EP4158341A1 - Procédé et système pour la détermination de la présence et/ou de la quantité d'au moins un analyte susceptible d'être contenu dans un échantillon - Google Patents
Procédé et système pour la détermination de la présence et/ou de la quantité d'au moins un analyte susceptible d'être contenu dans un échantillonInfo
- Publication number
- EP4158341A1 EP4158341A1 EP21728199.7A EP21728199A EP4158341A1 EP 4158341 A1 EP4158341 A1 EP 4158341A1 EP 21728199 A EP21728199 A EP 21728199A EP 4158341 A1 EP4158341 A1 EP 4158341A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- probe
- affinity tag
- complex
- primary
- amplification
- 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.)
- Withdrawn
Links
- 239000012491 analyte Substances 0.000 title claims abstract description 92
- 238000000034 method Methods 0.000 title claims abstract description 77
- 239000000523 sample Substances 0.000 claims abstract description 315
- 230000003321 amplification Effects 0.000 claims abstract description 131
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 131
- 239000000700 radioactive tracer Substances 0.000 claims abstract description 71
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 57
- 238000011161 development Methods 0.000 claims abstract description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 73
- 238000001514 detection method Methods 0.000 claims description 47
- 230000027455 binding Effects 0.000 claims description 45
- 239000002105 nanoparticle Substances 0.000 claims description 41
- 235000020958 biotin Nutrition 0.000 claims description 40
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 39
- 229960002685 biotin Drugs 0.000 claims description 38
- 239000011616 biotin Substances 0.000 claims description 38
- 239000011324 bead Substances 0.000 claims description 19
- 239000000377 silicon dioxide Substances 0.000 claims description 18
- 230000015572 biosynthetic process Effects 0.000 claims description 16
- 241000700605 Viruses Species 0.000 claims description 15
- 239000000427 antigen Substances 0.000 claims description 15
- 108091007433 antigens Proteins 0.000 claims description 15
- 102000036639 antigens Human genes 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 230000002255 enzymatic effect Effects 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 108010090804 Streptavidin Proteins 0.000 claims description 11
- 125000006850 spacer group Chemical group 0.000 claims description 11
- 108090001008 Avidin Proteins 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 7
- 108010043595 captavidin Proteins 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 108010087904 neutravidin Proteins 0.000 claims description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 7
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 6
- 239000012678 infectious agent Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 5
- 239000003242 anti bacterial agent Substances 0.000 claims description 3
- 229940088710 antibiotic agent Drugs 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 3
- 229940088597 hormone Drugs 0.000 claims description 3
- 239000006249 magnetic particle Substances 0.000 claims description 3
- 244000045947 parasite Species 0.000 claims description 3
- 238000003118 sandwich ELISA Methods 0.000 claims description 3
- 150000003431 steroids Chemical class 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- 210000004881 tumor cell Anatomy 0.000 claims description 3
- 239000011258 core-shell material Substances 0.000 claims 1
- 108020004414 DNA Proteins 0.000 description 50
- 241000589876 Campylobacter Species 0.000 description 29
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 description 24
- 239000000243 solution Substances 0.000 description 20
- 239000012528 membrane Substances 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000012071 phase Substances 0.000 description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 13
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 13
- 239000002953 phosphate buffered saline Substances 0.000 description 13
- 238000012360 testing method Methods 0.000 description 12
- 241000287828 Gallus gallus Species 0.000 description 11
- 239000000872 buffer Substances 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 238000009396 hybridization Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 230000035945 sensitivity Effects 0.000 description 10
- 238000003556 assay Methods 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 239000005289 controlled pore glass Substances 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 229940098773 bovine serum albumin Drugs 0.000 description 8
- 125000005647 linker group Chemical group 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 241000589875 Campylobacter jejuni Species 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 6
- 241000197306 H1N1 subtype Species 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 239000000090 biomarker Substances 0.000 description 6
- 238000007306 functionalization reaction Methods 0.000 description 6
- -1 glycans Proteins 0.000 description 6
- 235000013594 poultry meat Nutrition 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 102000011931 Nucleoproteins Human genes 0.000 description 5
- 108010061100 Nucleoproteins Proteins 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 208000037797 influenza A Diseases 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 150000008300 phosphoramidites Chemical class 0.000 description 5
- 238000011896 sensitive detection Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 241001135528 Campylobacter upsaliensis Species 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 4
- 239000003298 DNA probe Substances 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 235000010633 broth Nutrition 0.000 description 4
- 238000011088 calibration curve Methods 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 244000144977 poultry Species 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 210000003296 saliva Anatomy 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 108091023037 Aptamer Proteins 0.000 description 3
- 240000000533 Capsicum pubescens Species 0.000 description 3
- 235000015855 Capsicum pubescens Nutrition 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 3
- 108020003215 DNA Probes Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 241000712431 Influenza A virus Species 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 150000001615 biotins Chemical class 0.000 description 3
- 238000010804 cDNA synthesis Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 241000589986 Campylobacter lari Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 239000007801 affinity label Substances 0.000 description 2
- 230000000721 bacterilogical effect Effects 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000006781 columbia blood agar Substances 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000003869 genetically modified organism Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- 238000004626 scanning electron microscopy Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 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
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- QWTBDIBOOIAZEF-UHFFFAOYSA-N 3-[chloro-[di(propan-2-yl)amino]phosphanyl]oxypropanenitrile Chemical compound CC(C)N(C(C)C)P(Cl)OCCC#N QWTBDIBOOIAZEF-UHFFFAOYSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- LELMRLNNAOPAPI-UFLZEWODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;aminophosphonous acid Chemical compound NP(O)O.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LELMRLNNAOPAPI-UFLZEWODSA-N 0.000 description 1
- LHEJDBBHZGISGW-UHFFFAOYSA-N 5-fluoro-3-(3-oxo-1h-2-benzofuran-1-yl)-1h-pyrimidine-2,4-dione Chemical compound O=C1C(F)=CNC(=O)N1C1C2=CC=CC=C2C(=O)O1 LHEJDBBHZGISGW-UHFFFAOYSA-N 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 241000589562 Brucella Species 0.000 description 1
- 241000589877 Campylobacter coli Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 229910052684 Cerium Inorganic materials 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229940127399 DNA Polymerase Inhibitors Drugs 0.000 description 1
- 230000000970 DNA cross-linking effect Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 229910052692 Dysprosium Inorganic materials 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 229910052693 Europium Inorganic materials 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 206010069767 H1N1 influenza Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000491226 Influenza A virus (A/WSN/1933(H1N1)) Species 0.000 description 1
- 229910002319 LaF3 Inorganic materials 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 239000006391 Luria-Bertani Medium Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000005062 Polybutadiene Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241001138501 Salmonella enterica Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 229910052771 Terbium Inorganic materials 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- 229910052769 Ytterbium Inorganic materials 0.000 description 1
- GHLOAXYKSYNBTL-UHFFFAOYSA-N [Nb].[Er] Chemical compound [Nb].[Er] GHLOAXYKSYNBTL-UHFFFAOYSA-N 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000001745 anti-biotin effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012131 assay buffer Substances 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
- 230000008901 benefit Effects 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- ZMIGMASIKSOYAM-UHFFFAOYSA-N cerium Chemical compound [Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce] ZMIGMASIKSOYAM-UHFFFAOYSA-N 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000005829 chemical entities Chemical class 0.000 description 1
- 210000000991 chicken egg Anatomy 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000014670 detection of bacterium Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- KBQHZAAAGSGFKK-UHFFFAOYSA-N dysprosium atom Chemical compound [Dy] KBQHZAAAGSGFKK-UHFFFAOYSA-N 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009149 molecular binding Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000009343 monoculture Methods 0.000 description 1
- 239000002159 nanocrystal Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 238000002161 passivation Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229920002857 polybutadiene Polymers 0.000 description 1
- 229920000867 polyelectrolyte Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002444 silanisation Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 201000010740 swine influenza Diseases 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- GZCRRIHWUXGPOV-UHFFFAOYSA-N terbium atom Chemical compound [Tb] GZCRRIHWUXGPOV-UHFFFAOYSA-N 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 239000003634 thrombocyte concentrate Substances 0.000 description 1
- XOLBLPGZBRYERU-UHFFFAOYSA-N tin dioxide Chemical compound O=[Sn]=O XOLBLPGZBRYERU-UHFFFAOYSA-N 0.000 description 1
- 229910001887 tin oxide Inorganic materials 0.000 description 1
- 229910052719 titanium Inorganic materials 0.000 description 1
- 239000010936 titanium Substances 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- BYMUNNMMXKDFEZ-UHFFFAOYSA-K trifluorolanthanum Chemical compound F[La](F)F BYMUNNMMXKDFEZ-UHFFFAOYSA-K 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- NAWDYIZEMPQZHO-UHFFFAOYSA-N ytterbium Chemical compound [Yb] NAWDYIZEMPQZHO-UHFFFAOYSA-N 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
-
- 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
-
- 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
Definitions
- the present invention relates to the field of techniques for the analysis of liquid samples.
