CN115541880A - 基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法、材料及应用 - Google Patents
基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法、材料及应用 Download PDFInfo
- Publication number
- CN115541880A CN115541880A CN202211229579.XA CN202211229579A CN115541880A CN 115541880 A CN115541880 A CN 115541880A CN 202211229579 A CN202211229579 A CN 202211229579A CN 115541880 A CN115541880 A CN 115541880A
- Authority
- CN
- China
- Prior art keywords
- solution
- hkust
- washing
- pbs
- well
- 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
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 31
- 239000000427 antigen Substances 0.000 title claims abstract description 31
- 108091007433 antigens Proteins 0.000 title claims abstract description 31
- 102000036639 antigens Human genes 0.000 title claims abstract description 31
- 239000000463 material Substances 0.000 title claims abstract description 30
- 108010029541 Laccase Proteins 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 21
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 32
- 238000002965 ELISA Methods 0.000 claims abstract description 31
- 239000000523 sample Substances 0.000 claims abstract description 14
- 239000011148 porous material Substances 0.000 claims abstract description 11
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 5
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000010949 copper Substances 0.000 claims description 86
- 239000000243 solution Substances 0.000 claims description 80
- 239000013148 Cu-BTC MOF Substances 0.000 claims description 65
- 238000005406 washing Methods 0.000 claims description 60
- 241001678559 COVID-19 virus Species 0.000 claims description 34
- 239000008055 phosphate buffer solution Substances 0.000 claims description 32
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 238000003756 stirring Methods 0.000 claims description 24
- QMKYBPDZANOJGF-UHFFFAOYSA-N benzene-1,3,5-tricarboxylic acid Chemical compound OC(=O)C1=CC(C(O)=O)=CC(C(O)=O)=C1 QMKYBPDZANOJGF-UHFFFAOYSA-N 0.000 claims description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 13
- 238000002835 absorbance Methods 0.000 claims description 12
- 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 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 8
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 8
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 8
- 239000002243 precursor Substances 0.000 claims description 8
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 5
- 239000011668 ascorbic acid Substances 0.000 claims description 5
- 229960005070 ascorbic acid Drugs 0.000 claims description 5
- 235000010323 ascorbic acid Nutrition 0.000 claims description 5
