CN113238041B - Beta-cyclodextrin modification-based immune biological bar code sensitization probe and method for rapidly detecting atrazine - Google Patents
Beta-cyclodextrin modification-based immune biological bar code sensitization probe and method for rapidly detecting atrazine Download PDFInfo
- Publication number
- CN113238041B CN113238041B CN202110438060.1A CN202110438060A CN113238041B CN 113238041 B CN113238041 B CN 113238041B CN 202110438060 A CN202110438060 A CN 202110438060A CN 113238041 B CN113238041 B CN 113238041B
- Authority
- CN
- China
- Prior art keywords
- beta
- atrazine
- aunps
- probe
- solution
- 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
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 title claims abstract description 60
- 229920000858 Cyclodextrin Polymers 0.000 title claims abstract description 57
- MXWJVTOOROXGIU-UHFFFAOYSA-N atrazine Chemical compound CCNC1=NC(Cl)=NC(NC(C)C)=N1 MXWJVTOOROXGIU-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 51
- 239000000523 sample Substances 0.000 title claims abstract description 50
- 239000001116 FEMA 4028 Substances 0.000 title claims abstract description 46
- 235000011175 beta-cyclodextrine Nutrition 0.000 title claims abstract description 46
- 229960004853 betadex Drugs 0.000 title claims abstract description 46
- 206010070834 Sensitisation Diseases 0.000 title claims abstract description 17
- 230000008313 sensitization Effects 0.000 title claims abstract description 17
- 230000004048 modification Effects 0.000 title claims abstract description 10
- 238000012986 modification Methods 0.000 title claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000010931 gold Substances 0.000 claims abstract description 21
- 229910052737 gold Inorganic materials 0.000 claims abstract description 21
- 108091081406 G-quadruplex Proteins 0.000 claims abstract description 18
- 239000002105 nanoparticle Substances 0.000 claims abstract description 16
- -1 cyclodextrin modified gold Chemical class 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000002253 acid Substances 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 7
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- 238000010438 heat treatment Methods 0.000 claims abstract description 5
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 23
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 claims description 17
- 229940025294 hemin Drugs 0.000 claims description 17
- 239000012086 standard solution Substances 0.000 claims description 13
- 230000005284 excitation Effects 0.000 claims description 11
- CTFKOMUXSHQLLL-UHFFFAOYSA-N 2,6-ditert-butyl-4-[[1-(hydroxymethyl)cyclopentyl]methyl]phenol Chemical compound CC(C)(C)C1=C(O)C(C(C)(C)C)=CC(CC2(CO)CCCC2)=C1 CTFKOMUXSHQLLL-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 9
- 238000007885 magnetic separation Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000427 antigen Substances 0.000 claims description 8
- 102000036639 antigens Human genes 0.000 claims description 8
- 108091007433 antigens Proteins 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 230000001900 immune effect Effects 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 3
- 125000005581 pyrene group Chemical group 0.000 claims description 3
- 238000004153 renaturation Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- QFLARMBBFKMMGK-UHFFFAOYSA-N [K].[K].O.O.S(=O)(=O)=C1CC=C(C=C1)P(C1=CC=CC=C1)C1=CCC(C=C1)=S(=O)=O Chemical compound [K].[K].O.O.S(=O)(=O)=C1CC=C(C=C1)P(C1=CC=CC=C1)C1=CCC(C=C1)=S(=O)=O QFLARMBBFKMMGK-UHFFFAOYSA-N 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
- 239000012498 ultrapure water Substances 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 14
- 238000004458 analytical method Methods 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 10
- 239000000975 dye Substances 0.000 description 7
- 230000003197 catalytic effect Effects 0.000 description 6
- 239000002086 nanomaterial Substances 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical group 0.000 description 5
- 230000009471 action Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 239000002122 magnetic nanoparticle Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000000635 electron micrograph Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- JOUDBUYBGJYFFP-FOCLMDBBSA-N thioindigo Chemical compound S\1C2=CC=CC=C2C(=O)C/1=C1/C(=O)C2=CC=CC=C2S1 JOUDBUYBGJYFFP-FOCLMDBBSA-N 0.000 description 2
- TZDMVWHGWNZKCQ-UHFFFAOYSA-N 1-(2,8-dihydroxy-10H-phenazin-5-yl)ethanone Chemical compound CC(=O)N1C2=CC=C(O)C=C2NC2=CC(O)=CC=C12 TZDMVWHGWNZKCQ-UHFFFAOYSA-N 0.000 description 1
- PKYCWFICOKSIHZ-UHFFFAOYSA-N 1-(3,7-dihydroxyphenoxazin-10-yl)ethanone Chemical compound OC1=CC=C2N(C(=O)C)C3=CC=C(O)C=C3OC2=C1 PKYCWFICOKSIHZ-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 230000000955 neuroendocrine Effects 0.000 description 1
- 230000006911 nucleation Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000027272 reproductive process Effects 0.000 description 1
- HSSLDCABUXLXKM-UHFFFAOYSA-N resorufin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3N=C21 HSSLDCABUXLXKM-UHFFFAOYSA-N 0.000 description 1
- 238000013215 result calculation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000004454 trace mineral analysis Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy 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
- G01N33/543—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
- G01N33/54346—Nanoparticles
-
- 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
- G01N33/18—Water
- G01N33/1826—Organic contamination in water
- G01N33/184—Herbicides, pesticides, fungicides, insecticides or the like
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nanotechnology (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of food safety detection and analysis, and relates to an immune biological bar code sensitization probe based on beta-cyclodextrin modification and a method for rapidly detecting atrazine. The probe is prepared by a method comprising the following steps: (1) synthesizing cyclodextrin modified gold nanoparticles by a one-step method: mixing buffer solution, chloroauric acid and beta-CD, and heating for reaction after intense stirring to obtain cyclodextrin modified gold nanoparticle beta-CD@AuNPs; (2) preparation of probes: mixing the beta-CD@AuNPs obtained in the step (1) with an atrazine monoclonal antibody, and adding a G-quadruplex DNA chain to obtain the biological bar code probe beta-CD@AuNPs@Ab@G4. Compared with the traditional preparation method of the biological bar code gold nano-probe, the method shortens the time by nearly five times and is simple and easy to implement.
