CN113820505A - Kit - Google Patents
Kit Download PDFInfo
- Publication number
- CN113820505A CN113820505A CN202011202912.9A CN202011202912A CN113820505A CN 113820505 A CN113820505 A CN 113820505A CN 202011202912 A CN202011202912 A CN 202011202912A CN 113820505 A CN113820505 A CN 113820505A
- Authority
- CN
- China
- Prior art keywords
- solution
- concentration
- reagent
- gold
- nitrogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 85
- 102000003896 Myeloperoxidases Human genes 0.000 claims abstract description 61
- 108090000235 Myeloperoxidases Proteins 0.000 claims abstract description 61
- 238000001514 detection method Methods 0.000 claims abstract description 53
- 238000000034 method Methods 0.000 claims abstract description 36
- 238000001069 Raman spectroscopy Methods 0.000 claims abstract description 15
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 5
- 229940127219 anticoagulant drug Drugs 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 260
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 160
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 105
- 229910052757 nitrogen Inorganic materials 0.000 claims description 80
- 238000001035 drying Methods 0.000 claims description 72
- 239000008213 purified water Substances 0.000 claims description 66
- 239000002073 nanorod Substances 0.000 claims description 60
- 238000001237 Raman spectrum Methods 0.000 claims description 56
- 238000002360 preparation method Methods 0.000 claims description 47
- 239000011259 mixed solution Substances 0.000 claims description 34
- 239000000203 mixture Substances 0.000 claims description 32
- 239000002244 precipitate Substances 0.000 claims description 32
- 238000011010 flushing procedure Methods 0.000 claims description 29
- 238000005406 washing Methods 0.000 claims description 29
- 230000000903 blocking effect Effects 0.000 claims description 28
- 230000009871 nonspecific binding Effects 0.000 claims description 28
- 238000001228 spectrum Methods 0.000 claims description 27
- 239000000523 sample Substances 0.000 claims description 23
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 22
- 239000008367 deionised water Substances 0.000 claims description 21
- 229910021641 deionized water Inorganic materials 0.000 claims description 21
- 238000012360 testing method Methods 0.000 claims description 21
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000012496 blank sample Substances 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 17
- 239000006228 supernatant Substances 0.000 claims description 16
- 238000007306 functionalization reaction Methods 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 14
- 230000005284 excitation Effects 0.000 claims description 14
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 claims description 11
- 229960003151 mercaptamine Drugs 0.000 claims description 11
- WOWJJHXUUVZDBE-UHFFFAOYSA-N 4-amino-3-pyrazin-2-yl-1h-1,2,4-triazole-5-thione Chemical compound N1C(=S)N(N)C(C=2N=CC=NC=2)=N1 WOWJJHXUUVZDBE-UHFFFAOYSA-N 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 239000000758 substrate Substances 0.000 claims description 7
- 238000012986 modification Methods 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 2
- 229910052709 silver Inorganic materials 0.000 claims description 2
- 239000004332 silver Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 abstract description 2
- 230000003287 optical effect Effects 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 description 57
- 238000003756 stirring Methods 0.000 description 47
- 102000012192 Cystatin C Human genes 0.000 description 42
- 108010061642 Cystatin C Proteins 0.000 description 42
- 108700028909 Serum Amyloid A Proteins 0.000 description 39
- 102000054727 Serum Amyloid A Human genes 0.000 description 39
- 102000008857 Ferritin Human genes 0.000 description 33
- 108050000784 Ferritin Proteins 0.000 description 33
- 238000008416 Ferritin Methods 0.000 description 33
- 239000003154 D dimer Substances 0.000 description 26
- 108010052295 fibrin fragment D Proteins 0.000 description 26
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 24
- 210000002966 serum Anatomy 0.000 description 24
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 24
- 101001097889 Homo sapiens Platelet-activating factor acetylhydrolase Proteins 0.000 description 19
- 102000047182 human PLA2G7 Human genes 0.000 description 19
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 12
- 229960005070 ascorbic acid Drugs 0.000 description 12
- 235000010323 ascorbic acid Nutrition 0.000 description 12
- 239000011668 ascorbic acid Substances 0.000 description 12
- 244000309466 calf Species 0.000 description 12
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 12
- 238000007865 diluting Methods 0.000 description 12
- 230000012010 growth Effects 0.000 description 12
- 229910001961 silver nitrate Inorganic materials 0.000 description 12
- 239000012279 sodium borohydride Substances 0.000 description 12
