims description 7
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 7
- 101710134784 Agnoprotein Proteins 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000002189 fluorescence spectrum Methods 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 4
- 229910052751 metal Inorganic materials 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 4
- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 3
- 239000003638 chemical reducing agent Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- 238000012921 fluorescence analysis Methods 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 2
- 230000005284 excitation Effects 0.000 claims 2
- 238000003287 bathing Methods 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 238000005259 measurement Methods 0.000 claims 1
- 108091008104 nucleic acid aptamers Proteins 0.000 claims 1
- -1 stirring for 1min Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 17
- 238000000034 method Methods 0.000 abstract description 16
- 239000000523 sample Substances 0.000 abstract description 11
- 238000001179 sorption measurement Methods 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 5
- 230000000295 complement effect Effects 0.000 abstract description 4
- 230000000171 quenching effect Effects 0.000 abstract description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N Thymine Natural products CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 abstract description 2
- 229940113082 thymine Drugs 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract 1
- 235000013336 milk Nutrition 0.000 description 7
- 239000008267 milk Substances 0.000 description 7
- 210000004080 milk Anatomy 0.000 description 7
- 230000003115 biocidal effect Effects 0.000 description 5
- 238000010791 quenching Methods 0.000 description 4
- 230000009881 electrostatic interaction Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000004100 Oxytetracycline Substances 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960003405 ciprofloxacin Drugs 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229960003722 doxycycline Drugs 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000004186 food analysis Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002073 nanorod Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229960000625 oxytetracycline Drugs 0.000 description 1
- IWVCMVBTMGNXQD-PXOLEDIWSA-N oxytetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3[C@H](O)[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-PXOLEDIWSA-N 0.000 description 1