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000006185 dispersion Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 239000008188 pellet Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 230000000007 visual effect Effects 0.000 abstract description 5
- 230000003197 catalytic effect Effects 0.000 abstract description 4
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 abstract 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 24
- 108090000790 Enzymes Proteins 0.000 description 20
- 102000004190 Enzymes Human genes 0.000 description 20
- 239000002131 composite material Substances 0.000 description 10
- 108091006197 SARS-CoV-2 Nucleocapsid Protein Proteins 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 239000002114 nanocomposite Substances 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 5
- 238000002441 X-ray diffraction Methods 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102100023321 Ceruloplasmin Human genes 0.000 description 2
- 208000000059 Dyspnea Diseases 0.000 description 2
- 206010013975 Dyspnoeas Diseases 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012800 visualization Methods 0.000 description 2
- HNXGGWNCFXZSAI-UHFFFAOYSA-N 2-morpholin-2-ylethanesulfonic acid Chemical compound OS(=O)(=O)CCC1CNCCO1 HNXGGWNCFXZSAI-UHFFFAOYSA-N 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000956 alloy Substances 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 239000011162 core material Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012621 metal-organic framework Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 208000013220 shortness of breath Diseases 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
- B01J31/1691—Coordination polymers, e.g. metal-organic frameworks [MOF]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J35/00—Catalysts, in general, characterised by their form or physical properties
- B01J35/20—Catalysts, in general, characterised by their form or physical properties characterised by their non-solid state
- B01J35/23—Catalysts, in general, characterised by their form or physical properties characterised by their non-solid state in a colloidal state
-
- 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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
- B01J2531/02—Compositional aspects of complexes used, e.g. polynuclearity
- B01J2531/0213—Complexes without C-metal linkages
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
- B01J2531/10—Complexes comprising metals of Group I (IA or IB) as the central metal
- B01J2531/16—Copper
-
- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
- G01N2470/04—Sandwich assay format
- G01N2470/06—Second binding partner specifically binding complex of analyte with first binding partner
-
- 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)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Materials Engineering (AREA)
- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Inorganic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法、材料及应用,其方法主要是使用96孔板依次将新型冠状病毒N蛋白抗体、牛血清白蛋白溶液、新型冠状病毒抗原溶液和Ab2生物探针孵育后,利用生物探针对2,4‑二氯苯酚和4‑氨基安替比林的催化显色反应制备出检测新型冠状病毒的ELISA生物免疫传感器;该生物传感器具有设备简单、无需专业人员操作、低成本、灵敏度较高、可视化检测、较宽的检测范围和较低的检出限等优点。对新型冠状病毒的检测具有重要的科学意义和临床应用价值。
Description
技术领域
本发明属于分析化学、材料和生物传感技术领域,具体涉及一种新型冠状病毒的可视化ELISA检测方法、免疫分析和生物传感技术领域,具体是 SARS-CoV-2作为目标分析物,采用Cu2O/HKUST-1的纳米复合材料标记二抗来构建出一种夹心型免疫传感器。
背景技术
新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2)是一种线性单链RNA(ssRNA)病毒,基因组全长约29903个核苷酸,共包含10个基因,人感染了冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中,感染可导致肺炎、严重急性呼吸综合征、肾衰竭,甚至死亡,同时,研究表明SARS-CoV-2具有高传染性和高隐蔽性,对公众安全存在极大的威胁。因此准确快速检测是否感染SARS-CoV-2对患者及早治疗、保护公众安全和控制疫情传播具有重要的意义。
目前实时荧光定量PCR(RT-PCR)仍然是最常使用检测是否感染 SARS-CoV-2方法,具有高灵敏度、低样本需求量、高通量等优点,但其所用设备昂贵,成本较高、且该方法需要专业技术人员,故应用可能受限。另一常用的检测方法是传统ELISA法,主要以抗原抗体反应为依据,利用二抗上偶联的辣根过氧化物酶(HRP)催化底物显色,实现可视化检测。但由于天然酶在非生理条件下不稳定,容易变性失活,同时天然酶可能存在价格较贵等问题,使得传统的ELISA方法在特异性和灵敏度方面有所欠佳。
在本发明中,Cu-MOFs复合材料Cu2O/HKUST-1作为一种新型复合纳米酶材料,合成简单,能很好分散为水中,稳定性好,且具有结合抗体、适配体、多肽等具有识别作用的生物分子等特点,可与SARS-CoV-2抗体结合,而且 Cu2O/HKUST-1具有很好的漆酶活性,可较快催化2,4-二氯苯酚(2,4-DP)和4- 氨基安替比林(4-AP)反应显色。
SARS-CoV-2Nucleocapsid Protein(SARS-CoV-2N蛋白)是SARS-CoV-2 的衣壳蛋白,主要负责RNA的复制,衣壳蛋白常作为冠状病毒诊断检测工具,是免疫学快速诊断试剂的核心原料。因此,我们利用N蛋白对抗体可以特异性识别结合SARS-CoV-2的特点与复合纳米酶材料共同作用构建一个操作方便、低成本、高灵敏度、可视化以及高通量检测SARS-CoV-2的ELISA检测方法。
发明内容
针对当前新型冠状病毒抗原检测成本高、需专业设备及操作人员、等问题,本发明提出一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法,所述的新冠病毒抗原检测方法包括如下步骤:
步骤1)将Ab1滴加到96孔板中,并在冰箱中孵育过夜,然后用PBS溶液洗去未结合的Ab1;
步骤2)将牛血清蛋白添加到步骤(1)处理好的孔板中,在25℃下孵育 10-150min;孵育结束后,然后用PBS溶液清洗孔板;
步骤3)在步骤(2)处理好的孔板中加入不同浓度的SARS-CoV-2,在25℃下孵育10-150min,然后用PBS溶液清洗96孔板;
步骤4)在步骤(3)处理好的孔板中加入Ab2/Cu2O/HKUST-1生物探针材料,并在25℃下反应10-150min,并将未结合的生物探针用PBS溶液去除;
步骤5)分别将2,4-DP和4-AP滴加到步骤(4)处理好的板中,室温下反应 0.5-3h,用紫外分光光度计测量其在510nm处的吸光度值。
本发明所述的新冠病毒抗原检测方法包括的步骤具体为:步骤1)将50μL 的0.1-1.5μg·mL-1Ab1滴加到96孔板中,并在冰箱中孵育过夜,然后用含0.05%-5%吐温-20的PBS溶液洗去未结合的Ab1;
步骤2)将50μL 0.1-3%的牛血清蛋白添加到步骤(1)处理好的孔板中,在25℃下孵育10-150min;孵育结束后,然后用含0.05%-5%吐温-20的PBS 溶液清洗孔板;
步骤3)在步骤(2)处理好的孔板中加入50μL不同浓度的SARS-CoV-2,在25℃下孵育10-150min,然后用含0.05%-5%吐温-20的PBS溶液清洗96孔板;
步骤4)在步骤(3)处理好的孔板中加入50μL的0.1-2.5mg·mL-1 Ab2/Cu2O/HKUST-1生物探针材料,并在25℃下反应10-150min,并将未结合的生物探针用0.05%-5%吐温-20的PBS溶液去除;
步骤5)分别将50μL的0.5-5mg·mL-1的2,4-DP和50μL的0.5-5mg·mL-1 4-AP滴加到步骤(4)处理好的板中,室温下反应0.5-3h,用紫外分光光度计测量其在510nm处的吸光度值。
本发明所述步骤(4)制备Ab2/Cu2O/HKUST-1生物探针材料的步骤包括:
将SARS-CoV-2特异性抗体加入到Cu2O/HKUST-1分散液中并在4℃条件下搅拌过夜;用PBS洗掉游离的抗体后,加入BSA反应1-15h;将所得溶液离心分离并将沉淀分散在PBS中以获得Ab2/Cu2O/HKUST-1生物探针材料,将其储存在4℃备用。
本发明所述步骤(4)制备Ab2/Cu2O/HKUST-1生物探针材料,包括以下具体步骤:
将10μL 1-5mg·mL-1的SARS-CoV-2特异性抗体加入到1mL 0.1-2.5 mg·mL-1的Cu2O/HKUST-1分散液中并在4℃条件下搅拌过夜;用PBS洗掉游离的抗体后,加入1%BSA100μL反应1-15h;将所得溶液离心分离并将沉淀分散在0.1-1.5mL的PBS中以获得0.1-2.5mg·mL-1的Ab2/Cu2O/HKUST-1生物探针材料,将其储存在4℃备用。
本发明所述Cu2O/HKUST-1的制备方法,包括如下步骤:
(1)将CuCl2·2H2O溶解在去离子水中以形成均匀的溶液,搅拌条件下,将溶液加热至25-75℃,并继续搅拌0.5-2h,随后加入NaOH溶液,并在25-75℃条件下继续搅拌0.5-2h,然后加入抗坏血酸溶液,并在25-75℃下继续搅拌1-5 h,离心收集,用无水乙醇洗涤干燥获得Cu2O NCs;