- It relates in particular to the field of methods and systems for determining the presence and / or quantity of at least one analyte likely to be contained in a sample.
- Analytical techniques are concerned with the identification and characterization of chemicals, known as "analytes", in a sample.
- the analysis carried out can be qualitative, when the technique allows a search for the presence of the analyte in the sample; this analysis can be quantitative, when the technique further allows a measurement of the amount of analyte in the sample.
- Such analysis techniques include in particular biological analyzes when the desired analyte consists of a biological analyte.
- the analytical technique must ensure a selective and sensitive assay of the analyte likely to be contained in the sample analyzed.
- testing for pathogens may be based on propagation by culture before isolation. These methods are effective and sensitive but are generally laborious and over a long period of time (results are usually only available after several days).
- biosensors Other analytical tools rely on the specific molecular binding properties that are encountered in biological molecules known generically as biological sensors or biosensors (also called biosensors or “biosensors”).
- biosensors are particularly advantageous for the rapid and efficient determination of markers, typically biological markers, on account of their simplicity of use, their possible miniaturization and their capacity for analysis in real time.
- a biosensor indeed has the ability to recognize a target biological marker (biomarker or “target biomarker”), characteristic of the target analyte (for example a pathogen of interest).
- a target biological marker biological marker
- the biosensor carries a detection element commonly called a “bioreceptor” (also called a biological receptor or “bioreceptor”).
- bioreceptors consist for example of monoclonal antibodies, RNA, DNA, glycans, lectins, enzymes, tissues or whole cells.
- the bioreceptor plays a key role because its biochemical properties provide sensitivity and selectivity in the detection of biomarkers and help to avoid interference with other compounds in the test sample.
- biomarker The specific biochemical interaction between the biomarker and the bioreceptor is then converted, thanks to a tracer element (also called transducer or “transductor”), into a measurable signal.
- tracer element also called transducer or “transductor”
- the recording and display of the signal allows a qualitative, even quantitative, identification of the analyte in the analyzed sample.
- the tracer is carried directly by the biosensor.
- an analyte molecule is generally able to cooperate with a small number of biosensor / tracer molecules, or even with a single biosensor / tracer molecule.
- some analysis systems rely on cascade detection in which the bioreceptor is linked to an enhancer compound capable of receiving multiple tracer molecules.
- the present invention thus provides methods and systems suitable for determining, with increased sensitivity, the presence and / or quantity of at least one analyte likely to be contained in a sample.
- a method for determining the presence and / or the quantity of at least one analyte capable of being contained in a sample.
- said at least one analyte is chosen from biological analytes, in particular proteins, peptides, antibodies, hormones, steroids, antigens derived from infectious agents or from tumor cells, infectious agents such as bacteria , viruses or parasites, nucleic acids (DNA or RNA), therapeutic compounds, drugs or even antibiotics.
- the method according to the invention comprises:
- amplification probe of the core-envelope type, in which said envelope comprises several molecules with an affinity tag
- a tracer intended to convert the secondary complex into a visible and / or measurable signal, said tracer being bound or intended to bind with the molecules of the affinity tag of said at least one amplification probe, so that , in said secondary complex, at least some of the molecules of the affinity tag of said at least one amplification probe are linked with said label.
- Such a method thus allows, by a simple adaptation of the cascade detection systems, a significant amplification of the detection signal.
- the secondary complex obtained comprises the amplification probe which advantageously constitutes an interface capable of receiving a plurality of tracer molecules.
- the method according to the invention achieves indirect / cascade binding, in which a plurality of tracer molecules are linked to an analyte molecule.
- This new technology provides improved detection of all types of analytes, in particular biological analytes (microorganisms, virulence markers, antibiotic resistance markers, etc.).
- the addition of the amplification probe in a cascade detection system allows a significant increase in the signal.
- the affinity tag of the envelope of said at least one amplification probe consists of: - a first affinity tag, identical to the first affinity tag of said at least one primary probe, or
- a second affinity tag capable of binding specifically with said first affinity tag, at least part of said second affinity tag being in the form of a secondary probe consisting of a conjugate comprising, of a on the one hand, said second affinity tag and, on the other hand, a tracer, and
- amplification probe of the nucleus-envelope type, in which said envelope comprises several molecules of said first affinity tag, so as to obtain, starting from said primary complex, a secondary complex comprising analyte: primary probe: second affinity tag: amplification probe: secondary probes, said amplification probe of said secondary complex being linked to several molecules of said at least one secondary probe.
- a part of said second affinity tag of the revealing step is advantageously devoid of said tracer.
- the first affinity tag is chosen from biotin or homolog
- the second affinity tag is chosen from streptavidin or homolog, for example avidin, NeutrAvidin or Captavidin
- said at least one amplification probe comprises a core formed by a nanoparticle preferably having a size ranging from 10 to 100 nm; the nanoparticle is advantageously chosen from nanoparticles having a surface composed of silica, more preferably from nanoparticles composed of silica, advantageously also from silica beads; said at least one amplification probe comprises at least 20 molecules of said affinity tag;
- the envelope of said at least one amplification probe comprises spacers, connecting the heart with a first affinity tag molecule; the spacers are for example chosen from nucleic spacers, for example DNA sequences;
- the tracer is chosen from enzymes, fluorophores, radioisotopes, redox (or electroactive) molecules, magnetic, photoacoustic or photothermal particles; where appropriate, said at least one secondary probe is advantageously chosen from enzymatic conjugates or photoactive conjugates (colored, fluorescent, luminescent conjugates); the enzymatic conjugates are advantageously chosen from streptavidin-HRP or streptavidin-PA conjugates, and the reagents for the revelation step comprise a substrate of said enzymatic conjugates for a chemiluminescence reaction in the presence of said secondary complex;
- the revelation step is implemented in two successive phases with a first phase during which is added said second affinity tag, optionally at least a part in the form of said at least one secondary probe, to form a first sub-complex comprising analyte: primary probe: second affinity tag, then a second phase during which said at least one amplification probe and said at least one secondary probe are added to form said secondary complex, or a single phase during which the reagents are simultaneously added for the formation of the secondary complex;
- the binding site of said at least one primary probe is chosen from biomolecules, for example nucleic probes (RNA, DNA, aptamers sequences), protein probes (antibodies, enzymes, antigens, peptides);
- said at least one analyte is fixed on a support, for example for a method of the dot blot type, or on a capture probe, carried for example by a magnetic bead for a MELISA technique or by a support for a sandwich ELISA technique,
- the amplification step is carried out so as to obtain a secondary complex comprising several amplification probes in cascade / in series (for example two or three amplification probes); the secondary complex advantageously comprises at least two amplification probes linked together by at least a second affinity tag.
- the present invention also relates to the use of a core-envelope type probe as a signal amplification probe in a cascade detection method for determining the presence and / or the quantity of at least one.
- analyte capable of being contained in a sample
- said amplification probe comprising an envelope which comprises several molecules of an affinity tag.
- the amplification probe comprises an envelope which comprises several molecules of a first affinity tag each capable of specifically binding to a second affinity tag, at least part of said second affinity tag being intended in the form of a secondary probe consisting of a conjugate comprising, on the one hand, said second affinity tag and, on the other hand, a tracer.
- the nucleus-envelope type amplification probe comprises an envelope which comprises several molecules of said second affinity tag, at least some of which are linked to at least one molecule of said tracer.
- the present invention also relates to the reagent system, for implementing the method according to the invention.
- the reagent system includes:
- At least one primary probe which at least one primary probe is a conjugate comprising, on the one hand, a binding site capable of binding specifically with said analyte and, on the other hand, a first affinity tag, and
- At least one revelation reagent capable of forming a complex starting from the primary probe which at least one revelation reagent comprises:
- nucleus-type amplification probe - envelope in which said envelope comprises several molecules with an affinity tag
- a tracer intended to convert the complex into a visible and / or measurable signal, said tracer being bound or intended to bind with the molecules of the affinity tag of said at least one amplification probe, so that, in said complex, at least some of the molecules of the affinity tag of said at least one amplification probe are linked to at least one molecule of said tracer.