- 229910000033 sodium borohydride Inorganic materials 0.000 description 12
- 229910004042 HAuCl4 Inorganic materials 0.000 description 11
- 108010024976 Asparaginase Proteins 0.000 description 10
- 102000016752 1-Alkyl-2-acetylglycerophosphocholine Esterase Human genes 0.000 description 9
- 238000012544 monitoring process Methods 0.000 description 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 230000000630 rising effect Effects 0.000 description 7
- 102100032752 C-reactive protein Human genes 0.000 description 6
- 208000024172 Cardiovascular disease Diseases 0.000 description 6
- 108010010234 HDL Lipoproteins Proteins 0.000 description 6
- 102000015779 HDL Lipoproteins Human genes 0.000 description 6
- 206010061218 Inflammation Diseases 0.000 description 6
- BCKXLBQYZLBQEK-KVVVOXFISA-M Sodium oleate Chemical compound [Na+].CCCCCCCC\C=C/CCCCCCCC([O-])=O BCKXLBQYZLBQEK-KVVVOXFISA-M 0.000 description 6
- 238000001816 cooling Methods 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 230000004054 inflammatory process Effects 0.000 description 6
- 238000003760 magnetic stirring Methods 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 238000009210 therapy by ultrasound Methods 0.000 description 6
- 102000007330 LDL Lipoproteins Human genes 0.000 description 5
- 108010007622 LDL Lipoproteins Proteins 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 230000002526 effect on cardiovascular system Effects 0.000 description 5
- 230000024924 glomerular filtration Effects 0.000 description 5
- 238000011065 in-situ storage Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 201000001320 Atherosclerosis Diseases 0.000 description 4
- 208000015710 Iron-Deficiency Anemia Diseases 0.000 description 4
- 208000029078 coronary artery disease Diseases 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- 229910052742 iron Inorganic materials 0.000 description 4
- 239000003607 modifier Substances 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000006447 Phospholipases A2 Human genes 0.000 description 3
- 108010058864 Phospholipases A2 Proteins 0.000 description 3
- 206010040047 Sepsis Diseases 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 208000026106 cerebrovascular disease Diseases 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 208000017169 kidney disease Diseases 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 208000019423 liver disease Diseases 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 2
- 208000004476 Acute Coronary Syndrome Diseases 0.000 description 2
- 102000011767 Acute-Phase Proteins Human genes 0.000 description 2
- 108010062271 Acute-Phase Proteins Proteins 0.000 description 2
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 2
- 108010074051 C-Reactive Protein Proteins 0.000 description 2
- 206010008479 Chest Pain Diseases 0.000 description 2
- 102000015833 Cystatin Human genes 0.000 description 2
- 206010051055 Deep vein thrombosis Diseases 0.000 description 2
- 208000005189 Embolism Diseases 0.000 description 2
- 206010014522 Embolism venous Diseases 0.000 description 2
- 101000889990 Homo sapiens Apolipoprotein(a) Proteins 0.000 description 2
- 102000004895 Lipoproteins Human genes 0.000 description 2
- 108090001030 Lipoproteins Proteins 0.000 description 2
- 102000015439 Phospholipases Human genes 0.000 description 2
- 108010064785 Phospholipases Proteins 0.000 description 2
- 208000010378 Pulmonary Embolism Diseases 0.000 description 2
- 108010000499 Thromboplastin Proteins 0.000 description 2
- 102000002262 Thromboplastin Human genes 0.000 description 2
- 206010052779 Transplant rejections Diseases 0.000 description 2
- 108010062497 VLDL Lipoproteins Proteins 0.000 description 2
- 206010047249 Venous thrombosis Diseases 0.000 description 2
- 206010000891 acute myocardial infarction Diseases 0.000 description 2
- 206010002022 amyloidosis Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 208000020832 chronic kidney disease Diseases 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 108050004038 cystatin Proteins 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 102000045903 human LPA Human genes 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 229940012957 plasmin Drugs 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 208000023504 respiratory system disease Diseases 0.000 description 2
- 238000012502 risk assessment Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 2
- 201000005665 thrombophilia Diseases 0.000 description 2
- 208000004043 venous thromboembolism Diseases 0.000 description 2
- ASWBNKHCZGQVJV-UHFFFAOYSA-N (3-hexadecanoyloxy-2-hydroxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C ASWBNKHCZGQVJV-UHFFFAOYSA-N 0.000 description 1
- 102100025573 1-alkyl-2-acetylglycerophosphocholine esterase Human genes 0.000 description 1
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 1
- 208000002699 Digestive System Neoplasms Diseases 0.000 description 1