(2)称取Cu2O NCs和1,3,5-苯三甲酸分散于二甲基亚枫中,超声得到均匀的前体溶液;取上述前体溶液,加入到甲醇溶液中,室温下搅拌5-20h,离心收集,用甲醇洗涤干燥获得Cu2O/HKUST-1。
本发明所述Cu2O/HKUST-1的制备方法,包括以下具体步骤:
(1)将150-200mg的CuCl2·2H2O溶解在50-100mL去离子水中以形成均匀的溶液,搅拌条件下,将溶液加热至25-75℃,并继续搅拌0.5-2h,随后加入 1-10mL 0.2-2M的NaOH溶液,并在25-75℃条件下继续搅拌0.5-2h,然后加入1-10mL 0.2-0.6M的抗坏血酸溶液,并在25-75℃下继续搅拌1-5h,离心收集,用无水乙醇洗涤干燥获得Cu2O NCs;
(2)称取30-100mg Cu2O NCs和100-200mg的1,3,5-苯三甲酸分散于1-5 g的二甲基亚枫中,超声得到均匀的前体溶液,取上述50-300μL的前体溶液,加入到5-20mL的甲醇溶液中,室温下搅拌5-20h,离心收集,用甲醇洗涤干燥获得Cu2O/HKUST-1。
随后对上述制备得到的Cu2O NCs和Cu2O/HKUST-1纳米材料进行透射电子显微镜(TEM,图1A、B)、扫描电子显微镜(SEM,图2A、B)、X射线衍射 (XRD,图3A、B)、Zeta电位(图4A)、X射线光电子能谱(XPS,图4B) 和SEM的Mapping(图5)的表征,分别获得Cu2O/HKUST-1纳米材料的形貌以及结构、三维形貌及尺寸、晶体结构、颗粒表面电荷密度、材料表面元素及其化学态和元素分布的信息,从而证明Cu2O/HKUST-1纳米材料的制备成功。
一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测材料,所述的材料为Cu2O/HKUST-1。
本发明所述材料具有漆酶活性;与SARS-CoV-2抗体结合;催化2,4-二氯苯酚(2,4-DP)和4-氨基安替比林(4-AP)反应显色。
本发明基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法的应用,该应用检测新冠病毒抗原的范围为0-100ng·mL-1,检出限(LOD)达到0.105 fg/mL(S/N=3)。
本发明应用为制备ELISA传感器,且该ELISA传感器的使用pH为6.8。
本发明的有益成果
(1)本发明通过以CuCl2·2H2O、NaOH溶液、抗坏血酸溶液、1,3,5-苯三甲酸作为原料,在温和条件下制备了Cu2O/HKUST-1,然后利用Cu元素作为锚定生物识别物质特异性抗体的位点,合成了探针材料Ab2/Cu2O/HKUST-1,以 Cu2O/HKUST-1对2,4-DP和4-AP的催化显色反应,使用96孔板成功构建了三明治型ELISA传感器,实现了对SARS-CoV-2的检测。改进ELISA传感器的检测范围为0-100ng·mL-1,检出限(LOD)达到了0.105fg/mL(S/N=3),表现出了较高的灵敏度,为SARS-CoV-2的检测提供了一个低廉、快速、灵敏和可视化的方法。
(2)本发明使用的酶促比色信号读数。
(3)本发明使用了Cu2O/HKUST-1,对2,4-DP和4-AP的显色有很好的催化效果。
(4)本发明把改进ELISA与比色相结合,用来检测SARS-CoV-2。该方法具有操作方便、低成本、高灵敏度、可视化以及高通量检测等优点,有望用于实际样品的快速检测。
(5)本设备简单、无需专业人员操作、低成本、灵敏度较高、可视化检测、较宽的检测范围和较低的检出限等优点。对SARS-CoV-2的检测具有重要的科学意义和临床应用价值。
附图说明
图1为Cu2O NCs(A)、Cu2O/HKUST-1(B)的TEM图;
图2为Cu2O NCs(A)、Cu2O/HKUST-1(B)的SEM图;
图3为Cu2O NCs(A)、Cu2O/HKUST-1(B)的XRD图;
图4为Cu2O/HKUST-1的Zeta电位图(A)和XPS图(B);
图5为Cu2O/HKUST-1的Mapping图;
图6为传感器的pH筛选图;
图7为不同浓度SARS-CoV-2的紫外吸收谱图(A)和标准曲线(B)。
具体实施方式
实施例中所使用的化学试剂和溶剂均为分析纯;原材料均可在化学试剂公司或生物制药公司购买;所述搅拌采用磁力搅拌器搅拌方式。
实施例1:
一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法具体步骤如下:
(1)用PBS缓冲液稀释SARS-CoV-2N蛋白的一抗(Ab1)至1μg/mL,每孔50μL包被于96孔酶标板,4℃孵育过夜;
(2)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 1%BSA,室温封闭30min;
(3)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL浓度为0-100 ng/mL不同浓度的SARS-CoV-2溶液,室温孵育1h;
(4)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 1mg/mL Ab2@ Cu2O/HKUST-1纳米复合探针,室温孵育1h;
(5)用PBST洗涤液清洗孔酶标板3次,每孔加入适量2-吗啉乙磺酸缓冲溶液(MES),2,4-DP,4-AP,总体积200μL,室温下反应0.5h,用紫外分光光度计检测510nm处的吸光度值,绘制标准曲线。
实施例2:
一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法的具体步骤如下:
(1)用PBS缓冲液稀释SARS-CoV-2N蛋白的一抗(Ab1)至2μg/mL,每孔50μL包被于96孔酶标板,4℃孵育过夜;
(2)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 2%BSA,室温封闭50min;
(3)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL浓度为20ng/mL 的SARS-CoV-2溶液,室温孵育1h;
(4)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 1mg/mL Ab2@ Cu2O/HKUST-1纳米复合探针,室温孵育1.5h;