Description
Technical Field
The invention belongs to the technical field of food safety detection and analysis, and particularly relates to an immune biological bar code sensitization probe based on beta-cyclodextrin modification and a method for rapidly detecting atrazine based on the immune biological bar code sensitization probe modified by beta-cyclodextrin.
Background
Atrazine (AT) is one of the most widely used triazine herbicides in agriculture worldwide. Since the global demand for grains is gradually increased, the atrazine is inevitably used in agriculture, but the increase of the grain yield in the use process is accompanied with the aggravation of environmental pollution, and the atrazine in the soil can pollute crops and water bodies due to the circulatory system in nature. The effects of atrazine at a certain concentration on neuroendocrine, reproductive and developmental processes in aquatic animals and humans are shown by many epidemiological studies through investigation of drinking water to have a certain carcinogenic potential. The most common techniques for measuring atrazine today are Gas Chromatography (GC), high Performance Liquid Chromatography (HPLC) and gas chromatography mass spectrometry (GC-MS). These methods are robust and mature, but sample pretreatment is complex and time consuming, equipment is expensive, and specialized technicians are required, thereby limiting their wide application in practical analysis.
The biological bar code is a novel nucleic acid signal amplification detection technology, can be used for detecting nucleic acid and protein, and has higher sensitivity and specificity. Biological barcode analysis techniques typically utilize a large number of nucleic acids modified with nanomaterials as signal amplifying output molecules, with common nanomaterials including gold nanoparticles, polystyrene microspheres, and other spherical nanoparticles, and Magnetic Nanoparticles (MNPs) for separating complexes formed in conjunction with barcode signals. After the magnetic separation and signal collection are completed, dithiothreitol (DTT) solution is added to release DNA bar code, and detection can be carried out by various methods such as fluorescence, colorimetry, electrochemistry and the like.
Gold nanomaterials are commonly used as nucleic acid signal-loaded nanomaterials in biological barcode detection due to their high stability and biocompatibility. In addition, the surface chemistry of AuNPs is diverse and various biofunctional groups (e.g., saccharides, peptides, lipids, proteins, nucleic acids, etc.) can be attached by Au-SH or Au-N bond bonding or physical adsorption. In the traditional biological bar code detection, a gold nano probe is coupled with a DNA bar code through an Au-SH bond, the coupling step is complicated, and the modification time is long. In recent years, supermolecule recognition based on the action of a host and a guest has been widely used, and beta-cyclodextrin (beta-CD) is a kind of cyclic polysaccharide substance with reducibility and has rich amino and sulfhydryl groups. According to the invention, the one-step coupling of the antibody and the DNA bar code probe can be realized by virtue of the excellent host-guest effect of the annular cavity, so that the coupling efficiency and the probe preparation stability are greatly improved.
The G-quadruplex DNase formed by heme and DNA G-quadruplex is a nuclease having peroxidase-like activity and can be efficiently used as H 2 O 2 2,2' -azido bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) or luminol is catalyzed for substrates.Red (10-acetyl-3, 7-dihydroxyphenazine) is currently known for HRP and H 2 O 2 The most sensitive and stable fluorescent probe. The complex of G-quadruplex and hemin catalyzes the fluorescence of AR dye.
Disclosure of Invention
The invention aims to provide a biological bar code probe for detecting atrazine and a preparation method thereof, which have the advantages of short time, simple preparation method, better stability and higher sensitivity compared with the preparation of the traditional biological bar code probe.
In order to achieve the above object, the present invention provides an immune bio barcode sensitization probe based on beta-cyclodextrin modification, which is prepared by a method comprising the steps of:
(1) One-step method for synthesizing cyclodextrin modified gold nanoparticles:
mixing buffer solution, chloroauric acid and beta-CD, and heating for reaction after intense stirring to obtain cyclodextrin modified gold nanoparticle beta-CD@AuNPs;
(2) Preparation of the probe:
mixing the beta-CD@AuNPs obtained in the step (1) with an atrazine monoclonal antibody, and adding a G-quadruplex DNA chain to obtain the biological bar code probe beta-CD@AuNPs@Ab@G4.
