CN112946266B - 一种具有荧光信号放大功能的三维dna微球及其制备方法与应用 - Google Patents
一种具有荧光信号放大功能的三维dna微球及其制备方法与应用 Download PDFInfo
- Publication number
- CN112946266B CN112946266B CN202110153261.7A CN202110153261A CN112946266B CN 112946266 B CN112946266 B CN 112946266B CN 202110153261 A CN202110153261 A CN 202110153261A CN 112946266 B CN112946266 B CN 112946266B
- Authority
- CN
- China
- Prior art keywords
- beta
- lactamase
- microsphere
- dimensional dna
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000004005 microsphere Substances 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title abstract description 14
- 230000003321 amplification Effects 0.000 title description 5
- 238000003199 nucleic acid amplification method Methods 0.000 title description 5
- 108090000204 Dipeptidase 1 Proteins 0.000 claims abstract description 78
- 102000006635 beta-lactamase Human genes 0.000 claims abstract description 78
- 238000001514 detection method Methods 0.000 claims abstract description 40
- 241000894006 Bacteria Species 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 31
- 238000004557 single molecule detection Methods 0.000 claims abstract description 28
- 150000003952 β-lactams Chemical class 0.000 claims abstract description 17
- 241000588724 Escherichia coli Species 0.000 claims description 35
- 239000007853 buffer solution Substances 0.000 claims description 21
- 231100000331 toxic Toxicity 0.000 claims description 17
- 230000002588 toxic effect Effects 0.000 claims description 17
- 230000000903 blocking effect Effects 0.000 claims description 15
- 239000011248 coating agent Substances 0.000 claims description 15
- 238000000576 coating method Methods 0.000 claims description 15
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 15
- 238000011534 incubation Methods 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 11
- 239000002245 particle Substances 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 9
- 230000000295 complement effect Effects 0.000 claims description 8
- 239000006166 lysate Substances 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 108091034117 Oligonucleotide Proteins 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 239000010931 gold Substances 0.000 claims description 5
- 229910052737 gold Inorganic materials 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 3
- 238000000197 pyrolysis Methods 0.000 claims description 3
- 241000283707 Capra Species 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- 238000005336 cracking Methods 0.000 claims 1
- 230000003252 repetitive effect Effects 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 13
- 238000004458 analytical method Methods 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 9