- 235000019366 oxytetracycline Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 description 1
- 229940072172 tetracycline antibiotic Drugs 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
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"
-
- 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"
- G01N2021/6432—Quenching
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开的是基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器。由8个碱基的适配体互补序列、5个胸腺嘧啶间隔子和银纳米簇模板序列相连接，制备无标记荧光信号探针DNA‑AgNCs。DNA‑AgNCs与核酸适配体杂交互补形成双螺旋结构，表面暴露出负电荷，通过静电作用吸附到GNR表面，通过偶极子‑表面之间的表面能量转移，DNA‑AgNCs荧光被猝灭。四环素存在时，适配体与四环素紧密结合，DNA‑AgNCs从GNR表面释放，荧光恢复。利用GNR对单双链静电吸附的差异，以及对DNA‑AgNCs的荧光猝灭作用，本方法能够特异、灵敏、快速的检测四环素(检测限106.3pM，线性范围5～500nM)。本发明可通过替换相应的适配体和互补链推广到其他目标物检测中，对食品中有害物质的快速检测具有潜在的应用价值。
Description
技术领域
本发明属于生物传感器技术领域，具体涉及带正电的金纳米棒(GNR)和DNA银纳米簇(DNA-AgNCs)的制备方法，建立了无标记检测四环素的荧光适配体传感器，并应用于牛奶样品中四环素的检测，为无标记简便检测四环素提供一种新方法及可靠依据。
背景技术
四环素是20世纪40年代通过土壤收集发现的一种广谱抗生素，属于四环素类抗生素。四环素不仅对许多致命细菌感染有明显的治疗效果，而且成本低廉。它能促进生产，减少集约化养殖造成的相互感染，因此受到畜牧和水产养殖从业者的青睐。然而，由于四环素使用不当，肉类、鸡蛋、海鲜、乳制品、土壤等日常食品中存在抗生素残留，对人体健康构成严重威胁，也使许多细菌产生耐药性。为了食品安全，许多国家制定了食品中最大残留限量(maximum residue limit,MRL)，以防止人们接触这些抗生素残留。特别是世界卫生组织(WHO)、欧盟(EU)和中国农业部均限定牛奶中四环素的MRL为0.10mg/kg。传统四环素的检测方法主要有高效液相色谱、液相色谱-质谱等，然而这些方法往往费时费力，依靠大型昂贵的仪器和专业的操作人员，并需要有害的有机溶剂，严重影响检测的效率和安全性。此外，四环素除了分子结构小外，与其他抗生素结构也非常相似，这导致四环素的特异性检测存在困难。因此，开发一种省时、操作简便、成本低、灵敏度高和特异性强的检测食品中四环素残留的方法十分必要。本方法采用核酸适配体作为识别探针能够特异性识别小分子四环素，灵敏度高，特异性强。
DNA银纳米簇(DNA-templated silver nanoclusters,DNA-AgNCs)通常由几个到几十个金属原子组成，直径小于2nm，尺寸接近电子的费米波长。它们具有分子级的能级结构，通过能级之间的电子跃迁实现光的吸收和发射。它具有合成简单、量子产率高、光稳定性强、荧光发射强度可调、毒性低、生物相容性好等优点。此外，DNA-AgNCs的形成不需要繁琐和昂贵的DNA标记程序。因此，DNA-AgNCs可以作为一种理想的荧光材料，应用于各种目标物质检测、细胞标记或成像等无标记荧光传感器。
金纳米棒(gold nanorods,GNR)具有良好的水溶性、生物相容性和可见光区域强的局域表面等离子体共振，已被用作生物分析的荧光受体。金纳米棒是一种具有独特光学性质的纳米粒子，其光学性质取决于其大小和尺寸(长径比)。金纳米棒有两种主要的等离子体吸附，在520nm可见区域的横向局域表面等离子体共振吸附和在更长波长区域的纵向局域表面等离子体共振吸附。与球形金纳米颗粒相比，各向异性形状的GNR具有更高的稳定性，可以进行快速的电子转移，并在宽光谱范围内表现出可调谐的形状依赖光学特性，使其具有制备荧光生物传感器的多样化潜力。基于未修饰GNR的生物传感器主要依赖于带正电的GNR与带负电的寡核苷酸之间的静电相互作用。与单链DNA相比，GNR与双链DNA之间的静电吸收大大增强，相应的，随着GNR被强烈吸附，吸光率也逐渐增大。因此，可以利用单、双链和GNR之间静电相互作用的差异，将GNR荧光猝灭能力和静电吸附能力联合使用可开发“猝灭-恢复”型荧光传感器实现对目标物的检测。
发明内容
本发明的目的是为了解决现有技术存在的操作复杂、成本高、实际样品前处理复杂等问题，提供了一种利用DNA银纳米簇作为无标记荧光探针，基于DNA银纳米簇和金纳米棒之间的表面能量转移，开发了一种操作简单的无标记荧光适配体传感器检测四环素，实现了对四环素的可靠、灵敏检测，为无标记荧光传感器在抗生素残留检测中的应用提供新方法，同时本方法前处理简单，为四环素在实际样品重的检测提供一种新思路。
本发明的目的可通过如下技术方案实现：
一种基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器，其步骤如下：
A、金纳米棒；简称GNR的制备：
金种子的制备：将0.60mL 0.010M新鲜冰凉的NaBH4加入到5mL of 0.20M CTAB和5mL 0.00050M HAuCl4·4H2O的混合物中，剧烈搅拌2min，在25℃避光反应2h；