According to the present invention, preferably, the method of step (1) comprises: adding 20-40 mL of ultrapure water, 4-6 mL of PB buffer solution with the pH of 0.08-0.12M and the pH of 7.0, 0.8-1.2 mL of 0.008-0.012M chloroauric acid and 5-10 mL of 0.04-0.06M beta-CD into a round bottom flask, and heating to 95-100 ℃ for reaction for 40-80 min after intense stirring until the solution turns from light yellow to colorless and then turns into light red gradually to turn into reddish wine, so as to indicate that gold nanoparticles are gradually nucleated; naturally cooling to room temperature, centrifuging the solution at low temperature, and re-suspending with pure water for later use.
According to the present invention, preferably, the method of step (1) further comprises: further treatment of the β -cd@aunps with BSPP:
after adding di-potassium bis (p-sulfonylphenyl) phenylphosphinate dihydrate to the beta-CD@AuNPs solution, the reaction was carried out by vertical suspension, then sodium chloride solution was added dropwise, when the color changed from red to green, the supernatant was removed by centrifugation, resuspended in BSPP and methanol, and after repeating the centrifugation operation, the BSPP was added for resuspension.
According to the present invention, preferably, the method of step (2) comprises:
200-400 mu L of beta-CD@AuNPs is taken, 100-200 mu L of beta-CD@AuNPs is added, and 0.008-0.012M PBS is used for dilution to 5-15 mu g.ml -1 The atrazine monoclonal antibody is gently mixed and spun at the temperature of 4 ℃, added with a G-quadruplex DNA chain subjected to 0.4-0.6OD pre-renaturation treatment, and continuously and gently mixed at the temperature of 4 ℃; then the mixture is centrifugally washed under the condition of 4 ℃; finally, the suspension was resuspended in PBS buffer.
According to one embodiment of the invention, the probe is made by a method comprising the steps of:
(1) Synthesizing cyclodextrin modified gold nanoparticles by a one-step method; to a round bottom flask was added 20-40 mL of aqueous solution, 5mL of PB buffer (0.1M, pH 7.0), 1mL of chloroauric acid (0.01M) and 5-10 mL of beta-CD (0.05M), and after vigorous stirring, the solution was heated to 100deg.C and reacted for 60min, and it was seen that the solution changed from light yellow to colorless and then to light red, and gradually changed to reddish wine, indicating gradual nucleation of gold nanoparticles. After naturally cooling to room temperature, 100mL of the above solution was centrifuged at 10000rpm for 20min at 4℃and resuspended in 100mL of pure water for use. Subsequently 5-10 mg of bis (p-sulfonylphenyl) phenylphosphine dipotassium dihydrate (enhancing the dispersibility and stability of the AuNPs system) was added to the beta-CD@AuNPs solution (10 nm,20 mL), the reaction was suspended vertically for 3d, then 5M sodium chloride solution was added dropwise, and when the color changed from red to green, 1,600rcf was centrifuged for 30min and the supernatant was removed using 0.8mL of 2.5mM BSPP and 0.8mL methanol. After the end of the resuspension, centrifugation was repeated and 0.2mL of 2.5mM BSPP was added. Using ultraviolet radiationSee spectrophotometric scanning at 400-650 nm wavelength to determine beta-CD@AuNPs concentration (15 nm AuNPs molar extinction coefficient of 3.6X10) 8 cm -1 M -1 )。
(2) Preparation of gold nanoprobe: mu.L of BSPP-treated beta-CD@AuNPs was taken, 150. Mu.L was added and diluted to 10. Mu.g.mL with 0.01MPBS -1 The atrazine monoclonal antibody is gently mixed for 2 hours at 4 ℃, added with a G quadruplex (G-quad) DNA strand subjected to 0.5OD pre-90 DEG renaturation treatment, and continuously gently mixed for 3 hours at 4 ℃. The mixture was then centrifuged at 10000rpm for 20min at 4℃and washed 3 times with PBS buffer. Finally, the suspension was resuspended to 1mL with PBS buffer (0.01M, pH 7.4).
According to the present invention, preferably, the sequence of the G-quadruplex DNA strand is as shown in SEQ ID NO:1 is shown as follows: 5' -Pyrene-TTTTTTTTTTGGGTAGGGCGGGTTGGG-3' (SEQ ID NO: 1) modified at its 5' end with a Pyrene group.
The invention also provides a method for rapidly detecting atrazine based on the beta-cyclodextrin modified immune biological bar code sensitization probe, which is carried out by adopting the beta-cyclodextrin modified immune biological bar code sensitization probe and comprises the following steps:
(1) Mixing atrazine standard solutions with different concentrations with beta-CD@AuNPs@Ab@G4 at room temperature, incubating at 37 ℃, adding magnetic beads modified with atrazine antigens, incubating at 37 ℃ in an oscillating way, magnetically separating and washing, and adding into a fluorescent reaction system K + Solution, hemin, AR dye and H 2 O 2 Oscillating reaction at 20-30 deg.c, measuring fluorescence intensity after the reaction, and making standard curve based on the fluorescence intensity and the concentration of the corresponding atrazine standard solution;
(2) And (3) processing the sample to be tested containing atrazine according to the method of the step (1), measuring the fluorescence intensity of the sample, and obtaining the concentration of atrazine in the sample to be tested according to a standard curve.