- 239000007850 fluorescent dye Substances 0.000 abstract description 4
- 239000000427 antigen Substances 0.000 abstract description 3
- 102000036639 antigens Human genes 0.000 abstract description 2
- 108091007433 antigens Proteins 0.000 abstract description 2
- 230000008105 immune reaction Effects 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000004043 dyeing Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 56
- 239000000872 buffer Substances 0.000 description 28
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 16
- 229960000723 ampicillin Drugs 0.000 description 16
- 230000004044 response Effects 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000000178 monomer Substances 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 229940098773 bovine serum albumin Drugs 0.000 description 7
- 239000004098 Tetracycline Substances 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 229960000318 kanamycin Drugs 0.000 description 5
- 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 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 150000003951 lactams Chemical class 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 229960002180 tetracycline Drugs 0.000 description 5
- 229930101283 tetracycline Natural products 0.000 description 5
- 235000019364 tetracycline Nutrition 0.000 description 5
- 150000003522 tetracyclines Chemical class 0.000 description 5
- 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 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 229940121657 clinical drug Drugs 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- CNHYKKNIIGEXAY-UHFFFAOYSA-N thiolan-2-imine Chemical compound N=C1CCCS1 CNHYKKNIIGEXAY-UHFFFAOYSA-N 0.000 description 4
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 108010015776 Glucose oxidase Proteins 0.000 description 3
- 239000004366 Glucose oxidase Substances 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229940116332 glucose oxidase Drugs 0.000 description 3
- 235000019420 glucose oxidase Nutrition 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 102100038517 Pyridoxal kinase Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 1
- 239000011654 magnesium acetate Substances 0.000 description 1
- 229940069446 magnesium acetate Drugs 0.000 description 1
- 235000011285 magnesium acetate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012465 retentate Substances 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
-
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- G01N2333/986—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)
-
- 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
Abstract