生长液的制备：在25℃下，将0.2mL 0.0040M AgNO3溶液加入到25mL 0.20M CTAB溶液中，再加入25.0mL 0.0010M HAuCl4 4H2O溶液和350μL 0.0788M抗坏血酸(AA)，轻轻搅拌，由于AA为还原剂，加入混合溶液后，生长液由淡黄色变为无色；
金纳米棒的制备：将上述生长液与60μL的金种子混合，在30℃避光反应24h，溶液颜色逐渐变为深紫色；将合成的金纳米棒溶液离心(10,000rpm,15min)，去除上层的CTAB后重溶于纯净水，重复两次，置于4℃避光保存。
B、DNA银纳米簇；简称DNA-AgNCs的制备：
将10μM银纳米簇模板序列1(ccccccgggggcccccctttttcaccaccg)在95℃加热10min，然后冰水浴10min；31μL 10μM模板序列被溶解，混合着56.1μL PB缓冲液(pH 7.2)，然后加入10μL 930μM硝酸银(AgNO3)溶液，搅拌1min，冰水浴30min；加入10μL 930μM新鲜硼氢化钠(NaBH4)溶液5min，避光冰水浴3h以制备DNA银纳米簇。
C、四环素荧光分析：
107.1μL cDNA-AgNCs与31μL 10μM四环素核酸适配体序列2(cggtggtg)混合后，加入171.9μL的超纯水得到1μM dsDNA，避光放置在25℃金属浴种反应1h，形成双链结构；然后取30μL 1μM dsDNA加入30μL不同浓度的四环素在25℃避光反应30min；加入20μL GNR，在25℃避光反应10min；用超纯水将反应体系定容至300μL；在室温下使用荧光分光光度计测定溶液的荧光发射光谱。
本发明机理如下：
本发明，由8个碱基的四环素核酸适配体的互补序列、5个胸腺嘧啶间隔子和合成银纳米簇的序列相连接作为模板，制备无标记荧光信号探针DNA-AgNCs。在没有四环素的情况下，DNA-AgNCs与四环素核酸适配体杂交互补形成双螺旋结构，由于双链表面暴露出负电荷，通过静电作用吸附到金纳米棒上，通过偶极子-表面相互作用的表面能量转移(surfaceenergy transfer,SET)，金纳米棒能够猝灭DNA-AgNCs的荧光。当四环素存在时，适配体与四环素紧密结合，DNA-AgNCs从金纳米棒表面释放，导致荧光强度恢复。利用金纳米棒对单链和双链核酸静电吸附的差异，以及对DNA-AgNCs荧光猝灭的作用，建立了一种快速、灵敏检测四环素的无标记荧光适配体传感器，本方法能够特异、灵敏、快速的检测四环素。
本发明提供的这种基于DNA银纳米簇和金纳米棒之间的表面能量转移的荧光检测方法，为DNA银纳米簇无标记荧光探针和金纳米棒猝灭材料在四环素快速检测中的实际应用提供技术手段，该策略可通过替换相应的适配体和互补链推广到其他目标物检测中，在环境检测、食品分析方面展现出巨大的应用前景。
附图说明
图1：实施例3所述，为基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器的原理示意图。
图2：实施例1所述，金纳米棒的透射电镜图。
图3：实施例2所述，DNA银纳米簇的透射电镜图。
图4：实施例3所述，为本方法的可行性验证，DNA银纳米簇、DNA银纳米簇+核酸适配体、DNA银纳米簇+核酸适配体+金纳米棒、DNA银纳米簇+核酸适配体+四环素+金纳米棒的荧光光谱。
图5：实施例3所述，基于DNA银纳米簇和金纳米棒的无标记荧光适配体传感器检测四环素，为DNA银纳米簇+核酸适配体+四环素+金纳米棒体系存在不同浓度四环素(5,10,50,75,100,200,300,400,500nM)的荧光发射光谱。
图6：实施例3所述，为荧光(F645)与四环素浓度的线性关系图。
图7：实施例3所述，为本分析方法检测四环素的特异性(抗生素浓度均为300nM)。
图8：实施例4所述，基于DNA银纳米簇和金纳米棒的无标记荧光适配体传感器检测牛奶中四环素，为DNA银纳米簇+核酸适配体+四环素+金纳米棒体系存在不同浓度四环素(5,10,50,100,200,300,400nM)下，荧光(F645)与四环素浓度的线性关系图。
具体实施方式
下面结合附图对本发明做进一步地说明。
实施例1：金纳米棒的制备
金种子的制备：将0.60mL0.010M新鲜冰凉的NaBH4加入到5mLof 0.20M CTAB和5mL0.00050M HAuCl4·4H2O的混合物中，剧烈搅拌2min，在25℃避光反应2h；
生长液的制备：在25℃下，将0.2mL 0.0040M AgNO3溶液加入到25mL 0.20M CTAB溶液中，再加入25.0mL 0.0010M HAuCl4 4H2O溶液和350μL0.0788M抗坏血酸(AA)，轻轻搅拌，由于AA为还原剂，加入混合溶液后，生长液由淡黄色变为无色；
金纳米棒的制备：将上述生长液与60μL的金种子混合，在30℃避光反应24h，溶液颜色逐渐变为深紫色；将合成的金纳米棒溶液离心(10,000rpm,15min)，去除上层的CTAB后重溶于纯净水，重复两次，置于4℃避光保存；
实施例2：DNA银纳米簇的制备
将10μM银纳米簇模板序列1(ccccccgggggcccccctttttcaccaccg)在95℃加热10min，然后冰水浴10min；31μL 10μM模板序列被溶解，混合着56.1μLPB缓冲液(pH 7.2)，然后加入10μL 620μM硝酸银(AgNO3)溶液，搅拌1min，冰浴30min；加入10μL 620μM新鲜硼氢化钠(NaBH4)溶液5min，避光冰浴3h以制备DNA银纳米簇；
实施例3：基于DNA银纳米簇和金纳米棒的无标记荧光适配体传感器检测四环素：
107.1μL cDNA-AgNCs与31μL 10μM四环素核酸适配体序列2(cggtggtg)混合后，加入171.9μL的超纯水得到1μM dsDNA，避光放置在25℃金属浴种孵育1h形成双链结构；然后取30μL1μM dsDNA加入30μL不同浓度的四环素在25℃避光反应30min；加入20μL GNR，在25℃避光反应10min；用超纯水将反应体系定容至300μL；在室温下使用荧光分光光度计测定溶液的荧光发射光谱。