(5)用PBST洗涤液清洗孔酶标板3次,每孔加入适量2-吗啉乙磺酸缓冲溶液(MES),2,4-DP,4-AP,总体积200μL,室温下反应1h,用紫外分光光度计检测510nm处的吸光度值,根据绘制的标准曲线计算得到新冠病毒抗原的浓度为19.81ng/mL。
实施例3:
一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法的具体步骤如下:
(1)用PBS缓冲液稀释SARS-CoV-2N蛋白的一抗(Ab1)至0.5μg/mL,每孔50μL包被于96孔酶标板,4℃孵育过夜;
(2)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 1%BSA,室温封闭40min;
(3)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL浓度为10ng/mL 的SARS-CoV-2溶液,室温孵育1h;
(4)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 1mg/mL Ab2@ Cu2O/HKUST-1纳米复合探针,室温孵育1h;
(5)用PBST洗涤液清洗孔酶标板3次,每孔加入适量2-吗啉乙磺酸缓冲溶液(MES),2,4-DP,4-AP,总体积300μL,室温下反应1h,用紫外分光光度计检测510nm处的吸光度值,根据绘制的标准曲线计算得到新冠病毒抗原的浓度为9.75ng/mL。
实施例4:
一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法的具体步骤如下:
(1)用PBS缓冲液稀释SARS-CoV-2N蛋白的一抗(Ab1)至2.5μg/mL,每孔50μL包被于96孔酶标板,4℃孵育过夜;
(2)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 4%BSA,室温封闭20min;
(3)用PBST洗涤液清洗96孔酶标板3次,每孔加入100μL浓度为0.1ng/mL 的SARS-CoV-2溶液,室温孵育1h;
(4)用PBST洗涤液清洗96孔酶标板3次,每孔加入100μL 1mg/mL Ab2@ Cu2O/HKUST-1纳米复合探针,室温孵育1h;
(5)用PBST洗涤液清洗孔酶标板3次,每孔加入适量2-吗啉乙磺酸缓冲溶液(MES),2,4-DP,4-AP,总体积300μL,室温下反应1h,用紫外分光光度计检测510nm处的吸光度值,根据绘制的标准曲线计算得到新冠病毒抗原的浓度为0.105ng/mL。
实施例5:
一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法的具体步骤如下:
(1)用PBS缓冲液稀释SARS-CoV-2N蛋白的一抗(Ab1)至2.5μg/mL,每孔50μL包被于96孔酶标板,4℃孵育过夜;
(2)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 1%BSA,室温封闭50min;
(3)用PBST洗涤液清洗96孔酶标板3次,每孔加入150μL浓度为80ng/mL 的SARS-CoV-2溶液,室温孵育1h;
(4)用PBST洗涤液清洗96孔酶标板3次,每孔加入150μL 1mg/mL Ab2@ Cu2O/HKUST-1纳米复合探针,室温孵育1.5h;
(5)用PBST洗涤液清洗孔酶标板3次,每孔加入适量2-吗啉乙磺酸缓冲溶液(MES),2,4-DP,4-AP,总体积300μL,室温下反应1h,用紫外分光光度计检测510nm处的吸光度值,根据绘制的标准曲线计算得到新冠病毒抗原的浓度为81.25ng/mL。
实施例6:
一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法的具体步骤如下:
(1)用PBS缓冲液稀释SARS-CoV-2N蛋白的一抗(Ab1)至0.8μg/mL,每孔50μL包被于96孔酶标板,4℃孵育过夜;
(2)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 2.5%的BSA溶液,室温封闭30min;
(3)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL浓度为0.01ng/mL 的SARS-CoV-2溶液,室温孵育1h;
(4)用PBST洗涤液清洗96孔酶标板3次,每孔加入50μL 1mg/mL Ab2@ Cu2O/HKUST-1纳米复合探针,室温孵育0.5h;
(5)用PBST洗涤液清洗孔酶标板3次,每孔加入适量2-吗啉乙磺酸缓冲溶液(MES),2,4-DP,4-AP,总体积400μL,室温下反应1h,用紫外分光光度计检测510nm处的吸光度值,根据绘制的标准曲线计算得到新冠病毒抗原的浓度为0.0093ng/mL。
图1为Cu2O NCs(A)、Cu2O/HKUST-1(B)的透射电镜(TEM)图;其中Cu2O NCs正方体结构(图A);加入1,3,5-苯三甲酸,反应结束后,沿着Cu2O NCs立方体表面生长出很多明显的菱形结构(图B),这些结果也说明Cu2O/HKUST-1 复合材料的成功制备。
图2为Cu2O NCs(A)、Cu2O/HKUST-1(B)的扫描电子显微镜(SEM)图;其中Cu2O NCs正方体结构(图A);加入1,3,5-苯三甲酸,反应结束后,沿着Cu2O NCs立方体表面长出明显的八面体结构(图B)。这些结果也表明Cu2O/HKUST-1 复合材料的成功制备。
图3为Cu2O NCs(A)、Cu2O/HKUST-1(B)的X射线衍射技术(XRD)图。对照Cu2O的标准卡片,图A中29.6°,36.4°、42.3°和61.4°的衍射峰分别归属于Cu2O的(110),(111),(200)和(220)晶面,证明Cu2O的成功制备,图 B中,在6.7°,9.5°,11.6°,13.4°,19°的衍射峰分别归属于HKUST-1的(200), (220),(222),(400)和(600)晶面,同时存在Cu2O的典型晶面,表明Cu2O/HKUST-1材料的成功制备。
图4为Cu2O/HKUST-1的Zeta电位图(A)和XPS图(B),图A表明, Cu2O NCs的Zeta电位为负值,HKUST-1的Zeta电位为负值,Cu2O/HKUST-1 复合材料的电位也为负值,证明复合材料的成功制备;图B表明Cu2O/HKUST-1 复合材料中含有Cu、O和C元素,Cu和O属于Cu2O和HKUST-1材料,C元素属于HKUST-1,也进一步说明复合材料的成功制备。