The method according to the present invention, preferably, the method of step (1) comprises:
under the condition of room temperature, 80 to 120 mu L of atrazine standard solution with different concentrations and 8 to 12 mu L of atrazine standard solution are taken for preparationMixing the good beta-CD@AuNPs@Ab@G4, incubating for 20-40 min at 37 ℃, adding 8-12 mu L of magnetic beads modified with atrazine antigens, and incubating for 20-40 min at 37 ℃ in an oscillating way; magnetic separation, washing with PBST; finally adding 80-120 mu M K of fluorescent reaction system + 40-60 nM hemin, 80-120 mu M AR dye and 8-12 mu M H 2 O 2 And (3) carrying out oscillation reaction for 40-80 min at 20-30 ℃, measuring the fluorescence intensity after the reaction is finished, and preparing a standard curve according to the fluorescence intensity and the concentration of the corresponding atrazine standard solution.
According to the method of the invention, the fluorescence spectrophotometer settings parameters are as follows: the excitation wavelength is 533nm, the emission wavelength is 500-700 nm, and the width of the slit for excitation and emission is 10nm.
According to one specific embodiment of the invention, the method for rapidly detecting atrazine by using the beta-cyclodextrin modified immune bio-barcode sensitization probe comprises the following steps:
100 mu L of atrazine standard solution with different concentrations is taken and mixed with 10 mu L of prepared beta-CD@AuNPs@Ab@G4 at room temperature, incubated for 30min at 37 ℃,10 mu L of magnetic beads modified with atrazine antigen are added, and the mixture is subjected to shaking incubation for 30min at 37 ℃. Magnetic separation, washing 3 times with PBST. Finally adding 100 mu M K of fluorescent reaction system + Solution, 50nM hemin, 100. Mu.M AR dye and 10. Mu. M H 2 O 2 The total volume is 200 mu L, the reaction is carried out for 60min under the condition of 25 ℃, the fluorescence spectrophotometer is set with the following excitation wavelength of 533nm, the emission wavelength of 500-700 nm and the slit width of excitation and emission of 10nm, and the fluorescence intensity is measured under the condition.
According to the invention, based on the traditional immune biological bar code, the main guest action of cyclodextrin on beta-CD@AuNPs is utilized to simultaneously modify an antibody and a DNA bar code, an experimental principle is shown as shown in a figure 1, an antigen is modified on magnetic nano particles by an NHS agarose modification method, and a 5' -end modified pyrene group of a G quadruplex can generate the main guest action with a cavity of cyclodextrin by virtue of the main guest action of a beta-CD cavity on the beta-CD@AuNPs, so that the G quadruplex is modified on the beta-CD@AuNPs. Antibodies to atrazine are simultaneously modified at beta-CD@A by electrostatic adsorptionuNPs. When the target object exists, the magnetic nano probe modified with the atrazine antigen competes with small molecules and is combined with the gold nano probe modified with the atrazine antibody and the G quadruplex, and when the target object does not exist, the magnetic nano probe can be fully combined with the gold nano probe. After magnetic separation, G quadruplex on the gold nanoprobe forms a complex with hemin to catalyze the reaction by H 2 O 2 The mediated AR fluorescent dye fluoresces. When the target is present, the fluorescence intensity is reduced, and when the target is absent, the fluorescence intensity is maximized. The method can specifically and quantitatively detect the atrazine content in the water sample, when the atrazine content is higher, fewer MMPs-ATz Ag/Ab-AuNPs-DNA barcode complexes are formed, and the DNA barcode in the method is G-quad DNA with a catalytic function, can form a complex with hemin, and is catalyzed by H 2 O 2 Mediated duplex Red (fluorescent Red dye) fluoresces.
In the method for detecting atrazine by using the novel immune biological bar code sensitization probe based on beta-cyclodextrin modification, a magnetic probe coated with atrazine antigen competes with free atrazine small molecules and is combined with gold nano probes modified with atrazine monoclonal antibodies and a large number of G-quadruplexes. After magnetic separation, the G-quadruplex modified on the gold nanoprobe can form a complex with Hemin to form H 2 O 2 Catalyzing the reaction of the duplex Red fluorescent dye for the substrate produces the intense Red fluorescent substance resorufin.
The invention utilizes the host-guest function of beta-CD on beta-CD@AuNPs material to couple an antibody and a G-quadruplex on a gold nano probe by a one-step method, and constructs an ultrasensitive novel method for detecting atrazine by using immune biological bar code fluorescence. The method is rapid, simple and convenient to operate, high in sensitivity and strong in specificity, and can be used for trace analysis of atrazine in water quality, for example, detection of a sea and river water sample. Compared with the traditional biological bar code detection method, the method has the advantages of short preparation time, simple method, high detection sensitivity and detection range of 0.01ng/mL-100ng/mL.
In the invention, the biological bar code gold nano-probe is prepared by modifying the antibody and the DNA chain in the beta-CD@AuNPs in a shorter time, compared with the traditional preparation method of the biological bar code gold nano-probe, the method has the advantages that the time is shortened by nearly five times, and the method is simple and easy to implement. Therefore, the method has important practical significance for rapid detection of atrazine and has good guiding significance for realizing the field rapid detection technology.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 shows a schematic diagram of a method for rapidly detecting atrazine based on beta-cyclodextrin modified immunobiological barcode sensitization probes.
FIG. 2 shows electron microscopy images of cyclodextrin modified gold nanoparticles obtained with the addition of different molar concentrations of beta-CD.
FIG. 3 shows the results of the optimization of the pH of the reaction PBS buffer.