本方法原理可行(图4)，随着四环素的浓度增加，体系645nm处荧光逐渐升高(图5)。在四环素终浓度为5-500nM范围内，荧光强度(F645)与四环素浓度相关，线性方程为5-100nM：F645＝357.6+1.138C四环素(R2＝0.9937)，100-500nM：F645＝443.142+0.273C四环素(R2＝0.9917)，检出限(LOD)为106.3pM(图6)，可满足四环素的检测。本发明构建的方法对氯霉素、土霉素、多西环素、环丙沙星、卡那霉素均没有相应，只有四环素(300nM)可引起显著的变化(图8)，表明该方法对四环素具有较高选择性。
实施例4：实际样品中四环素含量的测定
利用本发明，采用标准加入法，对实际样品中的四环素进行检测，探讨其实用性。具体样品为牛奶，对原料奶样品进行预处理，具体步骤如下：将10mL牛奶与10mL水加入50mL离心管中混合均匀，加入5mL10％三氯乙酸，搅拌1分钟后在20℃下超声20分钟，在10000rpm离心10分钟，取上层清液通过0.45μM超滤膜过滤后，将滤液在10000rpm离心10分钟并去除沉淀，得到最终的上清液，此时已去除牛奶中的蛋白质、脂肪等有机物。向上清液中添加四环素标准液，终浓度分别为50、100和200nM，并利用本发明开发的荧光方法进行检测(图7)，在四环素终浓度为5-400nM范围内，荧光强度(F645)与四环素浓度相关，线性方程为5-400nM：F645＝267.87+0.54C四环素(R2＝0.9953)，可实现实际样品牛奶中四环素的检测分析。如表1所示，实际样品中四环素的添加回收率为97.94-99.78％，相对标准偏差(RSD)在2.37-5.28％之间，表明该检测方法可应用于实际样品检测中。
表1：应用本发明开发的荧光策略检测牛奶样品中的四环素(n＝3)
序列表
<110> 吉林大学
<120> 基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器
<141> 2021-10-20
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccccccgggg gccccccttt ttcaccaccg 30
<210> 2
<211> 8
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cggtggtg 8
Claims (7)
1.基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器，其特征在于，其步骤如下：
A、金纳米棒；简称GNR的制备：
金种子的制备：将0.60mL 0.010M新鲜冰凉的NaBH4加入到5mL of 0.20M CTAB和5mL0.00050M HAuCl4·4H2O的混合物中，剧烈搅拌2min，在25℃避光反应2h；
生长液的制备：在25℃下，将0.2mL 0.0040M AgNO3溶液加入到25mL 0.20M CTAB溶液中，再加入25.0mL 0.0010M HAuCl4 4H2O溶液和350μL0.0788M抗坏血酸，即AA，轻轻搅拌，由于AA为还原剂，加入混合溶液后，生长液由淡黄色变为无色；
金纳米棒的制备：将上述生长液与60μL的金种子混合，在30℃避光反应24h，溶液颜色逐渐变为深紫色；将合成的金纳米棒溶液离心，离心条件为：10,000rpm,15min；去除上层的CTAB后重溶于纯净水，重复两次，置于4℃避光保存；
B、DNA银纳米簇；简称DNA-AgNCs的制备：
将10μM银纳米簇模板序列1在95℃加热10min，然后冰水浴10min；31μL 10μM模板序列被溶解，混合着56.1μL PB缓冲液，PB缓冲液的pH值为7.2，然后加入10μL 620μM硝酸银溶液，搅拌1min，冰水浴30min；加入10μL 620μM新鲜硼氢化钠溶液5min，避光冰水浴3h以制备DNA银纳米簇；
C、四环素荧光分析：
107.1μL DNA-AgNCs与31μL 10μM四环素核酸适配体序列2混合后，加入171.9μL的超纯水得到1μM dsDNA，避光放置在25℃金属浴种孵育1h形成双链结构；然后取30μL 1μM dsDNA加入30μL不同浓度的四环素在25℃避光反应30min；加入20μL GNR，在25℃避光反应10min；用超纯水将反应体系定容至300μL；在室温下使用荧光分光光度计测定溶液的荧光发射光谱。
2.如权利要求1所述的一种基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器，其特征在于，步骤A所述的0.0040M AgNO3的体积为0.2mL。
3.如权利要求1所述的一种基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器，其特征在于，步骤B所述银纳米簇模板序列1为ccccccgggggcccccctttttcaccaccg。
4.如权利要求1所述的一种基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器，其特征在于，步骤B所述的31μL 10μM模板序列，56.1μL PB缓冲液，PB缓冲液的pH值为7.2，10μL 620μM硝酸银，10μL 620μM新鲜硼氢化钠。
5.如权利要求1所述的一种基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器，其特征在于，步骤C所述的四环素核酸适配体序列2为cggtggtg。