图5为Cu2O/HKUST-1材料的SEM的Mapping图,图5显示,Cu2O/HKUST-1 材料中含有Cu、O和C元素,与XPS的结果一致,同时,O元素在整个材料中都比较明显,因为Cu2O和HKUST-1中都含有O元素;C元素主要在八面体结构中,是由于C元素只在HKUST-1结构中出现,Cu元素在Cu2O立方体结构更为明显,可能是由于Cu2O中Cu元素含量更高,上述结果也表明 Cu2O/HKUST-1复合材料的成功制备。
图6为ELISA传感器的pH筛选图,pH对纳米酶的催化活性有很大的影响,为了获得最佳的漆酶活性,对反应的pH进行筛选,结果显示,pH为6.8时,纳米酶具有最优的催化活性。
图7为所制备的ELISA不同浓度的紫外吸收谱图以及标准曲线。将所构建的ELISA使用分光光度计在510nm条件下来对不同浓度的SARS-CoV-2进行检测,随着SARS-CoV-2浓度的增加,吸光度值逐渐增加(图7A)。如图7B所示,吸光度与SARS-CoV-2浓度的对数具有良好的线性关系,相关系数(R2)为0.9922, LOD为0.105fg·mL-1(S/N=3),显示出了良好的线性和较低的LOD值。
以上所述的仅是本发明的部分具体实施例(由于本发明的配方包含数值范围,故实施例不能穷举,本发明所记载的保护范围包含本发明的数值范围和其他技术要点范围),方案中公知的具体内容或常识在此未作过多描述(包括但不仅限于简写、缩写)。应当指出,上述实施例不以任何方式限制本发明,对于本领域的技术人员来说,凡是采用等同替换或等效变换的方式获得的技术方案均落在本发明的保护范围内。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (10)
1.一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法,其特征在于,所述的新冠病毒抗原检测方法包括如下步骤:
步骤1)将Ab1滴加到96孔板中,并在冰箱中孵育过夜,然后用PBS溶液洗去未结合的Ab1;
步骤2)将牛血清蛋白添加到步骤(1)处理好的孔板中,在25℃下孵育10-150min;孵育结束后,然后用PBS溶液清洗孔板;
步骤3)在步骤(2)处理好的孔板中加入不同浓度的SARS-CoV-2,在25℃下孵育10-150min,然后用PBS溶液清洗96孔板;
步骤4)在步骤(3)处理好的孔板中加入Ab2/Cu2O/HKUST-1生物探针材料,并在25℃下反应10-150min,并将未结合的生物探针用PBS溶液去除;
步骤5)分别将2,4-DP和4-AP滴加到步骤(4)处理好的板中,室温下反应0.5-3h,用紫外分光光度计测量其在510nm处的吸光度值。
2.根据权利要求1所述的一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法,其特征在于,所述的新冠病毒抗原检测方法包括的步骤具体为:步骤1)将50μL的0.1-1.5μg·mL-1Ab1滴加到96孔板中,并在冰箱中孵育过夜,然后用含0.05%-5%吐温-20的PBS溶液洗去未结合的Ab1;
步骤2)将50μL 0.1-3%的牛血清蛋白添加到步骤(1)处理好的孔板中,在25℃下孵育10-150min;孵育结束后,然后用含0.05%-5%吐温-20的PBS溶液清洗孔板;
步骤3)在步骤(2)处理好的孔板中加入50μL不同浓度的SARS-CoV-2,在25℃下孵育10-150min,然后用含0.05%-5%吐温-20的PBS溶液清洗96孔板;
步骤4)在步骤(3)处理好的孔板中加入50μL的0.1-2.5mg·mL-1Ab2/Cu2O/HKUST-1生物探针材料,并在25℃下反应10-150min,并将未结合的生物探针用0.05%-5%吐温-20的PBS溶液去除;
步骤5)分别将50μL的0.5-5mg·mL-1的2,4-DP和50μL的0.5-5mg·mL-14-AP滴加到步骤(4)处理好的板中,室温下反应0.5-3h,用紫外分光光度计测量其在510nm处的吸光度值。
3.根据权利要求1所述的一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法,其特征在于,所述步骤(4)制备Ab2/Cu2O/HKUST-1生物探针材料的步骤包括:
将SARS-CoV-2特异性抗体加入到Cu2O/HKUST-1分散液中并在4℃条件下搅拌过夜;用PBS洗掉游离的抗体后,加入BSA反应1-15h;将所得溶液离心分离并将沉淀分散在PBS中以获得Ab2/Cu2O/HKUST-1生物探针材料,将其储存在4℃备用。
4.根据权利要求2所述的一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法,其特征在于,所述步骤(4)制备Ab2/Cu2O/HKUST-1生物探针材料,包括以下具体步骤:
将10μL 1-5mg·mL-1的SARS-CoV-2特异性抗体加入到1mL 0.1-2.5mg·mL-1的Cu2O/HKUST-1分散液中并在4℃条件下搅拌过夜;用PBS洗掉游离的抗体后,加入1%BSA 100μL反应1-15h;将所得溶液离心分离并将沉淀分散在0.1-1.5mL的PBS中以获得0.1-2.5mg·mL-1的Ab2/Cu2O/HKUST-1生物探针材料,将其储存在4℃备用。
5.根据权利要求3所述的一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法,其特征在于,所述Cu2O/HKUST-1的制备方法,包括如下步骤:
(1)将CuCl2·2H2O溶解在去离子水中以形成均匀的溶液,搅拌条件下,将溶液加热至25-75℃,并继续搅拌0.5-2h,随后加入NaOH溶液,并在25-75℃条件下继续搅拌0.5-2h,然后加入抗坏血酸溶液,并在25-75℃下继续搅拌1-5h,离心收集,用无水乙醇洗涤干燥获得Cu2O NCs;
(2)称取Cu2O NCs和1,3,5-苯三甲酸分散于二甲基亚枫中,超声得到均匀的前体溶液;取上述前体溶液,加入到甲醇溶液中,室温下搅拌5-20h,离心收集,用甲醇洗涤干燥获得Cu2O/HKUST-1。
6.根据权利要求4所述的一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法,其特征在于,所述Cu2O/HKUST-1的制备方法,包括以下具体步骤:
(1)将150-200mg的CuCl2·2H2O溶解在50-100mL去离子水中以形成均匀的溶液,搅拌条件下,将溶液加热至25-75℃,并继续搅拌0.5-2h,随后加入1-10mL 0.2-2M的NaOH溶液,并在25-75℃条件下继续搅拌0.5-2h,然后加入1-10mL 0.2-0.6M的抗坏血酸溶液,并在25-75℃下继续搅拌1-5h,离心收集,用无水乙醇洗涤干燥获得Cu2O NCs;