Fig. 4 shows the catalytic TMB effect of gold nanoprobe addition volumes.
Fig. 5 shows experimental condition optimization results for the Hemin addition volume.
Figure 6 shows a standard curve of a method for rapid detection of atrazine based on beta-cyclodextrin modified immunobiological barcode sensitization probes.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein.
Embodiments of the present invention will be described in detail below with reference to examples, in which specific conditions not noted were performed according to conventional conditions or conditions suggested by manufacturers, using G-quad DNA synthesized by Shanghai Biotechnology (Beijing Synthesis), atrazine standards (1 mg/mL) used in the experiments were purchased from Polyboth Williams, anti-atrazine monoclonal antibodies (30 mg/mL) were prepared from laboratories, amplex Red (fluorescent Red dye) was purchased from Biyun Biotech Co., ltd., and F97 pro fluorescence spectrophotometer was purchased from Shanghai optical technologies Co., ltd. The sequence of the G-quadruplex DNA strand is shown in SEQ ID NO:1 is shown as follows: 5'-Pyrene-TTTTTTTTTTGGGTAGGGCGGGTTGGG-3' (SEQ ID NO: 1).
Example 1
(1) To a round bottom flask was added 35mL of aqueous solution, 5mL of PB buffer (0.1M, pH 7.0), 1mL of chloroauric acid (0.01M) and 10mL of beta-CD (0.01M, 0.03M, 0.05M) with vigorous stirring.
(2) When heated to 100deg.C and reacted for 60 minutes, the solution was seen to change from light yellow to colorless and then to light red, gradually changing to reddish wine.
(3) After naturally cooling to room temperature, 10mL of the above solution was centrifuged at 10000rpm at 4℃for 20min, and the pellet was resuspended in 1mL of pure water.
FIG. 2 shows electron microscopy images of cyclodextrin modified gold nanoparticles obtained with the addition of different molar concentrations of beta-CD.
Example 2
(1) PBS buffers with different pH values, namely pH6.0, pH6.5, pH7.0, pH7.5 and pH8.0, are prepared.
(2) 100. Mu.L of 20ng/mL atrazine standard solution diluted with different pH buffers was mixed with 10. Mu.L of prepared beta-CD@AuNPs@Ab@G4 at room temperature, incubated for 30min at 37℃and a corresponding negative control group was set.
(3) Then 10. Mu.L of immunomagnetic beads were added and incubated with shaking at 37℃for 30min.
(4) After magnetic separation for 1min, wash 3 times with PBST.
(5) Finally, the fluorescent reaction system (100 mu M K) + Solution, 50nM hemin, 100. Mu.M AR dye and 10. Mu. M H 2 O 2 ) The total volume was 200. Mu.L, and the reaction was carried out by shaking at 25℃for 60min.
(6) The fluorescence spectrophotometer was set with excitation wavelength of 533nm, emission wavelength of 500-700 nm, slit widths for excitation and emission of 10nm, and fluorescence intensity was measured under the conditions. And finally, subtracting the negative control group from the result calculation experiment group, and comparing the response difference change.
FIG. 3 shows the results of the optimization of the pH of PBS buffer. It can be seen that the reaction buffer has a relatively large influence on the experimental result, and the change value of the response fluorescence intensity is larger for the target object with the same concentration when the pH is 7.5.
Example 3
(1) mu.L, 10. Mu.L, 15. Mu.L, 20. Mu.L, 25. Mu.L, 30. Mu.L of gold nanoparticles (5.2 nM) were added to different 1.5mL centrifuge tubes and ddH was used 2 O was made up to 30. Mu.L.
(2) mu.L of sodium acetate buffer (0.01M, pH 4.5) was added to each centrifuge tube.
(3) 50. Mu.L of 2mM TMB and 50. Mu.L of H were added 2 O 2 。
(4) Oscillating reaction for 30min at 37 DEG C
(5) And measuring absorbance in the range of 500-700 nm by adopting an ultraviolet-visible spectrophotometer.
Fig. 4 shows the catalytic effect of gold nanoparticle addition volume. It can be seen that the addition of gold nano-materials has different catalytic effects on TMB, and the higher the addition of gold nano-materials, the better the catalytic effect.
Example 4
(1) 100. Mu.L of atrazine standard solution at a concentration of 20ng/mL was mixed with 10. Mu.L of prepared beta. -CD@AuNPs@Ab@G4 at room temperature, negative controls were set for each group, and incubated at 37℃for 30min.
(2) 10. Mu.L of immunomagnetic beads were added and incubated with shaking at 37℃for 30min.
(3) Magnetic separation, washing 3 times with PBST.
(4) Adding fluorescent reaction system (100 mu M K) containing hemin substrates with different concentrations + Solution, 25nM/50nM/100nM/150nM/200nM hemin, 100. Mu.M AR dye and 10. Mu. M H 2 O 2 ) The total volume was 200. Mu.L, and the reaction was carried out by shaking at 25℃for 60min.
(5) The fluorescence spectrophotometer was set with excitation wavelength of 533nm, emission wavelength of 500-700 nm, slit widths for excitation and emission of 10nm, and fluorescence intensity was measured under the conditions.
Fig. 5 shows the optimization results of the Hemin addition volume. It can be seen that the amount of hemin added can affect the catalytic effect on the fluorescent dye, indicating that too little amount of hemin added can affect the formation of complex.