6.如权利要求1所述的一种基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器，其特征在于，步骤C进行荧光光谱测定时，对DNA-AgNCs参数设置为激发波长为585nm，发射波长范围为620-750nm；激发与发射狭缝分别为10、10nm，响应时间为0.1s。
7.如权利要求1所述的一种基于DNA银纳米簇和金纳米棒检测四环素的适配体传感器，其特征在于，步骤C中DNA-AgNCs最佳发射波长位于645nm处。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111219184.7A CN113960003B (zh) | 2021-10-20 | 2021-10-20 | 基于dna银纳米簇和金纳米棒检测四环素的适配体传感器 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111219184.7A CN113960003B (zh) | 2021-10-20 | 2021-10-20 | 基于dna银纳米簇和金纳米棒检测四环素的适配体传感器 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113960003A true CN113960003A (zh) | 2022-01-21 |
| CN113960003B CN113960003B (zh) | 2023-10-24 |
Family
ID=79465597
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111219184.7A Active CN113960003B (zh) | 2021-10-20 | 2021-10-20 | 基于dna银纳米簇和金纳米棒检测四环素的适配体传感器 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113960003B (zh) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100111938A1 (en) * | 2008-10-31 | 2010-05-06 | Searete Llc, A Limited Liability Corporation Of The State Of Delaware | Compositions and methods for biological remodeling with frozen particle compositions |
| WO2015048429A1 (en) * | 2013-09-26 | 2015-04-02 | Massachusetts Institute Of Technology | Engineering dna assembly in vivo and methods of making and using the reverse transcriptase technology |
| CN105548108A (zh) * | 2015-12-18 | 2016-05-04 | 江南大学 | 一种基于金纳米棒核-量子点卫星状纳米结构组装体荧光信号超灵敏检测psa的方法 |
| JP2017095744A (ja) * | 2015-11-19 | 2017-06-01 | 大日本塗料株式会社 | 金ナノロッドを含む被験物質を検出するための組成物及びその用途 |
| WO2018054390A1 (zh) * | 2016-09-20 | 2018-03-29 | 江南大学 | 一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用 |
| CN108025082A (zh) * | 2015-07-28 | 2018-05-11 | 塔夫茨大学信托人 | 多肽整料 |
| CN108458998A (zh) * | 2018-01-29 | 2018-08-28 | 山西大学 | 一种基于免标记荧光增强的适配体dna银纳米簇测定铅离子的方法 |
| CN108486104A (zh) * | 2018-04-13 | 2018-09-04 | 长沙理工大学 | 基于dna-银纳米簇检测癌细胞的靶向荧光探针及应用 |
| KR20190052519A (ko) * | 2017-11-08 | 2019-05-16 | 주식회사 에이치피바이오 | Pg-1 펩타이드에 특이적으로 결합하는 dna 앱타머 및 이의 용도 |
| CN109991202A (zh) * | 2019-04-16 | 2019-07-09 | 南京医科大学 | 一种基于核酸适配体荧光传感器用于多目标物检测的方法 |
| CN110945120A (zh) * | 2017-04-07 | 2020-03-31 | 优迪有限合伙公司 | 测量纳米药物中受体-配体相互作用的效力的测定 |
| CN112893864A (zh) * | 2021-01-20 | 2021-06-04 | 江南大学 | 一种基于发夹模板制备的银纳米团簇及其在检测氯霉素中的应用 |
| CN113418884A (zh) * | 2021-06-21 | 2021-09-21 | 佛山科学技术学院 | 一种基于DNA-AuNPs体系的比色阵列传感器及其制备方法 |
| CN113418883A (zh) * | 2021-06-21 | 2021-09-21 | 佛山科学技术学院 | 一种基于DNA-AuNPs体系的比色阵列传感器的氨基糖苷类抗生素检测方法 |
-
2021
