CN116148457A - 一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用 - Google Patents
一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用 Download PDFInfo
- Publication number
- CN116148457A CN116148457A CN202210870746.2A CN202210870746A CN116148457A CN 116148457 A CN116148457 A CN 116148457A CN 202210870746 A CN202210870746 A CN 202210870746A CN 116148457 A CN116148457 A CN 116148457A
- Authority
- CN
- China
- Prior art keywords
- exonuclease
- carbon nanomaterial
- fluorescent
- cdna
- assisted
- 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
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 229910052799 carbon Inorganic materials 0.000 title claims abstract description 60
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 58
- 108060002716 Exonuclease Proteins 0.000 title claims abstract description 52
- 102000013165 exonuclease Human genes 0.000 title claims abstract description 52
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000002299 complementary DNA Substances 0.000 claims abstract description 41
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims abstract description 38
- 229930020125 aflatoxin-B1 Natural products 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000010791 quenching Methods 0.000 claims abstract description 15
- 235000013305 food Nutrition 0.000 claims abstract description 14
- 230000000171 quenching effect Effects 0.000 claims abstract description 14
- 230000003321 amplification Effects 0.000 claims abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 13
- 238000009396 hybridization Methods 0.000 claims abstract description 11
- 239000002115 aflatoxin B1 Substances 0.000 claims abstract description 7
- 238000001179 sorption measurement Methods 0.000 claims abstract description 5
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 3
- 230000001404 mediated effect Effects 0.000 claims abstract description 3
- 238000002372 labelling Methods 0.000 claims abstract 2
- 238000001514 detection method Methods 0.000 claims description 29
- 239000000523 sample Substances 0.000 claims description 25
- 239000002091 nanocage Substances 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 238000010008 shearing Methods 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 4
- 238000003760 magnetic stirring Methods 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 3
- 229910021389 graphene Inorganic materials 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000012103 Alexa Fluor 488 Substances 0.000 claims description 2