(2)称取30-100mg Cu2O NCs和100-200mg的1,3,5-苯三甲酸分散于1-5g的二甲基亚枫中,超声得到均匀的前体溶液,取上述50-300μL的前体溶液,加入到5-20mL的甲醇溶液中,室温下搅拌5-20h,离心收集,用甲醇洗涤干燥获得Cu2O/HKUST-1。
7.一种基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测材料,其特征在于,所述的材料为Cu2O/HKUST-1。
8.根据权利要求7所述的材料,其特征在于,所述材料具有漆酶活性;与SARS-CoV-2抗体结合;催化2,4-二氯苯酚2,4-DP和4-氨基安替比林4-AP反应显色。
9.根据权利要求2所述的基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法的应用,其特征在于,所述应用为,检测新冠病毒抗原的范围为0-100ng·mL-1,检出限LOD达到0.105fg/mL S/N=3。
10.根据权利要求9所述的应用,其特征在于,所述应用为制备ELISA传感器,且该ELISA传感器的使用pH为6.8。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211229579.XA CN115541880B (zh) | 2022-10-08 | 2022-10-08 | 基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法、材料及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211229579.XA CN115541880B (zh) | 2022-10-08 | 2022-10-08 | 基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法、材料及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115541880A true CN115541880A (zh) | 2022-12-30 |
CN115541880B CN115541880B (zh) | 2023-09-22 |
Family
ID=84732712
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211229579.XA Active CN115541880B (zh) | 2022-10-08 | 2022-10-08 | 基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法、材料及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115541880B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117599807A (zh) * | 2023-11-22 | 2024-02-27 | 云南大学 | 一种基于三金属纳米酶构建比色传感器检测新城疫病毒的方法 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107764763A (zh) * | 2017-10-03 | 2018-03-06 | 云南师范大学 | 碘离子信号增强的双氧水比色检测方法 |
CN111879920A (zh) * | 2020-08-06 | 2020-11-03 | 扬州大学 | 一种基于单金属Cu-MOF模拟酶的多组分无标记的免疫传感器 |
CN112213497A (zh) * | 2020-09-24 | 2021-01-12 | 杭州医学院 | 检测新型冠状病毒s蛋白独特性抗体的多肽-elisa试剂盒 |
CN112473750A (zh) * | 2020-11-12 | 2021-03-12 | 陕西科技大学 | 一种具有类漆酶活性的纳米酶及其制备方法和应用 |
CN112495444A (zh) * | 2020-12-18 | 2021-03-16 | 吉林师范大学 | 一种TiO2@HKUST-1复合光催化剂制备方法及应用 |
CN113349221A (zh) * | 2021-06-07 | 2021-09-07 | 中山大学 | 一种纳米复合材料Cu2O@HKUST-1及其制备方法和应用 |
CN114113582A (zh) * | 2021-12-20 | 2022-03-01 | 云南大学 | 金属有机框架纳米酶生物探针和elisa试剂盒 |
CN114289066A (zh) * | 2021-12-29 | 2022-04-08 | 云南大学 | 一种纳米模拟酶材料及其制备方法和应用、检测卵类粘蛋白的方法 |
-
2022
- 2022-10-08 CN CN202211229579.XA patent/CN115541880B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107764763A (zh) * | 2017-10-03 | 2018-03-06 | 云南师范大学 | 碘离子信号增强的双氧水比色检测方法 |
CN111879920A (zh) * | 2020-08-06 | 2020-11-03 | 扬州大学 | 一种基于单金属Cu-MOF模拟酶的多组分无标记的免疫传感器 |
CN112213497A (zh) * | 2020-09-24 | 2021-01-12 | 杭州医学院 | 检测新型冠状病毒s蛋白独特性抗体的多肽-elisa试剂盒 |
CN112473750A (zh) * | 2020-11-12 | 2021-03-12 | 陕西科技大学 | 一种具有类漆酶活性的纳米酶及其制备方法和应用 |
CN112495444A (zh) * | 2020-12-18 | 2021-03-16 | 吉林师范大学 | 一种TiO2@HKUST-1复合光催化剂制备方法及应用 |
CN113349221A (zh) * | 2021-06-07 | 2021-09-07 | 中山大学 | 一种纳米复合材料Cu2O@HKUST-1及其制备方法和应用 |
CN114113582A (zh) * | 2021-12-20 | 2022-03-01 | 云南大学 | 金属有机框架纳米酶生物探针和elisa试剂盒 |