- 2021-10-20 CN CN202111219184.7A patent/CN113960003B/zh active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100111938A1 (en) * | 2008-10-31 | 2010-05-06 | Searete Llc, A Limited Liability Corporation Of The State Of Delaware | Compositions and methods for biological remodeling with frozen particle compositions |
| WO2015048429A1 (en) * | 2013-09-26 | 2015-04-02 | Massachusetts Institute Of Technology | Engineering dna assembly in vivo and methods of making and using the reverse transcriptase technology |
| CN108025082A (zh) * | 2015-07-28 | 2018-05-11 | 塔夫茨大学信托人 | 多肽整料 |
| JP2017095744A (ja) * | 2015-11-19 | 2017-06-01 | 大日本塗料株式会社 | 金ナノロッドを含む被験物質を検出するための組成物及びその用途 |
| CN105548108A (zh) * | 2015-12-18 | 2016-05-04 | 江南大学 | 一种基于金纳米棒核-量子点卫星状纳米结构组装体荧光信号超灵敏检测psa的方法 |
| WO2018054390A1 (zh) * | 2016-09-20 | 2018-03-29 | 江南大学 | 一种用于胞内癌症标志物双重检测的卫星状纳米组装体的制备方法及应用 |
| CN110945120A (zh) * | 2017-04-07 | 2020-03-31 | 优迪有限合伙公司 | 测量纳米药物中受体-配体相互作用的效力的测定 |
| KR20190052519A (ko) * | 2017-11-08 | 2019-05-16 | 주식회사 에이치피바이오 | Pg-1 펩타이드에 특이적으로 결합하는 dna 앱타머 및 이의 용도 |
| CN108458998A (zh) * | 2018-01-29 | 2018-08-28 | 山西大学 | 一种基于免标记荧光增强的适配体dna银纳米簇测定铅离子的方法 |
| CN108486104A (zh) * | 2018-04-13 | 2018-09-04 | 长沙理工大学 | 基于dna-银纳米簇检测癌细胞的靶向荧光探针及应用 |
| CN109991202A (zh) * | 2019-04-16 | 2019-07-09 | 南京医科大学 | 一种基于核酸适配体荧光传感器用于多目标物检测的方法 |
| CN112893864A (zh) * | 2021-01-20 | 2021-06-04 | 江南大学 | 一种基于发夹模板制备的银纳米团簇及其在检测氯霉素中的应用 |
| CN113418884A (zh) * | 2021-06-21 | 2021-09-21 | 佛山科学技术学院 | 一种基于DNA-AuNPs体系的比色阵列传感器及其制备方法 |
| CN113418883A (zh) * | 2021-06-21 | 2021-09-21 | 佛山科学技术学院 | 一种基于DNA-AuNPs体系的比色阵列传感器的氨基糖苷类抗生素检测方法 |
Non-Patent Citations (3)
| Title |
|---|
| 孙亚兰;蔡伊娜;任冰雪;彭池方;: "基于铜/银纳米簇检测铜离子的方法研究", 分析测试学报, no. 02 * |
| 汪石;朱雨清;卢静荷;谭淑珍;: "基于核酸外切酶Ⅲ辅助信号放大的荧光法检测四环素", 分析试验室, no. 07 * |
| 高薇;吕功煊;: "碳量子点上转换材料的制备及其应用研究进展", 分子催化, no. 02 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113960003B (zh) | 2023-10-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ahmed et al. | Target specific aptamer-induced self-assembly of fluorescent graphene quantum dots on palladium nanoparticles for sensitive detection of tetracycline in raw milk | |
| Hu et al. | Green one-step synthesis of carbon quantum dots from orange peel for fluorescent detection of Escherichia coli in milk | |
| Mortazavi et al. | Bacterial biosynthesis of gold nanoparticles using Salmonella enterica subsp. enterica serovar Typhi isolated from blood and stool specimens of patients | |
| Wang et al. | Highly sensitive fluorometric determination of oxytetracycline based on carbon dots and Fe3O4 MNPs | |