- 239000012110 Alexa Fluor 594 Substances 0.000 claims description 2
- 241000701959 Escherichia virus Lambda Species 0.000 claims description 2
- 230000005284 excitation Effects 0.000 claims description 2
- CWQXQMHSOZUFJS-UHFFFAOYSA-N molybdenum disulfide Chemical compound S=[Mo]=S CWQXQMHSOZUFJS-UHFFFAOYSA-N 0.000 claims description 2
- 229910052982 molybdenum disulfide Inorganic materials 0.000 claims description 2
- 239000002135 nanosheet Substances 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 238000004729 solvothermal method Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 2
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 description 31
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 description 31
- 230000000694 effects Effects 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 244000105624 Arachis hypogaea Species 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 235000020232 peanut Nutrition 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 108700012359 toxins Proteins 0.000 description 4
- 229930195730 Aflatoxin Natural products 0.000 description 3
- 235000017060 Arachis glabrata Nutrition 0.000 description 3
- 235000010777 Arachis hypogaea Nutrition 0.000 description 3
- 235000018262 Arachis monticola Nutrition 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000005409 aflatoxin Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000013312 flour Nutrition 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 108091008104 nucleic acid aptamers Proteins 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 description 2
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 2
- 229960001082 trimethoprim Drugs 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 208000003643 Callosities Diseases 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- UVBUBMSSQKOIBE-DSLOAKGESA-N fumonisin B1 Chemical compound OC(=O)C[C@@H](C(O)=O)CC(=O)O[C@H]([C@H](C)CCCC)[C@@H](OC(=O)C[C@@H](CC(O)=O)C(O)=O)C[C@@H](C)C[C@H](O)CCCC[C@@H](O)C[C@H](O)[C@H](C)N UVBUBMSSQKOIBE-DSLOAKGESA-N 0.000 description 1
- QZIADBYRQILELJ-UHFFFAOYSA-N fumonisin B1 Natural products CCCCC(C)C(OC(=O)CC(CC(=O)O)C(=O)O)C(C)(CC(C)CC(O)CCCCC(O)CC(O)C(C)N)OC(=O)CC(CC(=O)O)C(=O)O QZIADBYRQILELJ-UHFFFAOYSA-N 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000111 isothermal titration calorimetry Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002636 mycotoxin Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- 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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及了一种碳纳米材料及核酸外切酶辅助检测黄曲霉毒素B1的荧光适配体传感器的制备方法和应用,包括以下步骤:DNA三链杂交结构的形成:将适配体HP1、HP2和cDNA在一定条件下孵育,得到HP1‑HP2‑cDNA三链杂交结构;核酸外切酶辅助双循环扩增模式(dRCA)的制备:使用核酸外切酶特异性剪切DNA三链杂交结构,产生两种酶解产物,参与双循环扩增放大信号;碳纳米材料介导cDNA共轭吸附进行荧光猝灭:加入一定量的碳纳米材料,将部分未反应的cDNA吸附并猝灭其荧光,得到碳纳米材料及核酸外切酶辅助的荧光适配体传感器。