本发明公开了一种具有荧光信号放大功能的三维DNA微球及其制备方法与应用,属于分析检测领域。一种三维DNA微球与β‑内酰胺酶抗体结合形成Ab‑DNF材料,能特异性识别耐药菌产生的β‑内酰胺酶。本发明还公开了一种利用上述Ab‑DNF材料构建单分子检测平台的方法,在384孔板上通过三明治夹心免疫反应,使Ab‑DNF材料与抗原结合并被荧光染料染色形成均匀的荧光点。荧光点数与β‑内酰胺酶浓度之间存在线性关系,从而实现对β‑内酰胺酶的定量检测。此方法灵敏度高,特异性好,简便快速,对β‑内酰胺酶的检测线性范围为100aM~1pM,最低检出限为100aM;对β‑内酰胺类耐药菌的检出限为10CFU/mL,检测时间30min,达到了快速检测的要求。
Description
技术领域
本发明属于分析检测领域,具体涉及一种具有荧光信号放大功能的三维DNA微球及其制备方法与应用。
背景技术
单分子检测是生物标志物检测领域的颠覆性技术,其最大特点是具有超高灵敏度,尤其在生命分析中,针对痕量样本或痕量分析物的精准测定方面发挥着不可替代的作用。但是单分子检测技术尚存在一些问题,如器件制造工艺和精度要求较高、设备昂贵、检测条件苛刻以及检测时间较长等,这极大限制了单分子检测在分析检测领域的应用,因此,亟需发展一种简单、低成本、快速的单分子检测技术。
近年来,DNA合成材料由于具有高特异性、高稳定性及易于合成等优势,在生物标志物检测方面发展迅速。其中,三维DNA微球(DNA Nanoflower,DNF)是由滚环扩增技术(Rolling Circle Amplification,RCA)产生的长链DNA自组装得到的花状DNA微球。由于荧光核酸染料可以特异性结合DNA,由长链DNA压缩团聚而成的DNF可结合大量染料形成高亮度的微米级荧光斑点,可在显微镜下直接观察计数。DNF表面有大量的寡核苷酸互补配对结合位点,通过与不同的抗体或DNA键合,实现对不同靶标分子的识别,包括小分子、蛋白质和RNA等。因此其可以作为生物分析检测和生物医学研究中重要的信号放大工具,弥补了单分子检测方法的不足。
β-内酰胺类耐药菌已被美国疾病控制中心列为具有严重威胁的一类抗性菌,针对此种耐药菌可通过检测其标志物(β-内酰胺酶)实现对抗性细菌的检测。常规的耐药菌检测方法是传统培养法,需要对病菌进行长时间培养,往往需要12~36h才能得出实验结果,较长的培养时间不利于耐药菌及相关疾病的检测和治疗。因此,建立准确、高灵敏、快速的检测方法对于耐药菌的检测和相关疾病的精准治疗至关重要。基于单分子的标志物检测方法可以用于耐药菌的快速检测,无需长时间培养,弥补了传统细菌检测方法的不足,有助于辅助疾病的精准治疗和预后控制。
发明内容
本发明为了解决现有的技术问题,利用三维DNA微球(DNF)与β-内酰胺酶抗体(Ab)结合获得对β-内酰胺酶具有特异性识别能力的材料(Ab-DNF),并提供一种快速检测β-内酰胺类耐药菌的方法。利用上述Ab-DNF建立一种简单的单分子检测方法,通过荧光计数法检测耐药菌产生的β-内酰胺酶。将具有特异性识别功能的Ab-DNF发展成单分子检测平台,可扩大其在生物分析检测及临床诊断中的实用价值。
本发明为解决上述技术问题,提供了一种具有β-内酰胺酶抗体的三维DNA微球,所述具有β-内酰胺酶抗体的三维DNA微球具有荧光信号放大的功能,β-内酰胺酶抗体通过连接的可以与三维DNA微球碱基互补配对的DNA单链连接到三维DNA微球表面,形成可以特异性识别β-内酰胺酶的β-内酰胺酶抗体-三维DNA微球;所述三维DNA微球为含有数量为108~109条的如SEQ ID NO.6所示的重复单链形成的花状结构微球。
进一步地,上述技术方案中,所述三维DNA微球的粒径为1~3μm。
进一步地,上述技术方案中,所述可以与三维DNA微球碱基互补配对的DNA单链的核苷酸序列如SEQ ID NO.3所示。
本发明还提供了具有β-内酰胺酶抗体的三维DNA微球的制备方法,其特征在于:包括如下步骤:
a、通过RCA反应制备三维DNA微球,所得三维DNA微球的粒径为1~3μm,呈花状结构,含稳定DNA单链及寡核苷酸互补配对结合位点;所得三维DNA微球为颗粒浓度为107~108个/mL,含重复单链数量108~109条的SEQ ID NO.6所示的核苷酸序列;
b、β-内酰胺酶抗体和三维DNA微球碱基互补配对的SEQ ID NO.3所示的DNA单链连接,得Ab-LD;
c、加入如SEQ ID NO.4所示的封闭DNA将三维DNA微球表面多余寡核苷酸碱基互补配对结合位点封闭,然后将Ab-LD中的DNA单链通过互补配对连接到三维DNA微球表面,形成可以特异性识别β-内酰胺酶的具有β-内酰胺酶抗体的三维DNA微球。
本发明还提供了一种具有β-内酰胺酶抗体的三维DNA微球在单分子检测中的应用。
进一步地,应用于β-内酰胺类耐药菌中的β-内酰胺酶的检测。
进一步地,上述技术方案中,β-内酰胺酶的检测方法,包括如下步骤:
(1)对β-内酰胺类耐药菌进行超声裂解,通过离心获得细菌裂解液,并用PBST缓冲液进行稀释;
(2)将2~15μg/mL包被缓冲液稀释的β-内酰胺酶抗体加入孔板中进行包被,包被完成后用PBST缓冲液洗涤;
(3)采用封闭缓冲液对孔板进行封闭,封闭完成后用PBST缓冲液洗涤;
(4)将步骤(1)所得含有β-内酰胺类耐药菌的稀释液、具有β-内酰胺酶抗体的三维DNA微球和1×SYBR-Gold染料的PBST缓冲液加入孔板中,孵育后用PBST缓冲液洗涤;
(5)采用倒置荧光显微镜对孔板进行荧光成像,然后通过ImageJ软件对荧光斑点计数。
建立荧光斑点数-抗原浓度的标准曲线:1)配制已知浓度的β-内酰胺酶标准溶液样品;2)按照上述方法获得该浓度蛋白标准溶液的荧光斑点数;3)依据标准溶液的浓度和荧光斑点的数量建立标准曲线。
进一步地,上述技术方案中,所述步骤(1)中超声裂解的时间为5~10min,离心的速度为8000~10000rpm/min;所述步骤(2)中的包被缓冲液为pH 9.6的碳酸盐缓冲液,包被的条件为4~5℃孵育12~14h或37~39℃孵育2~2.5h;所述步骤(3)中封闭缓冲液是体积分数为5%~10%的BSA溶液或山羊血清,体积为30~50μL,封闭的条件为37~39℃孵育60~120min;所述步骤(4)中孵育的条件为37~39℃孵育30~60min。
进一步地,上述技术方案中,洗涤次数均为至少3次,每次3~5min,PBST缓冲液为含有0.05%~0.1%(v/v)Tween-20的50mM~100mM PBS缓冲液,所用孔板为透明聚苯乙烯方孔平底板。
进一步地,上述技术方案中,所述β-内酰胺类耐药菌包括E.coli A、Toxic A、Toxic B、Toxic C。