| Liu et al. | Hybrid material for enrofloxacin sensing based on aptamer-functionalized magnetic nanoparticle conjugated with upconversion nanoprobes | |
| Ren et al. | Efficient preparation of nitrogen-doped fluorescent carbon dots for highly sensitive detection of metronidazole and live cell imaging | |
| Wang et al. | Efficient fabrication of ratiometric fluorescence imprinting sensors based on organic-inorganic composite materials and highly sensitive detection of oxytetracycline in milk | |
| Zhang et al. | Fluorescent carbon dots for probing the effect of thiram on the membrane of fungal cell and its quantitative detection in aqueous solution | |
| Guo et al. | A concise detection strategy of Staphylococcus aureus using N-Succinyl-Chitosan-dopped bacteria-imprinted composite film and AIE fluorescence sensor | |
| CN111690405B (zh) | 一种荧光碳点及其制备方法和在检测铜离子中的应用 | |
| Xu et al. | A unique dual-excitation carbon quantum dots: Facile synthesis and application as a dual-“on-off-on” fluorescent probe | |
| Mathew et al. | Carbon dots from green sources as efficient sensor and as anticancer agent | |
| Zhu et al. | β-Cyclodextrin functionalized N, Zn codoped carbon dots for specific fluorescence detection of fluoroquinolones in milk samples | |
| Bahavarnia et al. | Bio-assay of Acintobacter baumannii using DNA conjugated with gold nano-star: A new platform for microorganism analysis | |
| CN108982458B (zh) | 一种基于脱氧核酶修饰的磁珠颗粒用于锌离子检测的荧光方法 | |
| Lu et al. | On-site detection of multiple extracellular antibiotic resistance genes using SERS | |
| Guo et al. | Exogenous interference and autofluorescence-free ratiometric aptasensor for detection of OTA based on dual-colored persistent luminescence nanoparticles | |
| CN108680547B (zh) | 铜纳米簇作为荧光探针特异性检测溶液中利福平药物含量方面的应用 | |
| Na et al. | Fluorescence Sensor for Zearalenone Detection Based on Oxidized Single-walled Carbon Nanohorns/N-doped Carbon Quantum Dots-aptamer | |
| CN113960003A (zh) | 基于dna银纳米簇和金纳米棒检测四环素的适配体传感器 | |
| Chaudhary et al. | Rational hydrothermal-assisted green synthesis of blueish emitting carbon dots as an optical sensing platform for antibiotic detection in the milk sample | |
| Xu et al. | The dual nucleic acid amplification with dynamic light scattering strategy for ultrasensitive detection of Salmonella in milk | |
| Xia et al. | Development of ochratoxin a aptasensor based on Au nanoparticles@ g-C3N4 | |
| CN114058721B (zh) | 一种上转换荧光识别探针的制备方法及其产品和应用 | |
| CN113667720B (zh) | 一种检测miRNA-182的生物传感器及其制备方法和应用 |
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 |