随后,将传感器用于黄曲霉毒素B1的检测,按照上述步骤检测食品加标样品,分析其荧光变化。同其它用于黄曲霉毒素B1检测的荧光传感器相比,所制备的荧光适配体传感器具有灵敏度高、重复性好、准确度高的优点。
Description
技术领域
本发明涉及食品安全检测技术领域,具体涉及一种碳纳米材料和核酸外切酶辅助的检测黄曲霉毒素B1的荧光适配体传感器的制备方法和应用。
背景技术
黄曲霉毒素是一类日常生活中容易接触到的真菌毒素,主要由黄曲霉和某些寄生类曲霉产生。基于黄曲霉毒素的衍生物约有20种,其中黄曲霉毒素B1型(AFB1)的毒性最强,是目前人类已知的最易致癌的天然化合物。AFB1经常污染花生、玉米等粮油和谷物加工食品,分布较为广泛,预防难度大。由于AFB1的耐热性较强,通常在280℃以上才会分解,常规的食品加工方式难以消除AFB1的威胁。因此,建立一种快速、准确的检测和控制食品中AFB1的方法对食品安全和人体健康至关重要。
目前对于AFB1的检测以色谱分析方法为主,包括液相色谱法、液相色谱串联质谱、气相色谱串联质谱法。这些方法能够满足对于AFB1的高灵敏度检测,但由于色谱仪和质谱仪较为昂贵,并且需要技术娴熟的操作人员,样品处理过程复杂耗时,难以满足广泛背景下食品安全检测的需要。一些基于免疫的方法如酶联免疫吸附法(ELISA)的操作方法简便,成本较低,但在检测灵敏度方面存在限制,不能作为定量确证的手段。为了解决ELISA在定量检测上的不足,有许多新方法如荧光检测法被发展出来,现有文献张晓雨等人(《用于玉米中伏马毒素B1和玉米赤酶烯酮同时高灵敏检测的新型荧光免疫技术研究》)将荧光标记技术应用到ELISA检测上,构建了一种荧光免疫传感器,异硫氰酸荧光素作为示踪信号极大地提高了ELISA的灵敏度,克服了以上的缺点,然而ELISA使用抗原/抗体作为捕获探针,难以摆脱假阳性信号的干扰。为了解决以上问题,核酸适配体(aptamer)作为一种新型的识别分子被开发出来。Aptamer是一段结构化的单链寡核苷酸序列(DNA或RNA),由指数富集配体系统进化(SELEX)技术筛选而得的特异性靶标结合分子,对目标物分子具有严格的识别能力和高度的亲和力,可用于对低分子量毒素如AFB1的高特异性识别,现有技术段诺等人(《一种特异性识别甲氧苄啶的核酸适配体及其筛选和应用》)使用SELEX技术筛选出了能与甲氧苄啶特异性结合的一种aptamer,等温滴定量热法显示出较高的亲和力和特异性。与传统的抗体相比,核酸适配体的合成方法简单,生产批次间差异性小,稳定易保存,成本较抗体低,具有很大的发展潜力。本发明针对现有免疫法检测技术存在的问题,在荧光传感器高灵敏度识别的基础上,引入aptamer代替抗体作为识别分子,减小了假阳性信号的干扰,提高了检测灵敏度,又降低了生产成本;同时,本发明引入了几种不同的碳纳米材料和核酸外切酶,碳纳米材料的存在无需额外修饰的猝灭基团,在参与构建检测系统的同时,还起到了降低系统的背景信号的作用;核酸外切酶辅助构建的双循环扩增系统特异性剪切HP1-HP2-cDNA和部分HP2-cDNA双链,放大了目标物(AFB1)的信号,提高了检测系统的灵敏度;基于本发明的荧光传感策略在克服了以上现有技术存在问题的同时,具有操作步骤简单,无需复杂的处理过程,使用成本低的特点,有利于发明的推广应用。
发明内容
本发明的目的是克服目前常用检测方法中灵敏度低,成本高,容易出现假阳性信号,样品处理复杂等缺点,提供一种基于碳纳米材料和核酸外切酶辅助双循环放大信号的荧光适配体传感器对AFB1的检测方法,该方法能够简单快速,高灵敏度和高准确性地实现对食品样品中AFB1的检测。
为了达到上述目的,本发明采用以下技术方案:
一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用,包括以下步骤:
(1)在适配体互补链cDNA的3’端修饰荧光基团,得到荧光标记的信号探针;
(2)DNA三链杂交结构的形成:分别加入一定量的缓冲溶液,将经PCR仪梯度退火的适配体HP1、HP2,标记荧光基团的cDNA,以及不同浓度的AFB1共孵育,得到HP1-HP2-cDNA三链杂交结构;
(3)核酸外切酶辅助双循环扩增模式(dRCA)的制备:在步骤(2)中加入一定量的核酸外切酶,特异性剪切DNA三链杂交结构,使其产生两种酶解产物,酶解产物继续参与步骤(2)及第二步扩增的反应;
(4)碳纳米材料介导cDNA共轭吸附进行荧光猝灭,得到碳纳米材料和核酸外切酶辅助的荧光适配体传感器:向步骤(3)体系加入一定量的碳纳米材料共孵育,未参与dRCA的cDNA被碳纳米(猝灭)材料吸附进行荧光猝灭,参与dRCA的cDNA被核酸外切酶完全剪切,其3’端修饰的荧光基团被剪切后在溶液中游离,维持荧光信号。至此,一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器制备完成;
(5)应用于食品样品检测:按照步骤(1)-步骤(4)测得未知浓度的待测食品样品的荧光信号,其中步骤(2)加入的不同浓度的AFB1在实际样品检测时为食品提取液,检测结果带入标准曲线得到样品中AFB1浓度。