- the revelation reagents comprise:
- a second affinity tag capable of binding specifically with said first affinity tag, at least part of said second affinity tag being in the form of a secondary probe consisting of a conjugate comprising, of a on the one hand, said second affinity tag and, on the other hand, a tracer (enzyme, fluorophore, radioisotope, redox molecule, etc.), and
- amplification probe of the nucleus-envelope type, in which said envelope comprises several molecules of said first affinity tag, which reagent system is capable of forming, in the presence of said analyte, a complex comprising analyte: primary probe: second affinity tag: amplification probe: secondary probes, said amplification probe of said secondary complex being bound to several molecules of said at least one secondary probe.
- the amplification probe, of the nucleus-envelope type has an envelope which comprises several molecules of said second affinity tag, at least some of which are linked to at least one molecule of said tracer, so in obtaining, starting from said primary complex, a secondary complex comprising analyte: primary probe: amplification probe.
- FIG. 1 is a schematic view of a primary complex between an analyte and a primary probe (antibody type), which is obtained at the end of the recognition step according to the invention;
- FIG. 2 is a schematic view of a first amplification probe suitable for the method according to the invention.
- FIG. 3 is a schematic view of a secondary complex resulting from the revealing step, obtained by the cascade binding of the primary complex with in particular an amplification probe (according to FIG. 2) linked to several molecules of a secondary probe forming a tracer;
- FIG. 4 is a schematic view of a secondary complex in which the analyte is itself fixed by a magnetic bead during an assay in the form of enzymatic immuno-absorption on a magnetic support bead (also called “magnetic bead-based enzyme -linked immunosorbent assay ”or MELISA);
- FIG. 5 is a schematic view of a secondary complex, in which the analyte is itself supported on a support by a molecular dot blot hybridization technique
- FIG. 6 is a schematic view of a second amplification probe suitable for the method according to the invention
- FIG. 7 is a schematic view of a secondary complex resulting from the revealing step, obtained by the cascade binding of the primary complex with an amplification probe according to FIG. 6 carrying several tracer molecules;
- FIG. 8 is a figure illustrating the absorbance (450 - 550 mm) by Biot-NP MELISA technique as a function of plaque-forming units (PFU / mL), for whole viruses from different strains (H1 N1, H3N2, PRRSV ) in a buffer containing 10% saliva;
- FIG. 9 is a view comprising (A) Dot blot detection of Campylobacter DNA sequence with improved reading by biotin-Si-NP. It should be noted that no signal was obtained with a truncated Campylobacter (PR) sequence or with a control sequence of E. Coli (PE). (B) Conventional dot blot detection of the biotin-labeled CampyP3 probe using the dilution of the Campylobacter CP3 complementary sequence ranging from 1 ng / ⁇ l to 78 fg / ⁇ l.
- PR truncated Campylobacter
- PE E. Coli
- FIG. 10 is a view showing the results of the inclusivity and exclusivity tests of the enhanced dot blot sensor in pure cultures
- FIG. 11 illustrates a dot blot membrane obtained with naturally infected (C * 3 and C10) and uninfected (C4, C5 and C6) chicken samples; the CP3 template sequence (0.1 ng / pL) was used as a positive control.
- the present invention relates to methods and systems for determining the presence and / or amount of at least one analyte likely to be contained in a sample.
- analyte is understood to mean any chemical, biochemical or biological entity that it is desired to detect in a sample.
- This chemical entity advantageously consists of an entity resulting from the living world, preferably from the plant or animal world, or even present in humans.
- analytes detectable by the devices and methods according to the present invention there may be mentioned in particular biological analytes, in particular proteins, peptides, antibodies, hormones, steroids, antigens derived from infectious agents or from tumor cells. , infectious agents such as bacteria, viruses or parasites, nucleic acids (DNA or RNA), therapeutic compounds, drugs or even antibiotics.
- biological analytes in particular proteins, peptides, antibodies, hormones, steroids, antigens derived from infectious agents or from tumor cells.
- infectious agents such as bacteria, viruses or parasites, nucleic acids (DNA or RNA), therapeutic compounds, drugs or even antibiotics.
- sample is meant any sample in which the desired analyte is in solution or in suspension.
- This sample can in particular be any biological or bodily fluid.
- the sample may also have been obtained directly or indirectly from a biological or bodily fluid.
- the sample can also be a liquid extract from a solid or gaseous sample.
- the sample is for example obtained from water, air, soil, biological materials (tissues, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, genetically modified organisms, or materials derived from GMOs, etc.).
- biological materials tissues, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, genetically modified organisms, or materials derived from GMOs, etc.
- the method and the system according to the invention are advantageous in that they can be adapted both to the determination of the presence of at least one analyte, as to the determination of its quantity.
- detecting or “determining” is thus meant the qualitative determination (advantageously the presence or absence) of one or more analytes in a sample.
- detect or “determine” also includes the measurement and quantification of one or more analytes in the sample.
- the method according to the invention comprises the following successive steps:
- a revelation step B (FIGS. 3 and 7 in particular) during which is added at least one revelation reagent suitable for forming a secondary complex C2 starting from the primary probe 1 (and preferably from a first label d affinity 12 of said primary probe 1) of said primary complex C1.
- said at least one revealing reagent comprises:
- said at least one revelation reagent is intended to bind, in cascade, from said at least one primary probe 1 (and advantageously from its first affinity tag 12) to form the secondary complex C2.
- said at least one revelation reagent used makes it possible to obtain, starting from the primary probe 1 of the primary complex C1, a secondary complex C2 in which at least some of the molecules of the label d 'affinity 321 of said at least an amplification probe 3 are linked (directly or indirectly) with at least one molecule of the tracer 21 ( Figures 3 and 7).
- said tracer 21 can advantageously have two different forms:
- the tracer 21 is a separate reagent compared to said at least one amplification probe 3; the tracer 21 is intended to bind with the affinity tag 321 of said at least one amplification probe 3 (FIG. 3 in particular), or
- the tracer 21 and the amplification probe 3 form a single reagent; the tracer 21 is linked with the affinity tag 321 of said at least one amplification probe 3 ( Figures 6 and 7).
- the tracer 21 also called “marker”, “transducer” or “transductor”, is intended to convert the secondary complex C2 into a visible and / or measurable signal.
- tracer thus means any entity allowing direct or indirect detection with the naked eye, or with the aid of a device, due to the emission of a signal.
- the signal is, for example, fluorescence, coloring, the presence of isotope or a magnetic signal.
- the tracer 21 can thus be chosen from conventional tracers per se.
- this tracer 21 is chosen from enzymes, fluorophores, radioisotopes, redox (or electroactive) molecules, magnetic particles, photoacoustic particles or photothermal particles.
- any strong bond for example covalent (in particular for a single reagent), but also
- any weak binding for example of the antigen / antibody or analyte / anti-analyte type or affinity tags (in particular for separate reagents).
- the affinity tag 321 of the envelope 32 of said at least one amplification probe 3 is chosen from:
- the revealing step B leads to adding at least the following revealing reagents:
- the revelation reagents 2, 3, 5 used make it possible to obtain, starting from the primary probe 1 of the primary complex C1, a secondary complex C2 comprising analyte T: primary probe 1: second label d affinity 5: amplification probe 3: secondary probes 2.
- the amplification probe 3 of this secondary complex C2 is in particular linked to several molecules of said at least one secondary probe 2.
- a plurality of molecules of said at least one secondary probe 2 (carrying the tracer) is linked to an analyte T molecule.
- This structure offers a significant amplification of the signal obtained for each T analyte molecule present, thanks to the multiplication of the secondary probes 2 making up the secondary complex C2 (reported on the amplification probe 3).
- amplification probe 3 of the core 31-envelope 32 type in which said envelope 32 comprises several molecules of said second label.
- the amplification probe 3 used makes it possible to obtain, starting from the primary probe 1 of the primary complex C1, a secondary complex C2 comprising analyte T: primary probe 1: amplification probe 3.
- a plurality of tracer molecules 21 are linked to an analyte T molecule.
- This structure then offers a significant amplification of the signal obtained for each T analyte molecule present, thanks to the multiplication of the tracers 21 carried by the amplification probe 3.
- the revealing reagents are assembled in cascade by specific molecular bonds based on complementary affinity tags.
- the reagents used in the process advantageously comprise at least one pair of affinity labels, or even a single pair of affinity labels.
- affinity tags is advantageously meant a pair of chemical, biochemical or biological entities which are able to bind specifically with one another to form a complex.
- affinity tags include in particular the biological ligands chosen from peptides and oligonucleotides.