进一步地,上述技术方案中,步骤(2)中β-内酰胺酶抗体与步骤(4)中具有β-内酰胺酶抗体的三维DNA微球中的β-内酰胺酶抗体种类相同。
与现有技术相比,本发明的有益效果是:
本发明以制备的Ab-DNF作为识别元件构建单分子检测方法,在孔板上通过三明治夹心免疫反应,使Ab-DNF材料与抗原结合并被荧光染料染色形成均匀的荧光点。通过被测蛋白标志物的浓度与荧光斑点数目呈线性关系,实现对β-内酰胺酶和β-内酰胺类耐药菌的定量检测。该方法特异性高、操作简单、反应快速,可有效实现对临床耐药菌的快速检测,该方法对β-内酰胺酶的检测线性范围为100aM~1pM,最低检出限为100aM,对β-内酰胺酶耐药菌的检出限为10CFU/mL,β-内酰胺酶与Ab-DNF反应时间为30min。
附图说明
图1为特异性识别目标蛋白样品的Ab-DNF的制备方法(a)及单分子检测的流程图(b)。
图2为实施例2所制备DNF的扫描电镜图(a)与荧光倒置显微镜图(b)。
图3为实施例2所制备DNF的琼脂糖凝胶图(M为标记)。
图4为实施例2所制备DNF的单体消解dPAGE图,FR:51-nt和M条带的荧光强度之比(M为1pmol)。
图5为实施例2所制备DNF的表面寡核苷酸负载量分析图(a)及负载链控量分析图(b)。
图6为实施例2所制备的Ab-LD的SDS-PAGE和琼脂糖凝胶图;图中,M1为蛋白标记,M2为DNA标记,Ab-LD为抗体与DNA缀合物,Ab+LD为抗体和DNA混合。
图7为实施例3单分子检测时Ab-DNF对β-内酰胺酶、牛血清白蛋白、葡萄糖氧化酶、细胞色素C和对照的响应;图中,a为荧光定量图,b为荧光图(比例尺:200μm)。
图8为实施例3单分子检测结果图;图中,a为Ab-DNF与β-内酰胺酶的结合动力学,b为Ab-DNF对不同浓度β-内酰胺酶的响应,c为倒置荧光显微镜下不同浓度β-内酰胺酶的荧光成像(比例尺:200μm)。
图9为实施例3单分子检测方法中优化后Ab-DNF和对照Ab-DNF生物缀合物的线性范围,B-LD与Ab-LD添加比例分别为100:1、1000:1和1:1。
图10为实施例3中应用于单分子检测的耐药菌在不同抗生素条件下培养20h后的生长情况;图中,a为具有内酰胺抗性的E.coli A在含不同浓度氨苄青霉素培养基中的生长情况,b为无抗性的E.coli B在含不同浓度氨苄青霉素培养基中的生长情况及MIC值,c为具有卡那霉素抗性的E.coli K在含不同浓度卡那霉素培养基中的生长情况,d为具有卡那霉素抗性的E.coli K在含不同浓度氨苄青霉素培养基中的生长情况及MIC值,e为具有四环素抗性的E.coli T在含不同浓度四环素培养基中的生长情况,d为具有四环素抗性的E.coliT在含不同浓度氨苄青霉素培养基中的生长情况及MIC值。
图11为实施例3耐药菌单分子检测结果图;图中,a为Ab-DNF与细菌裂解液中β-内酰胺酶的结合动力学,b为Ab-DNF对不同稀释程度细菌裂解液的响应,c为Ab-DNF对E.coliA/K/T在添加与不添加相应抗生素培养下的响应(比例尺:200μm)。
图12为实施例3中应用于单分子检测的临床耐药在含不同浓度氨苄青霉素的培养基中培养20h后的生长情况;图中为细菌悬液在OD600下的吸光度,a为具有内酰胺抗性的Toxic A在含不同浓度氨苄青霉素培养基中的生长情况,b为具有内酰胺抗性的Toxic B在含不同浓度岸边青霉素培养基中的生长情况,c为具有内酰胺抗性的Toxic C在含不同浓度氨苄青霉素培养基中的生长情况,三种细菌在高浓度氨苄青霉素培养条件下的OD值没有降低,均未出现MIC值,表明三种细菌均具有内酰胺抗性。
图13为实施例3临床耐药菌单分子检测结果图;图中,a为Ab-DNF对Toxic A/B/C的响应荧光点计数结果,b为Ab-DNF对Toxic A/B/C的响应显微镜图;图b中:A+/B+/C+为含氨苄青霉素培养的Toxic A/B/C;A-/B-/C-/对照为不含氨苄青霉素培养的Toxic A/B/C/E.coli B(比例尺:200μm)。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案,下面结合实施例对本发明的具体实施方式作进一步详细说明,实施例包括Ab-DNF材料制备、表征和单分子检测法的构建等。
表1:本发明中使用的核酸序列
下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到;
下述实施例中的RCA缓冲液(10×):330mM Tris乙酸盐,100mM醋酸镁,660mM醋酸钾,1%(v/v)Tween-20,10mM DTT,pH 7.9;
下述实施例中的PNK缓冲液(10×):500mM Tris-HCl,100mM MgCl2,50mM DTT,1mMspermidine,pH 7.6;
下述实施例中的T4 DNA连接酶缓冲液(10×):400mM Tris-HCl,100mM MgCl2,100mM DTT,5mM ATP,pH 7.8;
下述实施例中的消解缓冲液(10×):100mM Tris-HCl,100mM MgCl2,1000mM KCl,1mg/mL BSA,pH 8.5;
下述实施例中的PCR缓冲液(10×):100mM Tris-HCl,500mM KCl,0.8%(v/v)NP-40,250mM Mg2+,pH 8.8;
下述实施例中的缓冲液:137mM NaCl and 3mM KCl,pH 7.4;
下述实施例中的包被缓冲液:15mM Na2CO3 and 34.8mM NaHCO3,pH 9.6;
下述实施例中的封闭缓冲液:含5%(w/v)BSA的50mM PBS,pH 7.5;
下述实施例中的结合缓和洗涤冲液:含0.05%(v/v)Tween-20的50mM PBS,pH7.4;
实施例1利用封闭手段合成识别β-内酰胺酶的Ab-DNF及单分子检测法的技术路线
a、特异性识别β-内酰胺酶Ab-DNF的制备表征;
b、单分子检测法的构建,测试Ab-DNF与β-内酰胺酶的结合能力及选择性,分析并优化检测的关键影响因素。用封闭缓冲液封闭后,将目标蛋白样品(β-内酰胺酶标准蛋白或细菌裂解液)、Ab-DNF及1×SYBR-Gold荧光染料加入预先包被捕获抗体的384孔板,通过抗原抗体识别形成三明治结构,Ab-DNF产生均匀的荧光斑点,在倒置荧光显微镜下观察并成像,应用ImageJ软件分析计数。分析并优化该单分子检测法的检测性能(如图1b)。