CN114289066A (zh) * | 2021-12-29 | 2022-04-08 | 云南大学 | 一种纳米模拟酶材料及其制备方法和应用、检测卵类粘蛋白的方法 |
Non-Patent Citations (1)
Title |
---|
FANGQI WANG等: "Nanozymes based on metal-organic frameworks: Construction and prospects", TRENDS IN ANALYTICAL CHEMISTRY, pages 1 - 50 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117599807A (zh) * | 2023-11-22 | 2024-02-27 | 云南大学 | 一种基于三金属纳米酶构建比色传感器检测新城疫病毒的方法 |
Also Published As
Publication number | Publication date |
---|---|
CN115541880B (zh) | 2023-09-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Chen et al. | “Three-in-one” multifunctional nanohybrids with colorimetric magnetic catalytic activities to enhance immunochromatographic diagnosis | |
Laocharoensuk | Development of Electrochemical Immunosensors towards Point‐of‐care Cancer Diagnostics: Clinically Relevant Studies | |
Wang et al. | Nanobody and nanozyme‐enabled immunoassays with enhanced specificity and sensitivity | |
Tan et al. | Ratiometric fluorescent immunoassay for the cardiac troponin-I using carbon dots and palladium-iridium nanocubes with peroxidase-mimicking activity | |
CN114594262B (zh) | 基于双功能融合蛋白的真菌毒素磁化学发光免疫分析试剂盒及其应用 | |
CN105891189B (zh) | 一种铜离子检测试剂盒及其应用 | |
Li et al. | Manganese dioxide nanoparticle-based colorimetric immunoassay for the detection of alpha-fetoprotein | |
CN103913573A (zh) | 一种基于纳米金和氧化石墨烯的双信号放大elisa检测方法 | |
CN115541880A (zh) | 基于铜金属有机框架纳米酶漆酶的新冠病毒抗原检测方法、材料及应用 | |
Zuo et al. | Rapid detection of severe fever with thrombocytopenia syndrome virus via colloidal gold immunochromatography assay | |
CN115469105B (zh) | 一种一步法非洲猪瘟elisa抗体快速检测试剂盒 | |
CN114113582B (zh) | 金属有机框架纳米酶生物探针和elisa试剂盒 | |
Sun et al. | A colorimetric method for determination of the prostate specific antigen based on enzyme-free cascaded signal amplification via peptide-copper (II) nanoparticles | |
Bu et al. | Immunoassay for foodborne pathogenic bacteria using magnetic composites Ab@ Fe 3 O 4, signal composites Ap@ PtNp, and thermometer readings | |
CN109444240B (zh) | 一种基于普鲁士蓝的电化学免疫传感器及基于该传感器所建立的电化学免疫传感方法和应用 | |
Wu et al. | Cascade amplified colorimetric immunoassay based on an integrated multifunctional composite with catalytic coordination polymers for prostate specific antigen detection | |
JP5473382B2 (ja) | 免疫測定方法 | |
CN114230804B (zh) | 一种过氧化铜/氢键有机骨架纳米材料及其探针、一种检测脂质运载蛋白-2试剂盒 | |
CN111693721A (zh) | 基于普鲁士蓝纳米酶标记物的酶联免疫吸附实验的制备方法及应用 | |
CN110987914A (zh) | 基于Zr-MOF纳米酶的磷酸化蛋白检测和区分的方法及α-酪蛋白定量检测 | |
CN114720515B (zh) | 一种线性范围可调、聚多巴胺介导的免修饰便携式电导率免疫传感器的构建方法及应用 | |
CN114381553B (zh) | 非洲猪瘟病毒检测用生物材料、试剂盒和非诊断目的的非洲猪瘟病毒的检测方法 | |
CN112557384B (zh) | 一种基于比色分析的硫化氢检测方法及应用 | |
Gao et al. | Significance of the antibody orientation for the lateral flow immunoassays: A mini-review | |
CN110333348B (zh) | 多肽与铜离子形成的纳米颗粒及制备方法及应用 |
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 |