- affinity tags are chosen from the couple:
- biotin also called “biotin” or “biot” in the examples
- biotin also called “biotin” or “biot” in the examples
- streptavidin or homolog for example avidin, NeutrAvidin, Captavidin
- sample E is thus brought into contact with said at least one primary probe 1 forming a biosensor.
- said at least one primary probe 1 therefore consists of a conjugate comprising:
- binding site 11 still forming a "bioreceptor" or recognition element, capable of binding specifically with the analyte T to form, if necessary, the primary complex C1, and
- the secondary complex C2 is advantageously formed, in cascade, from the first affinity tag 12.
- Said at least one primary probe 1, and in particular its binding site 11, consists of any chemical, biochemical or biological entity which is able to bind specifically to the analyte T to form the primary complex C1 (advantageously binary).
- bind or “binding” is meant any strong bond, for example covalent, but also any weak bond, for example of the antigen / antibody or analyte / anti-analyte type.
- Such a primary probe 1, and in particular its binding site 11, is thus advantageously chosen from biomolecules, namely for example:
- Figure 1 - protein probes ( Figure 1), including antibodies, enzymes, antigens, peptides, or
- FIG. 5 - nucleic acid probes (FIG. 5), encompassing the RNA sequences, the DNA sequences, the aptamers.
- the primary probe 1 (and in particular its binding site 11) is chosen for its specific binding properties to the T analyte, such as for example a ligand / anti-ligand pair, an antigen / antibody pair, a DNA / RNA pair. or a DNA / DNA pair.
- the binding site 11 is advantageously an antibody specific for the analyte.
- antibody specific for the analyte is meant an antibody capable of binding specifically with the analyte in a binding of the antigen / antibody type.
- It is typically a polyclonal antibody or a monoclonal antibody, having a high affinity for the analyte.
- it is a monoclonal antibody.
- the binding site 11 is advantageously a complementary DNA probe.
- the primary probe 1 consists of a probe capable of binding to the DNA of Gram-negative bacteria of the genus Campylobacter, and more preferably DNA probes specific for Campylobacter jejuni, Campylobacter coli, Campylobacter tari or Campylobacter upsaliensis as described. for example in the patent application IT102020000012496.
- the parameters (incubation time, rinsing, etc.) are adjusted so as to allow the formation of the primary complex C1 in the presence of the analyte T.
- the first affinity tag 12 of the primary probe 1 is advantageously chosen from biotin or homolog.
- binding site 11 and the first affinity tag 12 are advantageously linked by covalent bonds.
- said at least one analyte T Prior to bringing said at least one primary probe 1 together, said at least one analyte T can be fixed:
- a capture probe 6 carried for example by a magnetic ball B for a so-called MELISA technique or by a support for a sandwich ELISA technique, or
- the aforementioned capture probe 6 consists of any chemical, biochemical or biological entity which is able to bind specifically to the analyte T.
- bind or “binding” is meant any strong bond, for example covalent, but also any weak bond, for example of the antigen / antibody or analyte / anti-analyte type.
- Such a capture probe 6, and in particular its binding site is thus advantageously chosen from biomolecules, namely for example:
- - protein probes including antibodies, enzymes, antigens, peptides, or
- nucleic acid probes encompassing RNA sequences, DNA sequences, aptamers.
- the capture probe 6 (and in particular its binding site) is chosen for its specific binding properties to the T analyte, such as for example a ligand / anti-ligand cut, an antigen / antibody pair, a DNA / RNA pair. or a DNA / DNA pair.
- dot blot encompasses techniques for transferring molecules which make it possible to verify the presence of specific molecules in a medium, including the Southern blot for the DNA, the northern blot for the RNAs or the western blot for the proteins.
- dot blot also encompasses the transfer of nucleic acids (DNA or RNA) from a liquid medium directly onto a membrane, without prior separation (therefore without prior electrophoresis).
- the transfer can be carried out by simple diffusion, by diffusion in an electric field (electrodiffusion) or by vacuum suction.
- Revelation step B
- the revelation step B is carried out so as to detect, by means of at least one revelation reagent, any C1 primary complexes generated during the recognition step A.
- said at least one revelation reagent comprises:
- the second affinity tag 5 is adapted to bind specifically with the first affinity tag which is carried by the primary probe 1 or, as developed later, by said at least one amplification probe 3.
- the second affinity tag 5 is advantageously chosen from streptavidin or homologous, for example avidin or NeutrAvidin or Captavidin.
- the second affinity tag 5 may appear:
- the second affinity tag 5 in free form may represent 10 to 50%, more preferably 20 to 50%, of the second affinity tag molecules 5.
- the aforementioned secondary probe 2 consists of a detection reagent in the form of a conjugate comprising:
- an affinity tag 22 which consists of the aforementioned second affinity tag 5.
- this affinity tag of the secondary probe 2 is designated indifferently by the references 5 or 22 and can also be referred to as “second affinity tag”.
- the tracer 21 can thus be chosen from conventional tracers per se.
- this tracer 21 is chosen from enzymes, fluorophores, radioisotopes, redox (or electroactive) molecules, magnetic particles, photoacoustic particles, or photothermal particles.
- Said at least one secondary probe 2 is thus advantageously chosen from enzymatic conjugates or photoactive conjugates.
- enzyme conjugates is understood to mean conjugates with the enzymes H RP (horseradish peroxidase) or PA (alkaline phosphatase).
- the reagents for development step B advantageously comprise a substrate 4 for the enzymatic conjugates for a chemiluminescence reaction in the presence of the secondary complex C2.
- the substrate 4 can thus be chosen from, for example, the following compounds:
- TMB - chromogenic
- ADHP fluorescent
- Luminol chemiluminescent
- pNPP - chromogenic
- MUP fluorescent
- FPD chemiluminescent
- photoactive conjugates includes colored conjugates, fluorescent conjugates and luminescent conjugates.
- said at least one secondary probe 2 consists of an enzymatic conjugate chosen for example from streptavidin-HRP (horseradish peroxidase) or streptavidin conjugates.
- -PA alkaline phosphatase
- said at least one amplification probe 3 is of the core 31 (also called “heart”) - envelope 32 type.
- Envelope 32 includes multiple molecules of an affinity tag 321 (also referred to as "first affinity tag 321") which is identical to the first affinity tag 12 of primary probe 1.
- said at least one amplification probe 3 advantageously comprises at least 20 molecules of said first affinity tag 321.
- These molecules of said first affinity tag 321 are able to bind specifically with at least one molecule of the second affinity tag 5, 22, namely:
- the envelope 32 of said at least one amplification probe 3 also advantageously comprises spacers 322 which connect the nucleus 31 with a first affinity tag molecule 321.
- Such spacers 322 aim to separate the molecules of the first affinity tag 321 with respect to the nucleus 31 and with respect to each other, thus limiting the problems of steric hindrance during the revealing step B.
- This structure thus makes it possible to free the environment from the first affinity tags 321, and to increase the number of molecules of said at least one secondary probe 2 which is bound simultaneously with the amplification probe 3.
- This arrangement also allows a better presentation of the first affinity tags 321, thus promoting the recognition reaction (advantageously increase in the probability of reaction) with said at least one secondary probe 2.
- the spacers 322 are advantageously chosen from nucleic spacers, for example DNA sequences (for example a dTio-PEG 2 -dTio sequence).
- the core 31 is for its part advantageously formed by a nanoparticle 31 (also called “NP” in the examples) preferably having a size ranging from 10 to 100 nm.
- the size of the particles is defined as their largest size and can be measured using a scanning electron microscope or an optical microscope.
- the size of heterogeneously shaped objects is defined as the length of the larger side of the object.
- the nanoparticle 31 is advantageously chosen from nanoparticles having a surface composed of silica (including silicate).
- such nanoparticles are also chosen from nanoparticles composed of silica (or even silicate), advantageously also from silica beads.
- the nanoparticles can be chosen from silica nanoparticles (also called “Si-NP”), gold, silver, graphite, fluorescent nanocrystals (also called quantum dots), polymer nanoparticles of the type polystyrene, polypropylene or polybutadiene, or metal oxide nanoparticles, in particular of tin oxide, gadolinium, aluminum, titanium, cerium, erbium niobium, ytterbium, terbium, dysprosium , europium, or a mixture of these oxides, optionally coated to allow their functionalization.