实施例2材料的制备表征
特异性识别β-内酰胺酶Ab-DNF制备的具体步骤(图1a),包括DNF制备,Ab-LD制备,Ab-DNF制备等详细过程,该实验的流程为:
a、通过RCA反应制备DNF,4μM环状模版前体CDT-DNF(SEQ ID NO.1)、2mM ATP、5μL10×PNK缓冲液和10U PNK酶,在37℃孵育40min。加入3μM模版引物SEQ ID NO.2(TP),90℃加热5min变性,冷却至室温,向上述混合物中加入10μL的10×T4 DNA连接酶缓冲液和5U T4DNA连接酶,所得混合物(100μL)室温孵育1h,90℃加热5min使连接酶灭活。用标准乙醇沉淀和10%dPAGE电泳浓缩纯化环状模版;0.35μM环状模板、0.7μM模版引物SEQ ID NO.2(TP)、10μL 10×RCA缓冲液,1mM dNTPs和30U酶,在30℃孵育20h,65℃加热10min使聚合酶灭活,冷却后用300K滤膜清洗回收。如图2a所示为三维DNA微球的扫描电镜图,呈球形花状结构;如图2b所示为三维DNA微球通过荧光染料染色后在倒置荧光显微镜下的图片,证明其可以被染色观察;如图3所示为三维DNA微球的琼脂糖凝胶电泳图,图中DNF条带高于10kb DNA标准条带,证明三维DNA微球制备成功。
b、将步骤a中获得的DNF原液进行稀释(1/2.5、1/5、1/10、1/20)后使用血球计数板计算DNF的颗粒浓度。然后使用公式计算DNF的量(A/mL=B/80×400×104×C,B:计数,C:稀释倍数):
A1/2.5/mL=400/80×400×104×2.5=5.0×107个/mL;
A1/5/mL=210/80×400×104×5=5.25×107个/mL;
A1/10/mL=105/80×400×104×10=5.25×107个/mL;
A1/20/mL=55/80×400×104×20=5.5×107个/mL;
A=(A1/2.5+A1/5+A1/10+A1/20)/4=5.25×107个/mL。
c、计算单个DNF中的单体量,将步骤a中获得的DNF原液进行稀释(1/10、1/20、1/30、1/40)后进行消解反应:1μL DNF,5μL消解模板Dt(SEQ ID NO.5)(100μM),2μL 10×消解缓冲液和9μL ddH2O,90℃5min变性,冷却至室温后添加3μL EcoRV(20U/μL),在37℃孵育24h,90℃加热10min使EcoRV灭活;然后利用dPAGE对消解产物(单体)进行分析(以CDT-DNF作为标记)。用1×SYBR Gold对dPAGE凝胶室温染色10min,利用Amersham Typhoon扫描成像,如图4所示;通过ImageJ计算不同稀释倍数DNF中单体的荧光强度,使用以下公式计算单体的量:
N1/20=1.17×1pmol×20=23.4pmol;
N1/30=0.79×1pmol×30=23.7pmol;
N1/40=0.61×1pmol×40=24.4pmol;
N=(N1/20+N1/30+N1/40)/3=23.8pmol;
Nm=N×NA/5.25×107个/mL×1μL=2.73×108单体/个。
所得三维DNA微球(DNF)为颗粒浓度为107~108个/mL,含重复单链数量108~109条如SEQ ID NO.6所示的核苷酸序列。
d、制备Ab与DNA单链(Ab-LD)生物缀合物,将2mg MBS(6.4μmol)溶解在1mL二甲基亚砜(DMSO)中制得MBS溶液(6.4mM),将1mg2-亚氨基硫杂环戊烷(7.3μmol)溶解在1mLddH2O中制得2-亚氨基硫杂环戊烷溶液(7.3mM)。100pmolβ-内酰胺酶抗体(Ab)和1.4μL 2-亚氨基硫杂环戊烷溶液(10nmol)溶至50μL包被缓冲液,室温孵育20min,用10K滤膜除去过量的2-亚氨基硫杂环戊烷。1nmol LD(SEQ ID NO.3)和3.2μL MBS(20nmol)溶至50μL PBS缓冲液调,室温孵育1h,用3K滤膜除去过量的MBS。将两管离心柱上层截流物混合重悬于100μLPBS缓冲液中,室温孵育1h,用10K滤膜除去未结合的LD然后重悬于100μL PBS缓冲液中。Ab-LD生物缀合物的浓度约为1μM。如图6SDS PAGE所示,Ab与DNA单链(Ab-LD)生物缀合物成功合成。
e、确定B-LD的封闭作用并制备Ab-DNF生物缀合物,首先确定DNF表面DNA链的最大负载量:封闭DNA(B-LD)(SEQ ID NO.4)(0.5/1/1.5/2/2.5/3pmol)、2μL DNF(3500颗粒/1.6pmol单体)和5μL 10×PCR缓冲液,室温孵育1h,用300K滤膜除去过量的B-LD后将B-DNF(已封闭DNF)重悬于50μL ddH2O中,90℃加热5min变性后冷却。用300K滤膜除去DNF后将下层流出液用ddH2O补齐至200μL,用标准乙醇沉淀和10%dPAGE电泳浓缩纯化B-LD,如图5adPAGE图所示,DNF表面DNA链的最大负载量为2.5pmol。然后验证B-LD的封闭作用:B-LD/LD(摩尔比:1/5、1/1、5/1、10/1、50/1、100/1,总摩尔量:2.5pmol)、2μL DNF(3500颗粒/1.6pmol单体)和5μL 10×PCR缓冲液,与上述步骤相同,如图5b dPAGE图所示,当B-LD的量增多时LD的量减少,证明B-LD可以起到封闭的作用。最后制备Ab-DNF生物缀合物:B-LD/Ab-LD(摩尔比:100/1,B-LD:2.5pmol)、2μL DNF和5μL 10x PCR缓冲液,室温孵育1h,用300K滤膜除去过量的B-LD/Ab-LD后将截流物重悬于50μL PBS缓冲液中。
f、制备对照Ab-DNF(C-Ab-DNF)生物缀合物,B-LD/Ab-LD(摩尔比:1000/1,1/1;B-LD:2.5pmol,1.25pmol)、2μL DNF和5μL 10×PCR缓冲液。重复步骤e。
实施例3单分子检测法建立应用
a、计数ELISA程序。将20μL Ab(包被缓冲液稀释的10μg/mL捕获抗体)加入384孔板中,4℃孵育过夜,弃去多余Ab,PBST洗涤3次,每次3min;加入30μL含有5%BSA的封闭缓冲液,37℃孵育1h,弃去多余封闭缓冲液,PBST洗涤3次,每次3min;加入含目标蛋白样本(β-内酰胺酶标准蛋白或大肠杆菌裂解液)、Ab-DNF和1×SYBR-Gold的PBST20μL,37℃孵育30min后用PBST洗涤3次,每次3min;用倒置荧光显微镜成像(激发波长490nm,发射波长520nm),然后通过ImagJ软件计数;