- 206010048554 Endothelial dysfunction Diseases 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 208000007530 Essential hypertension Diseases 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010058861 Fibrin Fibrinogen Degradation Products Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 102000008015 Hemeproteins Human genes 0.000 description 1
- 108010089792 Hemeproteins Proteins 0.000 description 1
- 101000912205 Homo sapiens Cystatin-C Proteins 0.000 description 1
- 101001099460 Homo sapiens Myeloperoxidase Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- 102000019298 Lipocalin Human genes 0.000 description 1
- 108050006654 Lipocalin Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 208000031662 Noncommunicable disease Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 241000634212 Penia Species 0.000 description 1
- 102100026918 Phospholipase A2 Human genes 0.000 description 1
- 101710096328 Phospholipase A2 Proteins 0.000 description 1
- 108010003541 Platelet Activating Factor Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102000004987 Troponin T Human genes 0.000 description 1
- 108090001108 Troponin T Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- HVVWZTWDBSEWIH-UHFFFAOYSA-N [2-(hydroxymethyl)-3-prop-2-enoyloxy-2-(prop-2-enoyloxymethyl)propyl] prop-2-enoate Chemical compound C=CC(=O)OCC(CO)(COC(=O)C=C)COC(=O)C=C HVVWZTWDBSEWIH-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000003470 adrenal cortex hormone Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000000923 atherogenic effect Effects 0.000 description 1
- 230000003143 atherosclerotic effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- RKHQGWMMUURILY-UHRZLXHJSA-N cortivazol Chemical compound C([C@H]1[C@@H]2C[C@H]([C@]([C@@]2(C)C[C@H](O)[C@@H]1[C@@]1(C)C2)(O)C(=O)COC(C)=O)C)=C(C)C1=CC1=C2C=NN1C1=CC=CC=C1 RKHQGWMMUURILY-UHRZLXHJSA-N 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 208000033679 diabetic kidney disease Diseases 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000009266 disease activity Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000008694 endothelial dysfunction Effects 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 210000001263 extrapyramidal tract Anatomy 0.000 description 1
- 230000006624 extrinsic pathway Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 239000000208 fibrin degradation product Substances 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 108091005996 glycated proteins Proteins 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 102000049632 human CST3 Human genes 0.000 description 1
- 102000051251 human MPO Human genes 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000002650 immunosuppressive therapy Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000010438 iron metabolism Effects 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 108010071584 oxidized low density lipoprotein Proteins 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000002947 procoagulating effect Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003904 radioactive pollution Methods 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/745—Assays involving non-enzymic blood coagulation factors
- G01N2333/75—Fibrin; Fibrinogen
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
- G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nanotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention provides an application of a kit in Myeloperoxidase (MPO) detection, wherein the kit comprises: reagent I, reagent II, BSA solution and EDTA anticoagulant; in the application process, the reagent in the kit and MPO in a detection sample form a sandwich structure, and a strong optical signal is generated under Raman laser to realize the detection of Myeloperoxidase (MPO).
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to a kit.
Background
Serum Amyloid A (SAA), an acute phase protein and binding to plasma High Density Lipoprotein (HDL), has been described in association with cardiovascular and cerebrovascular diseases, tumors, renal diseases, amyloidosis, respiratory diseases and its use in related diseases: 1. the application in the aspect of tumor: SAA plays an important role in the occurrence, development, invasion and metastasis processes of tumors, and has high-concentration expression in metastatic patients of the tumors, so that the SAA has important application value in the aspects of curative effect observation and prognosis evaluation of the tumor patients; 2. aspect of cardiovascular disease: can be used as risk assessment index 3 of cardiovascular diseases and application in kidney diseases: particularly in the aspect of detection of renal transplant patients, SAA of renal transplant rejection patients is abnormally increased, and indexes such as uric acid, creatinine, CRP and the like are more sensitive; 4. other diseases, such as amyloidosis, respiratory diseases, and rheumatoid arthritis also have certain application value.