本发明的检测机理为:所用碳纳米材料可以与体系中游离的单链aptamer通过π-π堆积作用吸附在一起,并通过荧光能量共振转移(FRET)作用将aptamer上标记的荧光基团猝灭,降低其荧光信号;所用的核酸外切酶可以特异性从DNA双链的5’平或凹末端剪切,而对3’端没有剪切作用,从而保护了循环产物。具体的,通过将适配体HP1、HP2退火形成发夹结构,有效避免了核酸外切酶的消化,当适配体HP1与AFB1特异性结合后,HP1发夹打开并触发HP2构象的变化,而后HP2与cDNA杂交,形成HP1-HP2-cDNA三链杂交结构。此时,核酸外切酶识别杂交链并从其5’端剪切,释放HP1和部分HP2链,实现HP1的循环利用;部分HP2链可继续结合其他cDNA并继续被核酸外切酶剪切,实现第二步循环。加入碳纳米材料后,cDNA通过π-π堆积效应被碳纳米材料吸附,其上标记的荧光基团通过FRET效应被猝灭。其中参与循环放大的cDNA被剪切,而后其5’端标记FAM游离,荧光信号保持不变,未参加循环的cDNA被吸附后荧光信号降低。通过荧光信号响应强度的变化来定量样品中AFB1的含量。该传感器在适配体特异性识别AFB1的基础上,通过核酸外切酶的辅助实现双重循环信号放大,提高了传感器的灵敏度,同时碳纳米材料介导FRET使cDNA的荧光猝灭,降低背景干扰的同时提高了传感器的检测范围和检测限,从而实现对AFB1的快速,高灵敏度和低成本检测。
在本发明中,步骤(1)所述的缓冲溶液为50mM Tris-HCl,1×NEBuffer中的一种;
优选地,缓冲溶液为1×NEBuffer,其中包含50mM KAc,20mM Tris-Ac,10mM Mg(Ac)2,1mM DTT,pH为7.9。
在本发明中,步骤(1)所述的互补链cDNA的核苷酸序列如SEQ ID NO:1或SEQ IDNO:2所示:
5’-CCCCAAAGGATCAACTGC-3’(SEQ ID NO:1),5’-CCCGAAAGGATCAACTGC-3’(SEQ IDNO:2),荧光基团标记位置为3’端,cDNA链3’端修饰的荧光基团分别为Cy3,Cy5,FAM,ROX,Alexa Fluor 488,Alexa Fluor 594中的一种;
在本发明中,步骤(2)所述的适配体HP1,HP2,cDNA的浓度比为1:1:2,用量范围在3~6μL,浓度范围1~3μM,恒温培养箱孵育时间90~180min。
在本发明中,步骤(3)所述的核酸外切酶为T7噬菌体基因6核酸外切酶(T7 EXO),λ噬菌体核酸外切酶(λEXO)中的一种;
在本发明中,步骤(4)所述的碳纳米材料为多孔碳纳米笼(MgO为模板制备),氮掺杂碳纳米笼(MgO为模板制备),氧化石墨烯(Hummers法制备),碳掺杂二硫化钼纳米片(溶剂热法制备)中的一种。
在本发明中,步骤(4)所述的磁力搅拌锅的转速为500-800r/min,混合孵育时间为15-30min。
与现有技术相比,本发明具有如下显著优点:
1.本发明利用碳纳米材料猝灭DNA链上修饰的荧光基团,无需额外修饰的猝灭基团,在参与完成检测系统的同时起到了降低系统背景信号的作用。
2.本发明利用特定的核酸外切酶(T7 EXO、λEXO),特异性剪切HP1-HP2-cDNA和部分HP2-cDNA双链,构建了双循环扩增反应,极大地提高了检测系统的灵敏度。
3.基于本发明的荧光传感策略,操作步骤简单,无需复杂的处理过程,使用成本较低,有利于发明的推广应用。
附图说明
图1为碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备示意图。
图2为本发明实施例1所构建的传感器在无AFB1(实线)、存在0.1ng/mL AFB1(点画线)、100ng/mL AFB1(虚线)时的FAM的荧光强度。
图3为本发明实施例1所构建的传感器在其他干扰毒素存在的情况下对AFB1的选择性比较图。
具体实施方式
下面结合附图和实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1:
一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用,其实现方法如图1所示。
一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,包括以下步骤:
(1)核酸适配体结构修饰:即将单链的核酸适配体转换为部分互补的发夹结构:分别将100μL 1μM的适配体HP1和适配体HP2置于梯度PCR仪中执行变性退火程序。预设的PCR降温程序为95℃下维持5min,然后逐渐冷却至室温,折叠成稳定的发夹结构,将其置于4℃条件下保存备用。
(2)适配体双循环放大体系的构建:分别加入上述制备好的发夹适配体HP1(3μL 1μM),HP2(3μL 1μM),标记FAM荧光素的cDNA(6μL 1μM),T7 EXO(1μL 10U),Tris-HCl(35μL50Mm),2μL不同浓度的AFB1置于37℃恒温培养箱中孵育1.5h。
(3)碳纳米材料共轭吸附进行荧光猝灭:待孵育完成后,加入10μL 80μg/mL的多孔碳纳米笼猝灭材料,并使用超纯水将体系补充至200μL,随后将样品置于磁力搅拌锅中37℃搅拌20min,使猝灭材料与样品充分混合,溶液中未被T7 EXO剪切的cDNA被多孔碳纳米笼吸附后,其上标记的FAM由于与多孔碳纳米笼表面的FRET效应造成荧光猝灭。
(4)标准曲线的建立:将2μL不同浓度的AFB1标准溶液加入步骤(2)中,得到不同浓度梯度的样品检测液,并经步骤(3)孵育后检测荧光强度信号,以AFB1浓度的对数值为横坐标,荧光强度信号为纵坐标进行线性拟合,建立传感器对AFB1的标准曲线。
其中,检测样品荧光强度的具体步骤为:使用微量比色皿盛装待测样,设置荧光分光光度计的激发波长为492nm,发射波长测量范围510~600nm,狭缝选择10nm,测量电压680V,记录517nm发射波长时的荧光强度。
如图2所示,为本发明实施例1所构建的传感器在无AFB1(实线)、存在0.1ng/mLAFB1(点画线)、100ng/mL AFB1(虚线)时的FAM的荧光强度。