- silica nanoparticles also called “Si-NP”
- gold silver
- graphite also called quantum dots
- polymer nanoparticles of the type polystyrene polypropylene or polybutadiene
- metal oxide nanoparticles in particular of tin oxide, gadolinium, aluminum, titanium, cerium, erbium niobium, ytterbium,
- the revelation step B is carried out under conditions suitable for generating the secondary complex C2 ( cascade), starting from the primary probe 1 of said primary complex C1, comprising analyte T: primary probe 1: second affinity tag 5 (free or conjugated): amplification probe 3: secondary probes 2.
- the amplification probe 3 of this secondary complex C2, carrying a plurality of molecules of first affinity tag 321, is thus advantageously linked to several molecules of said at least one secondary probe 2 (via their second affinity tags 22) .
- This configuration then allows a significant amplification of the signal, with a plurality of molecules of secondary probe 2 (intended to produce a signal) for one molecule of analyte T.
- disclosure step B can take two alternative embodiments.
- the revealing step B is implemented in two successive phases with:
- the second phase B2 can itself be divided into two successive operations (successive addition of the revelation reagents):
- this first embodiment comprises a preparation phase during which said at least one amplification probe 3 is linked with the secondary probe 2 (by means of their affinity tags 321, 22), to form an amplification probe 3 / secondary probes 2 complex.
- the second phase B2 is implemented by directly adding the amplification probe 3 / secondary probes 2 complex which is intended to bind with the second affinity tags 5 (free or conjugated) of the first sub-complex. .
- the revelation step B is carried out in a single phase during which the reagents are added simultaneously for the formation of the secondary complex C2.
- the method advantageously comprises a preparation phase during which said at least one amplification probe 3 is linked with several molecules of secondary probes 2 (by means of their affinity tags 321, 22), to form a Amplification probe 3 / secondary probe complex 2.
- revelation step B is carried out by directly adding the amplification probe 3 / secondary probes 2 complex which is intended to bind with the primary probe 1 if it is still present.
- the amplification probe 3 / secondary probes 2 complex binds with the first affinity tag 12 of the primary probe 1, this via the second affinity tags 5, 22 (free or conjugated ).
- the amplification probe 3 used is similar to that described above in relation to FIGS. 1 to 5 in that it is of the core 31 - shell 32 type with a shell 32 which comprises several molecules of an affinity tag 321.
- This amplification probe 3 is distinguished in that the envelope 32 comprises several molecules of a second affinity tag 321 which is complementary to the first affinity tag 12 of the primary probe 1.
- At least 50% of the molecules of the second affinity tag 321 are linked to at least one molecule of the tracer 21.
- bound is understood here advantageously to mean a strong bond, preferably a covalent bond.
- the second affinity tag 321 / tracer 21 pair is thus advantageously chosen from the enzymatic conjugates or the photoactive conjugates, grafted onto the amplification probe 3.
- said at least one amplification probe 3 advantageously comprises at least 20 molecules of said second affinity tag 321.
- the second affinity tag 321 is adapted to bind specifically with the first affinity tag 12 which is carried by the primary probe 1.
- the second affinity tag 321 is advantageously chosen from streptavidin or homologous, for example avidin or NeutrAvidin or Captavidin.
- the envelope 32 of said at least one amplification probe 3 also advantageously comprises spacers 322 which connect the nucleus 31 with a second affinity tag molecule 321.
- the revelation step B is carried out under conditions suitable for generating the secondary complex C2 (in cascade) comprising analyte T: primary probe 1: amplification probe 3.
- the amplification probe 3 of this secondary complex C2 carries a plurality of second affinity tag 321 / tracer 21 conjugates.
- This configuration then allows a significant amplification of the signal, with a plurality of tracer molecules 21 (intended to produce a signal) for an analyte molecule T.
- the revelation step B is then advantageously carried out in a single phase during which the amplification probe 3 is added for the formation of the secondary complex C2.
- revelation step B is carried out by directly adding the amplification probe 3 which is intended to bind with the primary probe 1 if it is still present.
- the amplification probe 3 binds with the first affinity tag 12 of the primary probe 1, through the second affinity tags 321.
- the amplification step could be implemented so as to obtain a secondary complex C2 comprising several amplification probes 3 in cascade / in series (for example two or three probes d. amplification 3), before adding the tracer 21.
- the secondary complex C2 then advantageously comprises at least two amplification probes 3 linked together by at least one second affinity tag 5. Such an approach would make it possible to multiply the amplification effect.
- the revelation step B also advantageously includes a final reading phase during which:
- the analyte T is detected (presence of the analyte T in the sample E), when a signal is detected due to the formation of the secondary complex C2 comprising the tracer 21, or
- the present invention also relates to the reagent system, or kit, for implementing the method according to the invention.
- said at least one primary probe 1 which at least one primary probe 1 is a conjugate comprising, on the one hand, the binding site 11 capable of binding specifically with said analyte T and, on the other hand, the first label affinity 12, and
- a tracer 21 intended to convert the complex C2 into a visible and / or measurable signal, said tracer 21 being bound or intended to bind with the molecules of the affinity tag 321 of said at least one amplification probe 3 , so that, in said complex C2, at least some of the molecules of the affinity tag 321 of said at least one amplification probe 3 are linked to at least one molecule of said tracer 21.
- the revelation reagents comprise:
- said second affinity tag 5 capable of binding specifically with said first affinity tag 12, at least part of said second affinity tag 5 being in the form of said secondary probe 2 consisting of a conjugate comprising, on the one hand, said second affinity tag 5 and, on the other hand, the tracer 21, and - said at least one amplification probe 3, of the core 31 type - envelope 32, in which said envelope 32 comprises several molecules of said first affinity tag 12.
- such a system of reagents is capable of forming, in the presence of said analyte T, a C2 complex according to the invention which comprises analyte T: primary probe 1: second affinity tag 5: amplification probe 3: secondary probes 2.
- Amplification probe 3 of this C2 complex then advantageously carries several molecules of said at least one secondary probe 2.
- the first affinity tag 12 is chosen from biotin or homolog (for example a silica nanoparticle carrying biotin molecules, also called “biotin-Si-NP” or “biotin-nanoparticle” in the examples).
- the second affinity tag 5 is selected from streptavidin or homolog, eg, avidin, NeutrAvidin or Captavidin.
- such a reagent system advantageously comprises:
- said at least one primary probe 1 which at least one primary probe 1 is a conjugate comprising, on the one hand, the binding site 11 capable of binding specifically with said analyte T and, on the other hand, the first label affinity 12, and
- the amplification probe 3 used makes it possible to obtain, starting from the primary probe 1 of the primary complex C1, a secondary complex C2 comprising analyte T: primary probe 1: amplification probe 3.
- a plurality of tracer molecules 21 are linked to an analyte T molecule.
- the first affinity tag 12 is chosen from biotin or homolog.
- the second affinity tag 321 is selected from streptavidin or homolog, eg, avidin, NeutrAvidin or Captavidin.
- the detection system according to the invention can very well be adapted to the desired T analyte.
- the detection system is advantageously in dot blot form for the search for an analyte of the nucleic acid type (see Figure 6).
- the binding site 11 of the primary probe 1 is advantageously a complementary DNA probe, preferably coupled to biotin.
- the detection system is advantageously in ELISA or MELISA form for the search for an analyte of the antigen or hapten type (see FIGS. 1 and 4).
- the binding site 11 of the primary probe 1 is advantageously an antibody specific for the analyte, preferably coupled to biotin.
- the system also advantageously comprises a above-mentioned capture probe 6, carried for example by a magnetic ball B.
- Example 1 Manufacturing process of an amplification probe
- Si-NPs silicon nanoparticles
- Si-NPs silicon nanoparticles
- an innovative solid-phase synthesis technology that allows the functionalization of nanometric-sized particles with DNA fragments, as previously reported (Bonnet et al, Analyst 2018, Issue 10, 2018; De Crozals et al, RSC Advances, 2012, 2, 11858-11866).
- CPG Controlled Pore Glass
- the linker which allows better accessibility of the functions of interest, has been synthesized.
- biotin incorporation was monitored at 498 nm using quantitation of dimethoxytrityl by a UV-visible spectrophotometer.
- the biotin-Si-NP were released from the CPG by incubating the support in 1mL of 0.1% (w / v) DBU in a 1/1 (v / v) water-acetonitrile mixture.
- the DBU solution was stirred in a thermomixer at 22 ° C for 1 hour before being collected.
- NP nanoparticles
- the release kinetics were followed by quantifying the concentration of NP (nanoparticles) in each DBU solution by measuring the U V-visible at 560 nm.
- the released nanoparticles were washed with milli-Q water (1x 4mL then 3x2 mL) and concentrated on a 30K Amicon Ultra filter (5000g, 10 min).