Serum amyloid A is an acute phase protein whose concentration in blood can rise sharply in hours, rising 1000-fold above the initial concentration, during inflammatory, infectious and non-infectious diseases; SAA is associated with HDL, which regulates HDL metabolism during inflammation, and one particularly important property of SAA is that its degradation products can be deposited in different organs in the form of amyloid a fibrils, which is a serious complication in chronic inflammatory diseases; serum amyloid a elevation is also seen in atherosclerosis, diabetic nephropathy, acute myocardial infarction, coronary heart disease, chronic kidney disease. SAA has a similar effect as CRP in evaluating inflammation, monitoring its activity, and treatment. The SAA assay is more accurate than the C-reactive protein assay in diagnosing patients with viral infections, renal transplant rejection (particularly those undergoing immunosuppressive therapy), and cystic fibrosis with corticoid therapy. It was found that serum amyloid a is most closely related to disease activity in cases with arthritis. The simultaneous detection of C-reactive protein and serum amyloid A can improve the diagnostic sensitivity to infection.
Ferritin is a soluble tissue protein in the body that stores iron, is found mainly in the nuclei of the extrapyramidal tract of the liver, spleen and brain, and constitutes two subtypes of ferritin: one is L subtype with relative molecular mass of 19000, and the other is H subtype with relative molecular mass of 21000, and has effects of regulating iron metabolism balance, resisting oxidation stress, and eliminating part of heavy metal and toxic molecule. Among chronic kidney disease patients who depend on dialysis maintenance for a long time, dialysis patients who have half of the serum ferritin level as high as 500ng/mb serum ferritin more than 800ng/ml have higher CRP level and worse nutritional status and infection possibility.
Clinically, coronary artery disease is one of the leading causes of death in patients with cardiovascular disease in developed countries, and iron overdose increases the risk of cardiovascular disease. Serum ferritin is a new risk factor for acute myocardial infarction. In addition, patients with essential hypertension and cerebral infarction have close relationship with serum ferritin level. Ferritin is used as a reliable index of tissue iron and participates in the generation and development process of acute cerebral infarction; iron deficiency anemia is common in both developed and developing countries. Although bone marrow puncture is the gold standard for diagnosing iron-deficiency anemia, most patients are difficult to accept due to traumatic operation and limited by good and bad material availability, so that an iron-deficiency anemia index which is simple, convenient and feasible and has high sensitivity still needs to be actively searched. Serum ferritin can reflect the iron storage condition of the body and is a routine examination item for anemia. Serum ferritin reduction is highly specific for iron deficiency anemia, and the detection is less damaging than gold standard bone marrow stab staining, with reference ranges for males: 30-300ng/ml female: 10-200 ng/ml; at present, a large number of clinical and epidemiological researches find that the increase of the storage amount of ferritin in the body can be related to the suffering of liver cancer, lung cancer, colon cancer, esophageal cancer, gastrointestinal tumor, pancreatic cancer and breast cancer, and particularly, when the AFP value of the liver cancer is low, the ferritin value can be used for supplementing so as to improve the diagnosis rate.
Myeloperoxidase (MPO), a heme protein, is enriched in neutrophils, synthesized in the bone marrow before entry into the circulation by granulocytes and stored in azure granules. The external stimulus may cause the neutrophils to aggregate, thereby releasing myeloperoxidase. MPO has a relative molecular weight of 150kDa and is a tetramer formed by covalent bonding of two subunits, each of which is composed of one heavy chain alpha (relative molecular weight of 60kDa) and one light chain beta (relative molecular weight of 15 kDa). MPO can kill microorganisms in phagocytes by catalyzing and oxidizing chloride ions to generate hypochlorous acid, destroy various target substances, and play a role in various aspects such as organism generation, inflammatory reaction regulation and the like. More importantly, its oxidative modification of Low Density Lipoprotein (LDL) can cause atherosclerosis, and MPO is therefore thought to be involved in the development of cardiovascular disease. Currently, MPO is considered to be the most promising cardiovascular marker, and elevated MPO levels in vivo are predictive of risk of arteriosclerosis and coronary heart disease, are early warnings of myocardial infarction, are more sensitive than other indicators such as troponin T, CK-MB and CRP, and are more early diagnostic and risk assessment. MPO level can be obviously increased within 2h of chest pain occurrence, so for chest pain patients, MPO has more important clinical significance for diagnosing Acute Coronary Syndrome (ACS).