b、确定β-内酰胺酶的检出限。首先验证单分子检测法的选择性:分别加入β-内酰胺酶、牛血清白蛋白(BSA)、细胞色素C(C-C)、葡萄糖氧化酶(GOX)和空白对照,37℃孵育30min,如图7a选择性测试结果所示,该检测方法只对β-内酰胺酶具有高特异性响应,而对其他三种蛋白及空白基本无响应。
然后验证β-内酰胺酶与Ab-DNF的最佳反应时间:20μL含β-内酰胺酶(1pM)的PBS缓冲液,37℃孵育(5、10、15、20、25、30、40、50min),如图8a分析结果证明在40min时结合效果最好。
如图9所示,最后验证β-内酰胺酶的检出限:加入20μL含β-内酰胺酶(100aM~1pM)的PBS缓冲液,37℃孵育30min。对于C-Ab-DNF(B-LD/Ab-LD为1000/1),将β-内酰胺酶用PBS连续稀释为1fM~10pM,对于C-Ab-DNF(B-LD/Ab-LD为1/1),将β-内酰胺酶用PBS连续稀释为1pM~10nM。评估每个点的数据准确性,获得的变异系数(CV)均小于15%。如图8c为不同β-内酰胺酶的浓度下的荧光斑点图,图中斑点计数后结果如图8b,分析结果表明荧光斑点数与β-内酰胺酶的浓度成正比,应用Ab-DNF单分子检测法对β-内酰胺酶的检出限为100aM。
c、细菌样品中的β-内酰胺酶分析所应用的细菌种类与耐药性,E.coli A/ToxicA/B/C可以表达β-内酰胺酶具有抗β-内酰胺的能力,四环素耐药菌E.coli T和卡那霉素耐药菌E.coli K不表达β-内酰胺酶没有抗β-内酰胺的能力,E.coli B为非耐药细菌。
d、检测细菌的耐药性,如图10,图12所示,通过生长抑制试验证明E.coli A/B/K/T和Toxic A/B/C的β-内酰胺抗性:在含梯度稀释氨苄青霉素(0.064、0.125、0.25、0.5、1、2、4、8、16、32和64μg/mL)的96孔板中培养的细菌,包括空白(0μg/mL)对照,所有实验均重复三次。37℃孵育过夜,用酶标仪测定细菌的OD600。MIC值定义为抑制细菌生长的最低抗生素浓度;验证E.coli K/T和Toxic A/B/C的具有β-内酰胺抗性:含连续稀释的卡那霉素(E.coliK)或四环素(E.coli T)(0.064、0.125、0.25、0.5、1、2、4、8、16、32和64μg/ml),包括空白(0μg/ml)。按照上述方法进行检测。
e、培养用于单分子检测法的细菌,对E.coli A进行检测,以E.coli K/T作为抗性对照,以E.coli B作为阴性对照。E.coli A在添加和不添加氨苄青霉素(4μg/mL)SOB培养基中培养,E.coli K/T在添加和不添加卡那霉素/四环素(4μg/mL)SOB培养基中培养,E.coliB在SOB培养基中培养,37℃培养至OD600=0.5。超声5min,8000rpm离心10min获得细胞裂解液,用于单分子检测。
f、确定耐药菌的检出限,首先验证了单分子检测法的选择性:加入E.coli A/K/T(104CFU/mL),37℃孵育30min,如图11c为不同目标物存在下的荧光斑点图,图中斑点计数后结果如图11b,选择性测试结果显示,该检测方法只对E.coli A(表达β-内酰胺酶)有高特异性响应,而对E.coli/K/T(不表达β-内酰胺酶)基本无响应;然后验证了细菌裂解液与Ab-DNF的最佳反应时间,E.coli A(4μg/mL氨苄青霉素培养,104CFU/mL)样品和阴性对照E.coli B(104CFU/mL),37℃孵育(5、10、15、20、25、30、40、50min),如图11a,分析结果证明在40min时结合效果最好;最后验证了耐药菌的检出限:加入20μL含E.coli A和E.coli B(100~104CFU/mL)的PBS缓冲液,37℃孵育30min。分析结果证明荧光斑点数与细菌裂解液的浓度成正相关,应用Ab-DNF单分子检测法对β-内酰胺类耐药菌的检出限为10CFU/mL。
g、临床耐药菌的检测。首先培养细菌:Toxic A/B/C在添加和不添加氨苄青霉素(4μg/mL)培养基中培养,37℃培养至OD600=0.5;然后应用于单分子检测法:加入Toxic A/B/C和阴性对照E.coli B(104CFU/mL),37℃孵育30min,如图13选择性测试结果显示,只对Toxic A/B/C(表达β-内酰胺酶)具有高特异性响应,而对E.coli B(不表达β-内酰胺酶)基本无响应。
SEQUENCE LISTING
<110> 大连理工大学
<120> 一种具有荧光信号放大功能的三维DNA微球及其制备方法与应用
<130> 2021
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 51
<212> DNA
<213> 人工序列(Artifical Sequence)
<400> 1
attcgtgtga gaaaacccaa cccgccctac ccaaaagata tcgtcagatg a 51
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artifical Sequence)
<400> 2
ctcacacgaa ttcatctgac 20
<210> 3
<211> 30
<212> DNA
<213> 人工序列(Artifical Sequence)
<400> 3
tttttttttt gtcagatgaa ttcgtgtgag 30
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artifical Sequence)
<400> 4
gtcagatgaa ttcgtgtgag 20
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artifical Sequence)
<400> 5
cccaaaagat atcgtcagat ga 22
<210> 6
<211> 51
<212> DNA
<213> 人工序列(Artifical Sequence)
<400> 6