- the amount of dT10-PEG2-dT10-10% Biotin strands per NP was estimated using a Varian Cary 100 Bio UV-visible spectrophotometer (Agilent Technologies) using a quartz cuvette with 1 cm optical path.
- the nanoparticles were released from the CPG by incubating the support in 1mL of a 0.1% (w / v) solution of DBU (1,8-Diazabicyclo [5.4.0] undec-7-ene) in a mixture. water- acetonitrile 1/1 (v / v).
- the DBU solution was stirred in a thermomixer at 22 ° C for 1 hour before being collected.
- a fresh solution of DBU was added to the CPG suspension every hour.
- the release kinetics were followed by quantifying the nanoparticle concentration of each DBU solution by measuring the UV-visible at 560 nm.
- the released nanoparticles were washed with water (1x 4mL then 3x2 mL) and concentrated on a 30K Amicon Ultra filter (5000g, 10 min).
- the amount of strands per NP was estimated using a Varian Cary 100 Bio UV-visible spectrophotometer (Agilent Technologies) using a quartz cuvette with 1 cm optical path.
- the amount of linker grafted onto the nanoparticles was quantified by measuring the absorbance in water (200pL) at 260nm and 560nm using a microplate reader (Perkin Elmer).
- the concentration of nanoparticles was estimated as described previously by Bonnet et al. (Analyst 2018). To correspond to this estimate, we established an approximate molar extinction coefficient at 163,200 M-1 cm-1 which takes into account the epsilons of the different parts of the sequence corrected by their corresponding coupling efficiency.
- the functionalized NPs were observed under a transmission electron microscope (TEM) using a Philips CM120 device operating at an acceleration voltage of 120kV (Center Technonova des Microstructures, Lyon).
- TEM transmission electron microscope
- Philips CM120 device operating at an acceleration voltage of 120kV (Center Technonova des Microstructures, Lyon).
- the Si-NPs were observed after depositing 5 ⁇ l of dilute solution on a copper grid covered with Formvar-carbon and evaporation to dryness.
- Biotin-Si-NP were prepared using an innovative solid phase synthesis technology which allowed very high functionalization (Crozals et al., RSC Adv. 2012, 2, 11858-11866).
- a hydroxyl group was attached to the surface of Si-NPs by silanization as previously reported (Farre et al., Langmuir, 2010; Bonnet et al, Analyst 2018).
- the hydroxyl functions served as a support for the synthesis of an oligonucleotide linker on the surface of the NPs.
- biotin groups were grafted to the linker as explained in Materials and Methods. MET observations showed that the NP morphology remained stable during synthesis.
- Functionalized biotin-Si-NP had an average size of 50 ⁇ 3 nm, as estimated from TEM images. According to the absorbance measurements, the amount of linker was about 500 per NP. Furthermore, quantification of dimethoxytrityl established the coupling efficiency of biotin at 10%, which allowed biotin to be quantified at about 50 by N P. A relatively narrow particle size distribution of the functionalized NPs was confirmed by DLS measurements.
- the Si-NPs formed monodisperse aqueous solutions of particles having a hydrodynamic diameter (RH) of about 90 nm. The conjugation of biotin + linker groups shifted the RH to about 120 nm. This increase is probably related to the hydration layer around the biotin units bound to the arms bearing negatively charged groups. Biotin-Si-NP solutions were stable at 4 ° C for at least two months.
- Example 2 Double recognition strategy for sensitive detection of influenza A virus using nanoparticles coated with biotin and immunomagnetic beads
- Bovine Serum Albumin (BSA), Bradford Serum, TMB ELISA Colorimetric Reagent (3,3 ', 5,5'-Tetramethylbenzidine), Tween 20, HRP-Streptavidin, 5X ELISA Assay Buffer B / Sample Thinner and high quality chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). 10x Phosphate Buffered Saline (PBS) was provided by Andrea Dutscher (Brumath, France). Sodium chloride was purchased from Laurylab (Brindas, France). The buffers were prepared with deionized water and sterilized by 0.22 ⁇ m filtration.
- the Dynabeads® M-280 Tosylactivated and DSB-XTM Biotin Protein Labeling Kit (D-20655) are respectively from Invitrogen and Molecular Probes (Thermo Fisher Scientific, Waltham, MA, USA). Influenza A (9G8) and (5D8) anti-NuP antibodies were obtained from Santa Cruz Biotechnology (St. Cruz, CA, USA). The rhodamine coated silica nanoparticles were supplied by Nano-H (Saint Quentin Fallavier, France). Oligonucleotides were synthesized using an Applied Biosystems 394 DNA / RNA synthesizer (Applied Biosystems, Foster City, CA).
- the phosphoramidite synthons Biotin-TEG-phosphoramidite (1-dimethoxytrityloxy-3-0- (N-biotinyl-3-aminopropyl) - triethylene glycolyl-glyceryl-2-0- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite); Phosphoramidite spacer 9 (9-0-dimethoxytrityl-triethylene glycol, 1 - [(2-cyanoethyl) - (N, N-diisopropyl)] - phosphoramidite); dT-CE phosphoramidite (5'-dimethoxytrityl-2'-deoxythymidine, 3 '- [(2 cyanoethyl) - (N, N-diisopropyl)] - phosphoramidite) and all synthesis reagents were purchased from Glen Research (Sterling , Virginia).
- nucleoprotein (NuP) gene of H1N1 influenza virus strain A / WSN / 33
- strain A / WSN / 33 The full length nucleoprotein (NuP) gene of H1N1 influenza virus (strain A / WSN / 33) with a 6-His tag at its C-terminus was cloned into Escherichia coli expression vector pET22 (Novagen) and expressed in E. coli BL-21 Rosetta DE3 (Stratagene).
- Transformed cells were grown at 37 ° C in 100 ml of Luria-Bertani medium containing ampicillin up to the mid-exponential phase, then induced for 4 hours by addition of 1 mM isopropyl ⁇ -D-thiogalactopyranoside at 37 ° C. with stirring. NuP was purified.
- RNAse A 0.15 mg / ml at 35 ° C for 20 min in the presence of 10 mM Mg 2+ ions.
- the protein was purified by affinity chromatography using magnetic Ni beads (PureProteome, Millipore), following the procedure recommended by the manufacturer. NuP was then dialyzed against Tris buffer (20mM Tris, 300mM NaCl, pH 7.5). After dialysis, the protein concentration was determined using an extinction coefficient e of 56200 M 1 cm -1 at 280 nm.
- influenza A / WSN / 33 (H1N1) virus was obtained by reverse genetics and amplified on MDCK cells. Viruses were titrated by the plaque method. Purified viruses were inactivated by UV light for 38 min. The inactivation efficiency was verified by re-infection of MDCK cells. Inactivated influenza A / H1 samples were stored at -80 ° C. Influenza A / UDORN / 72 (H3N2) was spread on an embryonic chicken egg and applied to MDCK cells. Viruses were titrated by the plaque method. Purified viruses were inactivated using binary ethyleneimine (BEI) and stored at -80 ° C.
- BEI binary ethyleneimine
- the Flanders13 (13V09) porcine respiratory reproductive syndrome virus (VSDRP) used in this study was kindly provided by Dr. Nicolas Bertho (INRA, France) and Dr. Fanny Blanc (INRA, France).
- viral concentrations were determined in terms of MDCK cell infection.
- the initial virus titers were 10 8 PFU / mL for H1N1 sample 1, 1.5 10 8 PFU / mL for H1N1 sample 2, 10 8 PFU / mL for H2N3.
- Control VSDRP virus was titrated at 10 7 PFU / mL.
- tosyl activated magnetic beads (Dynabeads TM M-280, Invitrogen, US) were coated with 10 ⁇ g of anti-NuP Influenza A antibody (9G8) per mg of beads for 1 hour. with moderate agitation, according to the protocol recommended by the manufacturer. Then, after 15 minutes of passivation in BSA buffer (0.1M sodium phosphate, 150mM sodium chloride, 0.5% (w / v) BSA, pH 7.6), the magnetic beads were washed. three times in 0.01 M sodium phosphate, 150 mM sodium chloride, 0.1% (w / v) BSA, pH 7.4 and stored at 4 ° C until testing.