Example 5
(1) At room temperature, 100. Mu.L of the atrazine standard solution with different concentrations (0.01 ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100 ng/mL) was mixed with 10. Mu.L of the prepared beta-CD@AuNPs@Ab@G4, and incubated at 37℃for 30min.
(2) 10. Mu.L of immunomagnetic beads were added and incubated with shaking at 37℃for 30min.
(3) Magnetic separation for 1min, washing with PBST 3 times.
(4) Adding the fluorescent reaction system (100 mu M K) + Solution, 50nM hemin, 100. Mu.M AR dye and 10. Mu. M H 2 O 2 ) The total volume was 200. Mu.L, and the reaction was carried out by shaking at 25℃for 60min.
(5) The fluorescence spectrophotometer settings were as follows: the excitation wavelength is 533nm, the emission wavelength is 500-700 nm, the width of the exciting and emission slits is 10nm, and the fluorescence intensity is measured under the condition.
(6) And (3) taking a fluorescence value as an ordinate and a wavelength as an abscissa, and manufacturing a standard curve. The results are shown in FIG. 6.
Test case
By adopting the method, the sea and river water sample is detected, and the detection result is shown in table 1.
TABLE 1
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.
Sequence listing
<110> environmental medical and working medical institute of military medical institute of academy of military science
<120> an immunobiological barcode sensitization probe based on beta-cyclodextrin modification and a method for rapidly detecting atrazine
<130> BJI2002140HBY
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tttttttttt gggtagggcg ggttggg 27
Claims (4)
1. An immunobiological bar code sensitization probe based on beta-cyclodextrin modification, characterized in that the probe is prepared by a method comprising the following steps:
(1) One-step method for synthesizing cyclodextrin modified gold nanoparticles:
mixing buffer solution, chloroauric acid and beta-CD, and heating for reaction after intense stirring to obtain cyclodextrin modified gold nanoparticle beta-CD@AuNPs; the method of step (1) comprises: adding 20-40 mL of ultrapure water, 4~6mL 0.08~0.12M pH7.0 PB buffer solution, 0.8-1.2 mL of 0.008-0.012M chloroauric acid and 5~10mL 0.04~0.06M beta-CD into a round bottom flask, vigorously stirring, heating to 95-100 ℃ for reacting for 40-80 min, and gradually changing from light yellow to colorless and then from light red to wine red until the solution becomes gradually nucleated to form gold nanoparticles; naturally cooling to room temperature, centrifuging the solution at low temperature, and re-suspending with pure water for later use; the method of step (1) further comprises: further treatment of the β -cd@aunps with BSPP: adding di-potassium bis (p-sulfonylphenyl) phenylphosphine dihydrate into beta-CD@AuNPs solution, performing vertical suspension reaction, then dropwise adding sodium chloride solution, centrifuging to remove supernatant when the color changes from red to green, re-suspending with BSPP and methanol, repeating centrifugation operation, and adding BSPP for re-suspending;
(2) Preparation of the probe:
mixing the beta-CD@AuNPs obtained in the step (1) with an atrazine monoclonal antibody, and adding a G-quadruplex DNA chain to obtain the biological barcode sensitization probe beta-CD@AuNPs@Ab@G4; the method of step (2) comprises: 200-400 mu L of beta-CD@AuNPs is taken, 100-200 mu L of beta-CD@AuNPs is added, and 0.008-0.012M PBS is used for diluting to 5-15 mu g.ml -1 The atrazine monoclonal antibody is gently mixed and spun at the temperature of 4 ℃, added with 0.4-0.6OD G-quadruplex DNA chain which is subjected to pre-renaturation treatment, and continuously and gently mixed at the temperature of 4 ℃; then the mixture is centrifugally washed under the condition of 4 ℃; finally, the suspension is resuspended by PBS buffer
The sequence of the G-quadruplex DNA strand is shown in SEQ ID NO:1 is shown as follows: 5' -Pyrene-TTTTTTTTTTGGGTAGGGCGGGTTGGG-3' (SEQ ID NO: 1) modified at its 5' end with a Pyrene group.
2. A method for rapidly detecting atrazine based on a beta-cyclodextrin modified immune bio-barcode sensitization probe, the method being performed by using the beta-cyclodextrin modified immune bio-barcode sensitization probe according to claim 1, comprising the steps of:
(1) Mixing atrazine standard solutions with different concentrations with beta-CD@AuNPs@Ab@G4 at room temperature, incubating at 37 ℃, adding magnetic beads modified with atrazine antigens, incubating at 37 ℃ in an oscillating way, magnetically separating and washing, and adding into a fluorescent reaction system K + Solution, hemin, AR dye and H 2 O 2 Carrying out oscillation reaction at 20-30 ℃, measuring fluorescence intensity after the reaction is finished, and preparing a standard curve according to the fluorescence intensity and the concentration of the corresponding atrazine standard solution;
(2) And (3) processing the sample to be tested containing atrazine according to the method of the step (1), measuring the fluorescence intensity of the sample, and obtaining the concentration of atrazine in the sample to be tested according to a standard curve.