tcatctgacg atatcttttg ggtagggcgg gttgggtttt ctcacacgaa t 51
Claims (9)
1.一种具有β-内酰胺酶抗体的三维DNA微球,其特征在于:所述具有β-内酰胺酶抗体的三维DNA微球具有荧光信号放大的功能,β-内酰胺酶抗体通过连接的可以与三维DNA微球碱基互补配对的DNA单链连接到三维DNA微球表面,形成可以特异性识别β-内酰胺酶的β-内酰胺酶抗体-三维DNA微球;所述三维DNA微球为含有数量为108~109条的如SEQ ID NO.6所示的重复单链形成的花状结构微球;所述可以与三维DNA微球碱基互补配对的DNA单链的核苷酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的具有β-内酰胺酶抗体的三维DNA微球,其特征在于:所述三维DNA微球的粒径为1~3μm。
3.权利要求1-2中任一项所述的具有β-内酰胺酶抗体的三维DNA微球的制备方法,其特征在于:包括如下步骤:
a、通过RCA反应制备三维DNA微球,所得三维DNA微球的粒径为1~3μm,呈花状结构,含稳定DNA单链及寡核苷酸互补配对结合位点;所得三维DNA微球为颗粒浓度为107~108个/mL,含重复单链数量108~109条的SEQ ID NO.6所示的核苷酸序列;
b、β-内酰胺酶抗体和与三维DNA微球碱基互补配对的SEQ ID NO.3所示的DNA单链连接,得Ab-LD;
c、加入如SEQ ID NO.4所示的封闭DNA将三维DNA微球表面多余寡核苷酸碱基互补配对结合位点封闭,然后将Ab-LD中的DNA单链通过互补配对连接到三维DNA微球表面,形成可以特异性识别β-内酰胺酶的具有β-内酰胺酶抗体的三维DNA微球。
4.权利要求1-2中任一项所述的具有β-内酰胺酶抗体的三维DNA微球在单分子检测中的应用。
5.根据权利要求4所述的应用,其特征在于:应用于β-内酰胺类耐药菌中的β-内酰胺酶的检测。
6.根据权利要求5所述的应用,其特征在于;β-内酰胺酶的检测方法,包括如下步骤:
(1)对β-内酰胺类耐药菌进行超声裂解,通过离心获得细菌裂解液,并用PBST缓冲液进行稀释;
(2)将2~15μg/mL包被缓冲液稀释的β-内酰胺酶抗体加入孔板中进行包被,包被完成后用PBST缓冲液洗涤;
(3)采用封闭缓冲液对孔板进行封闭,封闭完成后用PBST洗涤;
(4)将步骤(1)所得含有β-内酰胺类耐药菌的稀释液、具有β-内酰胺酶抗体的三维DNA微球和1×SYBR-Gold染料的PBST缓冲液加入孔板中,孵育后用PBST缓冲液洗涤;
(5)采用倒置荧光显微镜对孔板进行荧光成像,然后通过ImageJ软件对荧光斑点计数。
7.根据权利要求6所述的应用,其特征在于:所述步骤(1)中超声裂解的时间为5~10min,离心的转速为8000~10000rpm/min;所述步骤(2)中的包被缓冲液为pH 9.6的碳酸盐缓冲液,包被的条件为4~5℃孵育12~14h或37~39℃孵育2~2.5h;所述步骤(3)中封闭缓冲液是体积分数为5%~10%的BSA溶液或山羊血清,封闭的条件为37~39℃孵育60~120min;所述步骤(4)孵育的条件为37~39℃孵育30~60min。
8.根据权利要求5或6所述的应用,其特征在于:所述β-内酰胺类耐药菌包括E.coliA、Toxic A、Toxic B、Toxic C。
9.根据权利要求6所述的应用,其特征在于:步骤(2)中β-内酰胺酶抗体与步骤(4)中具有β-内酰胺酶抗体的三维DNA微球中的β-内酰胺酶抗体种类相同。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110153261.7A CN112946266B (zh) | 2021-02-03 | 2021-02-03 | 一种具有荧光信号放大功能的三维dna微球及其制备方法与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110153261.7A CN112946266B (zh) | 2021-02-03 | 2021-02-03 | 一种具有荧光信号放大功能的三维dna微球及其制备方法与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112946266A CN112946266A (zh) | 2021-06-11 |
CN112946266B true CN112946266B (zh) | 2024-03-12 |
Family
ID=76243682
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110153261.7A Active CN112946266B (zh) | 2021-02-03 | 2021-02-03 | 一种具有荧光信号放大功能的三维dna微球及其制备方法与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112946266B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5132242A (en) * | 1987-07-15 | 1992-07-21 | Cheung Sau W | Fluorescent microspheres and methods of using them |
CN103115903A (zh) * | 2013-01-16 | 2013-05-22 | 大连理工大学 | 一种微量四环素类抗生素的荧光检测方法 |
CN105603104A (zh) * | 2016-03-16 | 2016-05-25 | 福州大学 | 一种检测血清中循环microRNA的方法 |
CN112266915A (zh) * | 2020-10-22 | 2021-01-26 | 大连理工大学 | 一种用于检测艰难梭菌的核酸适配体及应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105764490B (zh) * | 2013-09-24 | 2020-10-09 | 加利福尼亚大学董事会 | 用于生物测定和诊断的胶囊封装的传感器和感测系统及其制造和使用方法 |