- BSA buffer 0.1M sodium phosphate, 150mM sodium chloride, 0.5% (w / v) BSA, pH 7.6
- MELISA protocol for the detection of disabled viruses 1.10 6 magnetic beads previously coated with anti-NuP antibodies for Influenza A (9G8) were first of all washed three times with PBS buffer. Solutions of deactivated virus (PBS, 10% saliva) were incubated with the beads with moderate agitation at room temperature for 1 hour. The beads were concentrated by means of a magnet and washed three times with Wash Buffer 0.1. Conjugated anti-biotin antibody (0.5 ⁇ g / ml) in Capture Buffer (PBS, 0.1% tween, 0.1% BSA) was added to the suspension before gentle stirring for 1 hour. .
- Streptavidin / HRP developing solution 100 ⁇ L, 1 ⁇ g / mL was added and the mixture was stirred at room temperature for 30 minutes at 1000rpm in a thermomixer. The beads were washed three times with Washing Buffer 0.1 before adding 100 ⁇ L of TMB and incubating in the dark for 30 minutes at room temperature. Finally, the supernatant was diluted with 100 ⁇ l of 2M sulfuric acid and the absorbance at 450 nm was read in a microplate reader (Perkin Elmer). A450 nm - Asso nm gave the correct value after removing the background signal.
- biotin-NP (1.10 11 NP / mL in PBS, Tween 20 at 0.1% (v / v)) were added to the suspension of magnetic beads before a incubation for 30 minutes at 1000 rpm in a thermomixer.
- a streptavidin / HRP developing solution 100 ⁇ L, 1 ⁇ g / mL was added before the suspension was incubated at room temperature for 30 minutes at 1000 rpm in a thermomixer.
- the beads were washed three times with Washing Buffer 0.1 before adding 100 ⁇ L of TMB and incubating in the dark for 5 minutes at room temperature. Finally, the supernatant was diluted with 100 ⁇ l of 2M sulfuric acid and the absorbance at 450 nm was read in a microplate reader (Perkin Elmer). A450 nm - Asso nm gave the correct value after removing the background signal.
- biotin-NPs to amplify the positive signal in the detection step.
- Silica nanoparticles 50 nm in diameter were surface functionalized with an oligonucleotide strand carrying a biotin group at its end.
- the NPs were first immobilized on a controlled porosity glass support (CPG).
- CPG controlled porosity glass support
- NOM Nano-on-Micro
- SEM Sccanning Electron Microscopy
- the NOM material was used in a DNA / RNA synthesizer to perform NP functionalization via phosphoramidite chemistry. This strategy has made it possible to obtain highly functionalized nanoparticles.
- a spacer oligonucleotide (ODN sequence: dTio-PEG2-dTio) was synthesized on the NPs in order to remove the biotins from the surface of the NPs.
- ODN per NP About 500 ODN per NP were estimated by measuring UV absorbance at 260 nm and 560 nm and quantifying the ODN per NP ratio in the suspension, a protocol described by Bonnet et al. (Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: Application to the sensitive detection of bacteria in human platelet concentrates. Analyst. 143 (10), 2293-2303 (2016)). Then the biotin group was incorporated. We decided to control the synthesis program on the device to obtain a coupling efficiency of 10% for the incorporation of biotin. Using this strategy, we incorporated about 50 biotins per N P. This amount was optimized to achieve efficient binding of the biotin group during the MELISA assay.
- the number of NPs in the suspension obtained was estimated from the fluorescent NP quantification curve.
- the structure of NPs after release was checked by TEM. This observation showed that the morphology of the NPs remained stable during the synthesis.
- the assay based on biotin-nanoparticles and immunomagnetic beads was studied for the detection of the H1N1 viruses (samples 1 and 2), H3N2 and VSDRP (Fig. 8).
- the viral solutions were diluted in lysis buffer supplemented with 10% saliva.
- the detection step was performed using highly biotinylated nanoparticles to increase the anchoring efficiency of streptavidin-HRP. This strategy allowed an increase in the signal in the event of a positive response (Fig. 8). This improved sensitivity proved the effectiveness of the amplification strategy.
- Detection limits of 3x10 3 PFU / mL, 6x10 4 PFU / mL and 4x10 4 PFU / mL were calculated for sample 1 and sample 2 of H1N1, and H3N2, respectively.
- biotin-NP in the detection step improved the sensitivity of the MELISA assay by a factor of 65 to 75.
- Si-NP silica nanoparticles
- Biotin-decorated Si-NPs were used to improve the readout of the streptavidin chemiluminescent signal - HRP.
- An improvement in signal intensity compared to the conventional test was obtained due to the conjugate effect of biotin-Si-NP compared to biotin alone.
- Our study illustrates the invention that the conjugate effect of biotin-Si-NP could become an effective way to increase the sensitivity of the cost-effective, specific and easy to perform dot blot technique.
- Streptavidin-HRP Proclin, acetonitrile, 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), controlled porosity glass (120-200 Mesh, CPG-3000 ⁇ ). .were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Fluorescent rhodamine B silica NPs (Si-NP, diameter ⁇ 50 nm) were supplied by Nano-H (Saint Quentin Fallavier, France). Phosphoramidite DNA building blocks and all DNA synthesis reagents were purchased from Glen Research (Sterling, Virginia, USA). Phosphate buffered saline (PBS) was purchased from Andrea Dutscher (Brumath, France).
- Triton, SDS, NaCl, Trizma base, phenol, chloroform, bacteriological peptone and isoamyl alcohol were purchased from SIGMA (Milan, Italy).
- SIGMA Tinucleotide
- Campylobacter from C. jejuni, C. edi, C. lari, C. upsaliensis and C. fétus (base localization: 72-108) was used as a recognition element (probe called CampyP3). CampyP3 has been labeled at its 5 ′ end with biotin for the development of a chemiluminescent dot blot assay. The probe was tested using the OligoAnalyzer3.1
- PR was designed by mismatching nucleic acid positions of the CP3 sequence, while PE corresponded to the E. coli sequence which shows some similarities to Campylobacter.
- Campylobacter strains were grown under microaerophilic conditions (5% C> 2, 10% CO2 and 80% N2) in anaerobic gas jars.
- Microaerophilic conditions were generated using an Oxoid TM CampyGen TM 2.5 I Sachet (Oxoid, Italy) at 37 ° C for 48h.
- DNA was extracted from Campylobacter isolates from Columbia blood agar plates (both pure strains and isolates were from chicken samples) after Gram staining and light microscopic observations of morphology and mobility. cells (Brucella broth, Thermofisher, Italy) after oxidase and catalase tests.
- DNA extraction from pure cultures and enrichment broths DNA was extracted from pure strains and isolates of chicken samples according to Manzano et al (2015). 2 ml of Bolton Enrichment Broth was harvested by centrifugation at 13,000 g and 4 ° C for 10 min. The bacterial pellets were washed three times with PBS, before being subjected to DNA extraction as previously reported (Manzano 2015). The extracted DNAs were rehydrated using 50 ⁇ l of sterile distilled water and treated with RNase enzyme at 37 ° C for 1 h. Finally, DNA was quantified using Nanodrop TM 2000C (ThermoFisher Scientific, Italy). The DNA concentration was adjusted to 100 ng / mL using sterile distilled water.
- the extracted DNA samples were denatured at 95 ° C for 10 min, immediately placed on ice and deposited as a fraction of 1 ⁇ l on the positively charged nylon membrane (Amersham Hybon TM -XL, GE Healthcare, France).
- the membrane which received the deposits was exposed to UV at 254 nm for 10 minutes to fix the DNA.
- the membrane was soaked in preheated hybridization buffer ( 0.5 M Na 2 HP0 4, 0.5 M NaH 2 PO 4, 10 mM EDTA, 1% SDS, pH 7.5) at 65 ° C for 30 min. with moderate agitation.
- the membrane was transferred to a new petri dish and incubated with 10 6 biotin-Si-NP / mL, PBS, pH 7 , 4 for 30 min, at room temperature, with moderate stirring. 0.7 mM streptavidin-HRP in blocking solution was added after washing and incubated for 30 min with shaking. The signal was then revealed using the improved chemiluminescent substrate luminol for HPR detection (Thermo Scientific, France). The membrane was removed from solution and observed using an imaging system.
- ChemiDoc MP Biorad, France. Detection signals were quantified using Image LabTM software (Biorad). The normalized value of the dot intensity was calculated using the equation (RI0 -Pln) / PI0, where RI0 and Pin represent the pixel intensity obtained for the detection of 0.1 ng / pL of CP3 probe (standard) and experimental sample, respectively.
- the target DNA was first immobilized on a porous nylon membrane by UV irradiation to allow DNA crosslinking to the positively charged surface.