Lipoprotein-associated phospholipase A2 (lipoprotein-associated phosphophospholipase A2, Lp-PLA2), also known as platelet activating factor acetylphthalate hydrolase (PAF-AH), is a phospholipase secreted by inflammatory cells that promotes the hydrolysis of oxidized phospholipids, is a member of the phospholipase A2(PLA2) superfamily and has a relative molecular mass of 45.4kD (441 amino acids). Lp-PLA2 is one of the subtypes in the phospholipase superfamily, also known as platelet activating factor acetylhydrolase, and is secreted by macrophages, T cells, and mast cells in the intima of blood vessels. Lp-PLA2 expression is upregulated in atherosclerotic plaques and strongly expressed in macrophages in the fibrous cap of vulnerable plaques. Lp-PLA2 can hydrolyze oxidized phospholipids in oxidized low density lipoprotein ox-LDL to produce lipidic pro-inflammatory substances, such as lysolecithin and oxidized free fatty acids, which in turn produce a variety of atherogenic effects, including endothelial cell death and endothelial dysfunction, stimulating the production of adhesion factors and cytokines. These substances can further produce self-reinforcing circulation by chemotactic inflammatory cells to generate more proinflammatory substances. Lp-PLA2 released into the blood circulation was mainly bound to apolipoprotein (Apo) B-rich lipoproteins, Low Density Lipoprotein (LDL) accounting for 80%, and the rest bound to High Density Lipoprotein (HDL), lipoprotein and Very Low Density Lipoprotein (VLDL). Lp-PLA2 levels were positively correlated with LDL subcomponent levels in patients with atherosclerotic disease.
Lipoprotein-associated phospholipase A2(LP-PLA2) is a vascular specific inflammatory marker, and LP-PLA2 is an independent risk factor marker for atherosclerotic cardiovascular disease, coronary heart disease, and ischemic stroke. The detection of LP-PLA2 can directly and accurately reflect the degree of inflammation in blood vessels, and reflects dynamic changes as a dynamic index. Lp-PLA2 is a high-specificity prediction index of cardiovascular and cerebrovascular malignant events; reflecting the inflammation degree of atheromatous plaques, and effectively identifying benign plaques and malignant plaques; independently predicting cardiovascular and cerebrovascular embolism diseases caused by atherosclerosis; the dynamic change reaction treatment effect plays an auxiliary guidance for the clinical treatment and the curative effect of the patients with the cardiovascular and cerebrovascular embolism diseases.
The Cystatin (CPI) was subsequently named cystatin C. The protein is called gamma-micro protein and gamma-metaglobulin, is widely present in nucleated cells and body fluids of various tissues, is a low molecular weight and alkaline non-glycated protein, has the molecular weight of 13.3KD, consists of 122 amino acid residues, can be produced by all nucleated cells of the body, and has constant production rate. Cystatin c in circulation is cleared only by glomerular filtration, is an endogenous marker reflecting the change of glomerular filtration rate, is reabsorbed in the proximal convoluted tubule, is completely metabolized and decomposed after reabsorption, and does not return to blood, so that the concentration in blood is determined by the glomerular filtration rate, does not depend on the influence of any external factors such as sex, age and diet, and is an ideal endogenous marker reflecting the change of glomerular filtration rate. Cystatin C is used for early diagnosis and disease monitoring of kidney disease, reflecting the most ideal endogenous marker of GFR. Cystatin C (Cys-C) is an endogenous substance which basically meets the requirements of an ideal endogenous Glomerular Filtration Rate (GFR) marker so far, and is an index which is newly developed and has good renal function evaluation sensitivity and high specificity.
D-Dimer (D-Dimer): the simplest fibrin degradation products, whose increase in mass concentration reflects hypercoagulable state and secondary hyperfibrinolysis in vivo, are mainly used in the diagnosis of Venous Thromboembolism (VTE), Deep Venous Thrombosis (DVT) and Pulmonary Embolism (PE).
The hypercoagulable state in tumor patients is related to tissue factor dependent extrinsic pathway and non-tissue factor related tumor procoagulant effect, and the D-dimer concentration is shown in a large amount of literature, and can be used as a judgment standard for tumor stage, prognosis and the like.
In liver diseases, the content of plasma D-dimer is obviously increased and is positively correlated with the severity of liver diseases, because plasmin resistance, AT-III resistance and the like are synthesized by the liver, the synthesis of liver diseases is reduced, hyperfibrinolysis is caused, fibrin and fibrinogen are degraded under the activation of plasmin, degradation products of D-dimer and the like are obviously increased, and the concentration of D-dimer can be used as a sign for judging the damage degree of the liver.