为了验证所制备的碳纳米材料和核酸外切酶辅助的荧光适配体传感器对AFB1有特异性识别,在50mM的Tris-HCl中加入AFB1标准品,使样品中AFB1的浓度为5ng/mL;采用50mM的Tris-HCl分别配置其他干扰毒素(OTA,ZEN,DON,T-2)标准品溶液,浓度均为100ng/mL。按照实施例1所构建的检测体系对上述几种不同干扰毒素标准品进行检测,检测结果如图3所示,说明本发明方法对AFB1具有较高的选择性。
对比例1:
一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,未特别说明的步骤与实施例1相同,不同之处在于,步骤(2)中,加入发夹适配体HP1、HP2和cDNA时的同时加入多孔碳纳米笼。与实施例1相比,测定相同浓度的AFB1(50ng/mL)时的ΔF值明显降低,表明在同时加入多孔碳纳米笼时直接吸附了体系中的cDNA,使体系中的cDNA浓度降低,反应效果减弱,因此得到结论:多孔碳纳米笼需要在双循环扩增完成之后加入,不能一步完成所有反应。
实施例2:
一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,未特别说明的步骤与实施例1相同,不同之处在于,步骤(3)所用的碳纳米材料为氧化石墨烯。与实施例1相比,测定相同浓度的AFB1(50ng/mL)时的荧光强度差值ΔF明显降低,表明实施例1所用的多孔碳纳米笼有更好的吸附和猝灭效果。
实施例3:
一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用,其实际应用,包括以下步骤:
(1)实际样品处理:取0.5g的食品加标样品(玉米粉、花生粉),加入到10mL甲醇/水溶液(比例7:3)中,置于摇床上震荡20分钟,随后取出置于离心机中离心10分钟,转速10000转/分钟,离心完成后取出上清液得到食品样品提取液。
(2)样品检测:按照实施例1的步骤(1)-步骤(3)测得荧光强度信号,带入标准曲线得到样品中AFB1的浓度。
(3)以花生粉为实际样品进行测定时,以10ng/mL加入量为基准,在花生粉中分别加入基准量0.1倍,10倍的AFB1标准品。取2μL样品溶液,按照实施例1步骤(1)-步骤(3)测定荧光强度信号,带入实施例1检测的标准曲线得到样品中的AFB1浓度,每份样品重复测定3次取平均值,计算RSD及回收率如下表所示:
经验证所制备的荧光适配体传感器对黄曲霉毒素B1的检测具有准确度高,线性范围宽,检测下限低的特点。同时,对实际样品(如玉米粉)的检测结果表明所制备的传感器具有非常好的实际应用价值。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (10)
1.一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,其特征在于,包括以下步骤:
(1)在适配体互补链cDNA的3’端修饰荧光基团,得到荧光标记的信号探针;
(2)DNA三链杂交结构的形成:分别加入一定量的缓冲溶液,经PCR仪梯度退火的适配体HP1、HP2,标记荧光基团的cDNA,以及不同浓度的黄曲霉毒素B1,置于37℃恒温培养箱中孵育一定时间,得到HP1-HP2-cDNA三链杂交结构;
(3)核酸外切酶辅助双循环扩增模式(dRCA)的制备:向上述体系中加入一定量的核酸外切酶,特异性剪切DNA三链杂交结构,使其产生两种酶解产物,酶解产物继续参与步骤(2)及第二步扩增的反应;
(4)碳纳米材料介导cDNA共轭吸附进行荧光猝灭,得到碳纳米材料和核酸外切酶辅助的荧光适配体传感器:向步骤(3)体系加入一定量的碳纳米材料,在磁力搅拌锅混合孵育一定时间,未参与dRCA的cDNA被碳纳米材料吸附进行荧光猝灭,参与dRCA的cDNA被核酸外切酶完全剪切,其3’端修饰的荧光基团被剪切后在溶液中游离,维持荧光信号,至此,荧光适配体传感器制备完成,设置固定的激发波长,测量范围和测量电压,在荧光分光光度计下记录发射波长的荧光强度。
2. 根据权利要求1所述的一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,其特征在于,步骤(1)中,所用的缓冲溶液为50mM Tris-HCl,1×NEBuffer中的一种;优选地,缓冲溶液为1×NEBuffer,其中包含50 mM KAc,20 mM Tris-Ac,10 mM Mg(Ac)2,1 mM DTT,pH为7.9。
3. 根据权利要求1所述的一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,其特征在于,步骤(1)中,所述的互补链cDNA的序列为5’- CCCCAAAGGATCAACTGC-3’或5’- CCCGAAAGGATCAACTGC -3’所示,荧光基团标记位置为3’端;优选地,cDNA的序列为5’- CCCCAAAGGATCAACTGC -3’。
4. 根据权利要求1所述的一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,其特征在于,步骤(1)中,cDNA链 3’端修饰的荧光基团分别为Cy3,Cy5,FAM,ROX,Alexa Fluor 488,Alexa Fluor 594中的一种;优选地,cDNA链 3’端修饰的荧光基团为FAM。
5. 根据权利要求1所述的一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,其特征在于,步骤(2)中所述的适配体HP1,HP2,cDNA的浓度比为1:1:2,用量范围在3~6 μL,浓度范围1~3 μM,恒温培养箱孵育时间90~180min。