- the biotin-labeled CampyloP3 complementary DNA probe was allowed to hybridize with the target DNA. Hybridization was detected using a streptavidin-HRP conjugate in combination with a chemiluminogenic luminol substrate in the presence of the H2O2 activator.
- the streptavidin-biotin sandwich made it possible to bind biotin-Si-NP to the DNA probe, and therefore, to amplify the detection signal compared to biotin alone.
- Standard curves were established using a CP3 DNA target in both dot blot configurations (without and with biotin-Si-NP) under optimized conditions.
- the calibration curves were obtained from the quantification of the intensity of the chemiluminescent light for each concentration of CP3, from at least three independent spots per concentration.
- biotin-Si-NP resulted in an almost 30-fold increase in the intensity of the chemiluminescent signal.
- the calculated LODs were similar or lower than those obtained with the sensors reported previously for the detection of Campylobacter, such as for example 0.5 ng / pL in Fontanot et al. (2014), 0.37 ng / pL in Manzano et al. (B & B2015) and 0.09 nM in Morant-Minana et al. (B&B, 2015).
- the reproducibility of the dot blot enhanced by biotin-Si-NP was estimated at 5% according to the signal obtained for the detection of the same concentration of CP3.
- the proposed biotin-Si-NP-enhanced dot blot method succeeded in detecting low levels of Campylobacter naturally present after enrichment, and could be considered for the determination and detection of Campylobacter spp. present in contaminated poultry meat.
- This work describes a simple dot blot method, improved with biotin-Si-NP, for rapid and reliable detection of Campylobacter spp. present in contaminated poultry meat.
- the dot blot test is widely used in molecular biology and genetics to detect a target DNA / RNA sequence.
- biotin-Si-NPs resist well to temperature of use and are stable over time.
- the LOD obtained was 0.006 ng / ⁇ l (0.2 nM). Such low concentrations of DNA detected are close to those that can be detected by qPCR (Manzano et al. 2018; Vidic et al., 2019).
- the developed system is a promising tool for rapid and inexpensive screening of poultry samples for the presence of Campylobacter because detection is performed on bacterial DNA without a prior amplification step.
- our dot blot can be applied to DNA extracted by different extraction methods and from various food matrices, since it is not sensitive to DNA polymerase inhibitors.
- the proposed multiplex, miniaturized and sensitive paper test is simple to design and could be used by the food industry and regulatory agencies for the detection of other pathogens to monitor food quality. We believe that in the future it could be integrated with a lab-based biosensor on a chip that includes an automated DNA extraction protocol, or even a cell phone.
- DNA was extracted from pure strains and isolates of chicken samples according to Manzano et al (2015). The concentration of total DNA was adjusted to 100 ng / mL using sterile distilled water. Before immobilization, the extracted DNA samples were denatured at 95 ° C for 10 min, immediately placed on ice and deposited as a fraction of 1 ⁇ l on the positively charged nylon membrane (Amersham Hybon TM -XL, GE Healthcare, France).
- the membrane which received the deposits was exposed to UV at 254 nm for 10 minutes to fix the DNA.
- the membrane was soaked in preheated hybridization buffer ( 0.5 M Na 2 HP0 4, 0.5 M NaH 2 P0 4 , 10 mM EDTA, 1% SDS, pH 7.5) at 65 ° C. for 30 min with moderate stirring. 4 ng / pL of the CampyP3 probe labeled with denatured biotin (100 ng / pL) was added to the hybridization buffer and left overnight at 65 ° C. with moderate stirring to allow hybridization. The membrane was washed twice with 0.1 SDS, SSC (sodium citrate saline,
- the signal was then revealed using the improved chemiluminescent substrate for HPR detection (Thermo Scientific, France).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR2005578A FR3110971A1 (fr) | 2020-05-27 | 2020-05-27 | Procédé et système pour la détermination de la présence et/ou de la quantité d’au moins un analyte susceptible d’être contenu dans un échantillon |
PCT/EP2021/063546 WO2021239588A1 (fr) | 2020-05-27 | 2021-05-20 | Procédé et système pour la détermination de la présence et/ou de la quantité d'au moins un analyte susceptible d'être contenu dans un échantillon |
Publications (1)
Publication Number | Publication Date |
---|---|
EP4158341A1 true EP4158341A1 (fr) | 2023-04-05 |
Family
ID=72885633
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP21728199.7A Withdrawn EP4158341A1 (fr) | 2020-05-27 | 2021-05-20 | Procédé et système pour la détermination de la présence et/ou de la quantité d'au moins un analyte susceptible d'être contenu dans un échantillon |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP4158341A1 (fr) |
FR (1) | FR3110971A1 (fr) |
WO (1) | WO2021239588A1 (fr) |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2855613B1 (fr) * | 2003-05-26 | 2005-08-19 | Biocytex | Procede de detection et de quantification multiplex d'analytes dans un echantillon a l'aide de microspheres |
FR2977811B1 (fr) | 2011-07-12 | 2019-07-05 | Universite Claude Bernard Lyon I | Materiau pour synthese supportee |
-
2020
- 2020-05-27 FR FR2005578A patent/FR3110971A1/fr not_active Withdrawn
-
2021
- 2021-05-20 EP EP21728199.7A patent/EP4158341A1/fr not_active Withdrawn
- 2021-05-20 WO PCT/EP2021/063546 patent/WO2021239588A1/fr unknown
Also Published As
Publication number | Publication date |
---|---|
FR3110971A1 (fr) | 2021-12-03 |
WO2021239588A1 (fr) | 2021-12-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Liu et al. | SERS-based lateral flow strip biosensor for simultaneous detection of Listeria monocytogenes and Salmonella enterica serotype enteritidis | |
Bhardwaj et al. | Fluorescent nanobiosensors for the targeted detection of foodborne bacteria | |
Fu et al. | Dramatically enhanced immunochromatographic assay using cascade signal amplification for ultrasensitive detection of Escherichia coli O157: H7 in milk | |
Song et al. | Aptamer-based detection methodology studies in food safety | |
Abdelhamid et al. | Selective biosensing of Staphylococcus aureus using chitosan quantum dots | |
KR101097882B1 (ko) | 과산화효소 활성을 가지는 자성 나노입자와 효소가 다공성 실리카의 기공 내에 고정되어 있는 다공성 실리카 복합체 및 그 제조방법 | |
Wu et al. | A sensitive aptasensor for the detection of Vibrio parahaemolyticus | |
EP0744028B1 (fr) | Surfaces hautement specifiques pour reactions biologiques, procede pour leur preparation et procede pour leur utilisation | |
D’Agata et al. | Ultrasensitive detection of non-amplified genomic DNA by nanoparticle-enhanced surface plasmon resonance imaging | |
Luan et al. | A POCT colorimetric aptasensor for streptomycin detection using porous silica beads-enzyme linked polymer aptamer probes and exonuclease-assisted target recycling for signal amplification | |
JP2012242394A (ja) | 標的物質検出用キット及びこれを利用した標的物質検出方法 | |
WO2015055708A1 (fr) | Dosage biologique qualitatif sensible ay moyen d'oxyde de graphène comme agent révélateur d'analyte | |
Bonnet et al. | Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates | |
Arshad et al. | Nanotechnology for therapy of zoonotic diseases: A comprehensive overview | |
Gao et al. | A sensitive colorimetric aptasensor for chloramphenicol detection in fish and pork based on the amplification of a nano-peroxidase-polymer | |
EP3207371B1 (fr) | Composition pour dosage immuno-enzymatique par immunofluorescence et ses utilisations | |
EP3126832A1 (fr) | Contrôles pour la mise en oeuvre de procédés d'analyse multiplexe | |
WO2021239588A1 (fr) | Procédé et système pour la détermination de la présence et/ou de la quantité d'au moins un analyte susceptible d'être contenu dans un échantillon | |
EP2082233B1 (fr) | Procede de diagnostic in vitro des staphylococcus aureus producteurs de pvl | |
Wang et al. | FRET-enhanced nanoflares for sensitive and rapid detection of ampicillin | |
JP2010172324A (ja) | アプタマーを用いて微生物を検出する方法及びキット | |
EP1247096B1 (fr) | Procede d'immobilisation de reactif(s) affin(s) sur phase solide hydrophobe | |
Bruce-Tagoe et al. | Advances in aptamer-based biosensors for monitoring foodborne pathogens | |
WO2017221875A1 (fr) | Complexe et ses utilisations | |
Su et al. | Magnetic bead-based mimic enzyme-chromogenic substrate and silica nanoparticles signal amplification system for avian influenza A (H7N9) optical immunoassay |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20221125 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20231201 |