3. The method of claim 2, wherein the method of step (1) comprises:
under the condition of room temperature, 80-120 mu L of atrazine standard solution with different concentrations is mixed with 8-12 mu L of prepared beta-CD@AuNPs@Ab@G4, incubated for 20-40 min at 37 ℃, 8-12 mu L of magnetic beads modified with atrazine antigens are added, and the mixture is subjected to shaking incubation for 20-40 min at 37 ℃; magnetic separation, washing with PBST; finally, adding 80-120 mu M K of a fluorescent reaction system + Solution of 40~60nM hemin,80~120 mu M AR dye and 8-12 mu M H 2 O 2 And (3) carrying out oscillation reaction for 40-80 min at 20-30 ℃, measuring the fluorescence intensity after the reaction is finished, and preparing a standard curve according to the fluorescence intensity and the concentration of the corresponding atrazine standard solution.
4. The method of claim 2, wherein the fluorescence spectrophotometer settings parameters are as follows: the excitation wavelength is 533nm, the emission wavelength is 500-700 nm, and the width of the slits for excitation and emission is 10nm.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110044888 | 2021-01-13 | ||
| CN2021100448889 | 2021-01-13 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113238041A CN113238041A (en) | 2021-08-10 |
| CN113238041B true CN113238041B (en) | 2024-01-16 |
Family
ID=77128925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110438060.1A Active CN113238041B (en) | 2021-01-13 | 2021-04-22 | Beta-cyclodextrin modification-based immune biological bar code sensitization probe and method for rapidly detecting atrazine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113238041B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114235791B (en) * | 2021-11-30 | 2023-11-24 | 上海应用技术大学 | Method for rapidly detecting anthracene in water by using cyclodextrin-modified nano gold particle sensor |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2846106A1 (en) * | 2011-08-26 | 2013-03-07 | Alan Marc Kleinfeld | Development and use of fluorescent probes of unbound bilirubin |
| WO2013070653A1 (en) * | 2011-11-09 | 2013-05-16 | Board Of Trustees Michigan State University | Metallic nanoparticle synthesis with carbohydrate capping agent |
| CN103293298A (en) * | 2013-07-05 | 2013-09-11 | 常熟理工学院 | Toxin detection method based on G-quadruplex nuclease |
| CN103305622A (en) * | 2013-06-28 | 2013-09-18 | 上海交通大学 | Method for detecting lead by G-quadruplex fluorescence method formed by using unlabeled functional nucleic acid |
| CN104459124A (en) * | 2014-12-04 | 2015-03-25 | 济南大学 | Preparation method and application of electrochemical immunosensor based on HS-beta-CD-Ag-GOD conjugate |
| CN104962615A (en) * | 2015-06-04 | 2015-10-07 | 西南大学 | DNA flexagon nano structure-nanogold biosensor based on adapter modification and preparing method and application of DNA flexagon nano structure-nanogold biosensor |
| CN105699650A (en) * | 2016-02-16 | 2016-06-22 | 中国农业科学院农业质量标准与检测技术研究所 | Carbofuran bio-barcode immunoassay kit and application thereof |
| CN107435063A (en) * | 2016-05-27 | 2017-12-05 | 首都师范大学 | A kind of quick method for preparing sulfydryl modification DNA nano-Au composites (DNA AuNP) |
| CN107976548A (en) * | 2017-11-21 | 2018-05-01 | 湖南工程学院 | Detection fibrin ferment kit based on tetra- serobilas of micro-fluidic chip and G--ferroheme DNA enzymatic and its preparation method and application |
| CN108459159A (en) * | 2018-02-11 | 2018-08-28 | 莎穆(上海)生物科技有限公司 | Supramolecular self assembly mediates the new method of mesh nano gold enhancing immuno-chromatographic test paper strip sensitivity |
| CN108535483A (en) * | 2018-04-02 | 2018-09-14 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Atrazine detection kit and application based on up-conversion fluorescence immunosensor and Atrazine detection method |
| CN109142704A (en) * | 2018-07-06 | 2019-01-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | A kind of kit and method based on bio-barcode and rolling circle amplification detection T-2 toxin |
| CN109852673A (en) * | 2019-01-17 | 2019-06-07 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
-
2021
- 2021-04-22 CN CN202110438060.1A patent/CN113238041B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2846106A1 (en) * | 2011-08-26 | 2013-03-07 | Alan Marc Kleinfeld | Development and use of fluorescent probes of unbound bilirubin |
| WO2013070653A1 (en) * | 2011-11-09 | 2013-05-16 | Board Of Trustees Michigan State University | Metallic nanoparticle synthesis with carbohydrate capping agent |
| CN103305622A (en) * | 2013-06-28 | 2013-09-18 | 上海交通大学 | Method for detecting lead by G-quadruplex fluorescence method formed by using unlabeled functional nucleic acid |
| CN103293298A (en) * | 2013-07-05 | 2013-09-11 | 常熟理工学院 | Toxin detection method based on G-quadruplex nuclease |
| CN104459124A (en) * | 2014-12-04 | 2015-03-25 | 济南大学 | Preparation method and application of electrochemical immunosensor based on HS-beta-CD-Ag-GOD conjugate |
| CN104962615A (en) * | 2015-06-04 | 2015-10-07 | 西南大学 | DNA flexagon nano structure-nanogold biosensor based on adapter modification and preparing method and application of DNA flexagon nano structure-nanogold biosensor |