6. 根据权利要求1所述的一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,其特征在于,步骤(3)中所述的核酸外切酶辅助的dRCA的制备,所用的核酸外切酶为T7噬菌体基因6核酸外切酶(T7 EXO),λ噬菌体核酸外切酶(λ EXO)中的一种;优选地,核酸外切酶为T7 EXO。
7.根据权利要求1所述的一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,其特征在于,步骤(4)中所述的碳纳米材料为碳纳米笼(MgO为模板制备),氮掺杂碳纳米笼(MgO为模板制备),氧化石墨烯(Hummers法制备),碳掺杂二硫化钼纳米片(溶剂热法制备)中的一种;优选地,碳纳米材料为碳纳米笼。
8. 根据权利要求1所述的一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法,其特征在于,步骤(4)中所述的磁力搅拌锅的转速为500-800 r/min,混合孵育时间为15-30min。
9.一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的应用,其特征在于,所述的荧光适配体传感器用于食品安全为目的的检测黄曲霉毒素B1。
10. 根据权利要求9所述的一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的应用,其特征在于,检测食品样品时包括以下步骤:分别在碳纳米材料和核酸外切酶辅助的荧光适配体传感器中加入2~4 μL的缓冲溶液和食品提取液;按照权利要求1步骤(1)-步骤(4)所述的方法构建传感体系,检测荧光变化。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210870746.2A CN116148457A (zh) | 2022-07-23 | 2022-07-23 | 一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210870746.2A CN116148457A (zh) | 2022-07-23 | 2022-07-23 | 一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116148457A true CN116148457A (zh) | 2023-05-23 |
Family
ID=86349476
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210870746.2A Pending CN116148457A (zh) | 2022-07-23 | 2022-07-23 | 一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116148457A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107988351A (zh) * | 2017-12-14 | 2018-05-04 | 福州大学 | 一种环状dna在正义rna的检测、成像及基因治疗中的应用 |
CN108251507A (zh) * | 2018-03-09 | 2018-07-06 | 上海市计量测试技术研究院 | 一种核酸的检测体系及其检测方法和应用 |
CN110592191A (zh) * | 2019-09-18 | 2019-12-20 | 南京邮电大学 | 一种基于酶催化循环及二硫化钼吸附介导可视化检测核酸的方法 |
-
2022
- 2022-07-23 CN CN202210870746.2A patent/CN116148457A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107988351A (zh) * | 2017-12-14 | 2018-05-04 | 福州大学 | 一种环状dna在正义rna的检测、成像及基因治疗中的应用 |
CN108251507A (zh) * | 2018-03-09 | 2018-07-06 | 上海市计量测试技术研究院 | 一种核酸的检测体系及其检测方法和应用 |
CN110592191A (zh) * | 2019-09-18 | 2019-12-20 | 南京邮电大学 | 一种基于酶催化循环及二硫化钼吸附介导可视化检测核酸的方法 |
Non-Patent Citations (3)
Title |
---|
DELUN CHEN等: "A universal constructing method for high performance DNA biosensors based on the optimized photoelectrode material and dual recycling amplification", APPLIED SURFACE SCIENCE, vol. 2022, no. 585, pages 1 - 9 * |
HAO WU等: "Target-triggered and T7 exonuclease-assisted cascade recycling amplification strategy for label-free and ultrasensitive fluorescence detection of aflatoxin B1", SENSORS AND ACTUATORS B: CHEMICAL, vol. 2020, no. 321, pages 1 - 9 * |