CN103994946A (zh) * | 2014-06-09 | 2014-08-20 | 厦门大学 | 基于气压检测的多种靶标的高灵敏定量分析方法 |
-
2021
- 2021-02-03 CN CN202110153261.7A patent/CN112946266B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5132242A (en) * | 1987-07-15 | 1992-07-21 | Cheung Sau W | Fluorescent microspheres and methods of using them |
CN103115903A (zh) * | 2013-01-16 | 2013-05-22 | 大连理工大学 | 一种微量四环素类抗生素的荧光检测方法 |
CN105603104A (zh) * | 2016-03-16 | 2016-05-25 | 福州大学 | 一种检测血清中循环microRNA的方法 |
CN112266915A (zh) * | 2020-10-22 | 2021-01-26 | 大连理工大学 | 一种用于检测艰难梭菌的核酸适配体及应用 |
Non-Patent Citations (3)
Title |
---|
Electrochemical immunoassay for thyroxine detection using cascade catalysis as signal amplified enhancer and multi-functionalized magnetic graphene sphere as signal tag;Jing Han 等;《Analytica Chimica Acta》;第790卷;第24-30页 * |
Ultrasensitive detection of T-2 toxin in food based on bio-barcode and rolling circle amplification;Man Zhang 等;《Analytica Chimica Acta》;第1043卷;第98-106页 * |
荧光探针信号放大策略及生物传感新方法研究;李春丽;《中国优秀硕士学位论文全文数据库 工程科技I辑》;正文第1-68页 * |
Also Published As
Publication number | Publication date |
---|---|
CN112946266A (zh) | 2021-06-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6457564B2 (ja) | エキソヌクレアーゼを用いた近接伸長アッセイ | |
EP1996941B1 (en) | Method for analyte detection using proximity probes | |
CN106170564B (zh) | 基于杂交链式反应(hcr)的检测的邻近试验 | |
EP2627781B1 (en) | Dynamic range methods | |
JP6043901B2 (ja) | 遮断試薬及びその使用のための方法 | |
Cai et al. | Single-digit Salmonella detection with the naked eye using bio-barcode immunoassay coupled with recombinase polymerase amplification and a CRISPR-Cas12a system | |
CN112725343A (zh) | 联合金纳米探针和CRISPR-Cas的蛋白标志物检测试剂盒及检测方法 | |
You et al. | A fast and ultrasensitive ELISA based on rolling circle amplification | |
CN108624653B (zh) | 一种用于血流感染病原体的量子点核酸检测的试剂盒 | |
CN112946266B (zh) | 一种具有荧光信号放大功能的三维dna微球及其制备方法与应用 | |
US6596496B1 (en) | Analytical system based upon spore germination | |
Gong et al. | Combination of functionalized nanoparticles and polymerase chain reaction-based method for SARS-CoV gene detection | |
JP2023171748A (ja) | 近接検出アッセイのための制御 | |
US20210261953A1 (en) | Method to perform high-throughput single cell genomic and phenotypic analyses | |
US20220090174A1 (en) | Detection method for a target nucleic acid and kit | |
KR20120055224A (ko) | 핵산효소-분자비콘을 이용한 핵산의 검출방법 | |
WO2023059731A2 (en) | Single molecule assays for ultrasensitive detection of analytes | |
CN117512072A (zh) | 一种核酸分子的免扩增检测方法、试剂盒及其应用 | |
CN117295947A (zh) | 用于检测分析的多重标记的聚合物构建体 | |
CN115290883A (zh) | 多种外泌体蛋白的同步检测方法 | |
US20080020382A1 (en) | Biological Microbeads for Various Flow Cytometric Applications | |
Eklund | Multiplex protein analysis by proximity ligation assay with microarray analysis |
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 |