| CN105699650A (en) * | 2016-02-16 | 2016-06-22 | 中国农业科学院农业质量标准与检测技术研究所 | Carbofuran bio-barcode immunoassay kit and application thereof |
| CN107435063A (en) * | 2016-05-27 | 2017-12-05 | 首都师范大学 | A kind of quick method for preparing sulfydryl modification DNA nano-Au composites (DNA AuNP) |
| CN107976548A (en) * | 2017-11-21 | 2018-05-01 | 湖南工程学院 | Detection fibrin ferment kit based on tetra- serobilas of micro-fluidic chip and G--ferroheme DNA enzymatic and its preparation method and application |
| CN108459159A (en) * | 2018-02-11 | 2018-08-28 | 莎穆(上海)生物科技有限公司 | Supramolecular self assembly mediates the new method of mesh nano gold enhancing immuno-chromatographic test paper strip sensitivity |
| CN108535483A (en) * | 2018-04-02 | 2018-09-14 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Atrazine detection kit and application based on up-conversion fluorescence immunosensor and Atrazine detection method |
| CN109142704A (en) * | 2018-07-06 | 2019-01-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | A kind of kit and method based on bio-barcode and rolling circle amplification detection T-2 toxin |
| CN109852673A (en) * | 2019-01-17 | 2019-06-07 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
Non-Patent Citations (5)
| Title |
|---|
| G-quadruplex DNAzyme-based microcystin-LR (toxin) determination by a novel immunosensor;Zhu Y 等;Biosens Bioelectron;第26卷(第11期);第4393-4398页 * |
| Potassium-lead-switched G-quadruplexes: a new class of DNA logic gates;Li T 等;J Am Chem Soc;第131卷;摘要、补充材料 * |
| Three-in-One: Sensing, Self-Assembly, and Cascade Catalysis of Cyclodextrin Modified Gold Nanoparticles;Zhao Y 等;J Am Chem Soc;第138卷;摘要，补充材料第1.3节 * |
| 基于生物条形码结合杂交链式反应双重信号放大检测蓖麻毒素;苑帅 等;分析化学;第47卷(第1期);第147-154页 * |
| 基于纳米材料比色传感器的构建及其应用;苏邵;李晶;汪联辉;;南京邮电大学学报(自然科学版)(第03期);第102-114页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113238041A (en) | 2021-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xiong et al. | Emerging strategies to enhance the sensitivity of competitive ELISA for detection of chemical contaminants in food samples | |
| US10895536B1 (en) | Method for preparing a ratiometric fluorescent sensor for phycoerythrin based on a magnetic molecularly imprinted core-shell polymer | |
| CN108344783B (en) | Electrochemical cell sensor and preparation method and application thereof | |
| CN109752536B (en) | Optical probe based on gold nanoparticle efficient assembly structure and preparation and application thereof | |
| CN107764784B (en) | Fluorescence method for detecting aflatoxin B1 based on copper nanoclusters | |
| CN110776915B (en) | Fluorescent/colorimetric bifunctional microsphere based on multi-level assembly structure and preparation method thereof | |
| CN107290519A (en) | The construction method of SERS aptamer sensors based on nanometer package assembly and application | |
| CN113238041B (en) | Beta-cyclodextrin modification-based immune biological bar code sensitization probe and method for rapidly detecting atrazine | |
| CN110346560B (en) | Multi-enzyme signal particle and preparation method and application thereof | |
| CN110658168B (en) | Method for detecting testosterone by gold nanocluster-gold nanorod immunosensor | |
| CN104914088A (en) | Method for detecting supersensitivity of Mucin-1 surface enhanced raman signal based on golden rod nuclear-silver satellite assembling body | |
| CN105036073B (en) | Preparation method of gold nucleus-quantum dot satellite structure assembly | |
| CN113758910B (en) | Determination of aflatoxin B in vinegar culture 1 By Raman enhanced spectroscopy | |
| Gao et al. | Significance of the antibody orientation for the lateral flow immunoassays: A mini-review | |
| CN109283333B (en) | Method for quantitatively analyzing drug resistance of escherichia coli based on gold shell-up-conversion chiral dimer | |
| CN112730338B (en) | Ag @ Au-based porous structure dual-signal nano amplification probe and SPR immunodetection method thereof | |
| CN113777297A (en) | Fluorescence differential rapid detection method based on magnetic nanoparticles | |
| CN113640274A (en) | Staphylococcus aureus detection method based on aptamer gated mesoporous silica | |
| CN107064485B (en) | Application of decahedral nano silver probe in screening aptamer by using surface plasmon resonance imaging technology | |
| CN110553991B (en) | Biological/chemical detection reagent and detection method based on hollow gold nanoparticle-DNA compound | |
| CN116067937A (en) | SERS aptamer sensor based on core-shell nano assembly structure and preparation and application thereof | |
| CN115825426A (en) | Acetamiprid lateral flow analysis method and test paper based on triple helix bivalent aptamer and nanoenzyme | |
| CN116237535A (en) | Platinum-coated gold nano bipyramid synthesized by DNA (deoxyribonucleic acid) mediation and preparation method and application thereof | |
| CN116148239A (en) | Multiple SERS signal enhanced nano sandwich bacteria detection system and preparation method thereof | |
| CN115837255A (en) | Preparation method of colloidal gold-silicon dioxide composite microspheres |
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 |