XIAOTING LI等: "A T7 exonuclease assisted dual-cycle signal amplification assay of miRNA using nanospheres-enhanced fluorescence polarization", TALANTA, vol. 2019, no. 202, pages 297 - 302 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wu et al. | Screening and development of DNA aptamers as capture probes for colorimetric detection of patulin | |
Hu et al. | An amplified graphene oxide-based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling forbioassays | |
Yang et al. | A novel fluorescent platform of DNA-stabilized silver nanoclusters based on exonuclease III amplification-assisted detection of Salmonella Typhimurium | |
CN112574998B (zh) | 检测黄曲霉毒素b1的探针组、试剂盒及其应用 | |
CN105675565B (zh) | 一种快速检测黄曲霉毒素b1的方法 | |
Liu et al. | Direct fluorescence anisotropy assay for cocaine using tetramethylrhodamine-labeled aptamer | |
CN110320169B (zh) | 一种牛奶中氧四环素残留的比色检测方法 | |
Qiu et al. | Label-free, homogeneous, and ultrasensitive detection of pathogenic bacteria based on target-triggered isothermally exponential amplification | |
CN111793622A (zh) | 基于酶辅助级联循环扩增的发夹探针组及制备方法、用途 | |
CN111662900A (zh) | 一种磺胺二甲嘧啶核酸适配体筛选方法、试剂盒及应用 | |
CN113804898B (zh) | 同时检测皮质醇、血清睾酮、肌酸激酶同工酶的荧光传感方法和试剂盒 | |
Fu et al. | Lateral flow strip biosensor based on streptavidin-coated gold nanoparticles with recombinase polymerase amplification for the quantitative point-of-care testing of Salmonella | |
CN113322256B (zh) | 一种探针组、传感器、检测方法及其用途 | |
CN113265447B (zh) | 一种用于检测肌酸激酶同工酶的滚环扩增-金四面体比色检测方法和试剂盒 | |
Qin et al. | Rapid detection of Pseudomonas aeruginosa using a DNAzyme‐based sensor | |
CN109402128A (zh) | 黄曲霉毒素b1的核酸适配体、含有该核酸适配体的黄曲霉毒素b1检测试剂盒及检测方法 | |
Zhu et al. | An immobilization-free electrochemical aptamer-based assay for zearalenone based on target-triggered dissociation of DNA from polydopamine nanospheres with strand displacement amplification | |
Ye et al. | An ultrasensitive sandwich immunoassay with a glucometer readout for portable and quantitative detection of Cronobacter sakazakii | |
Deng et al. | A novel ratiometric fluorescent aptasensor accurately detects patulin contamination in fruits and fruits products | |
CN112080551A (zh) | 一种双酶介导级联信号放大的氨苄西林检测适体传感器 | |
CN116148457A (zh) | 一种碳纳米材料和核酸外切酶辅助的荧光适配体传感器的制备方法和应用 | |
CN114807147B (zh) | 黄曲霉毒素b1的核酸适配体及其应用 | |
CN112763472B (zh) | 一种用于检测t-2毒素残留的检测系统及其制备方法和应用 | |
CN112304914B (zh) | 基于荧光光谱法检测四环素类抗生素的方法 | |
Zhao et al. | A novel fluorescent aptasensor based on dual-labeled DNA nanostructure for simultaneous detection of Ochratoxin A and Aflatoxin B1 |
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 | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20230523 |