Sepsis refers to systemic inflammatory reaction and immune dysfunction syndrome caused by infection, and is essentially a series of pathophysiological reactions of organisms to infectious factors, wherein coagulation system abnormality is one of the manifestations, inflammatory cells are activated in sepsis patients, the coagulation system in organisms is activated through various ways, then a human body generates an anticoagulant substance to start a fibrinolysis system, the levels of D-dimers in blood of sepsis patients with different infection types are different, the level of D-dimers can be used for evaluating the disease severity and prognosis of the patients, and the evaluation of treatment effect can also play a role.
However, the currently used serum amyloid A assay kit cannot simultaneously have the functions of low detection limit, simple operation and dynamic monitoring.
At present, the common detection modes of ferritin include an immunoassay, a turbidimetry and a chemiluminescence method, but the methods cannot simultaneously have the functions of low detection limit, simple operation and dynamic monitoring.
At present, common detection methods of myeloperoxidase include a continuous monitoring method, an enzyme-linked immunosorbent assay (ELISA) method and a flow cytometry method, but the methods cannot simultaneously have the functions of low detection limit, simple operation and dynamic monitoring.
Currently, there are two methods, active and concentration, for the detection of Lp-PLA 2. The activity method mainly adopts high performance liquid chromatography, radioactivity measurement method, enzyme hydrolysis substrate method and the like. The high performance liquid chromatography has low sensitivity and is easily interfered by various components in blood. The problems of radioactive pollution, low accuracy, poor repeatability and the like of a reagent exist in the radioactivity determination method; the enzyme hydrolysis substrate method mainly depends on imported reagents, and has the problems of high cost and the like. The method for detecting the concentration mainly adopted clinically is an ELISA method. However, the above method cannot simultaneously have the functions of low detection limit, simple operation and dynamic monitoring.
At present, the common detection modes of cystatin C include a one-way immunodiffusion method (RID), a Radioimmunoassay (RIA), a time-resolved fluoroimmunoassay (TRFIA), an Enzyme Immunoassay (EIA), a particle-enhanced transmission immunoturbidimetry (PETIA) and a particle-enhanced scattering immunoturbidimetry (PENIA), but the prior art cannot simultaneously have the functions of low detection limit, simple operation and dynamic monitoring.
At present, dry type immune scattering chromatography, immunoturbidimetry and quantitative analysis are commonly used as detection methods of the D-dimer, but the methods cannot simultaneously have the functions of low detection limit, simple operation and dynamic monitoring.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a kit, which comprises: 10ml of top layer reagent, 10ml of bottom layer reagent, 10ml of BSA solution and 10ml of anticoagulant LEDTA;
the top layer reagent comprises: the kit comprises a gold-core silver-shell nanorod substrate, cysteamine modified molecules, glutaraldehyde modified molecules, a serum amyloid A monoclonal antibody and a 1 XPBS buffer solution;
the bottom layer reagent comprises: a gold core silver shell nanorod substrate, 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole Raman detection molecules and glutaraldehyde functional modification molecules;
the concentration of the BSA solution was 1%.
Further, the preparation method of the kit comprises the following steps:
(1) preparation of Top layer reagent
(1.1) adding a Raman detection molecular ethanol solution into a gold-core silver-shell nanorod solution, gently shaking and centrifuging, and removing a supernatant to obtain a first mixture;
(1.2) dispersing the mixture I into deionized water, adding a functional modifier solution, gently shaking and centrifuging to obtain a mixture II;
(1.3) dispersing the mixture II into a serum amyloid A detection antibody solution to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12 hours, centrifuging, removing a supernatant, dispersing a precipitate into a 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) performing amino functionalization treatment on the gold-core silver-shell nanorod solution by using a cysteamine solution, washing with purified water after the treatment, and drying with nitrogen;
(2.2) adding a functional modifier solution for functional treatment, flushing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.1 was added to the serum amyloid A capture antibody solution, incubated at 4 ℃ for 12 hours, then washed with purified water and dried with nitrogen.
Further, in the step (1.1), the raman detection molecule is 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole raman detection molecule, the molar concentration of the raman detection molecule ethanol solution is 5mmol/L, and the dosage is 2 μ L;
the dosage of the gold-core silver-shell nanorod solution is 1 mL;
the time for the gentle shaking was 2h and the conditions for the centrifugation were 7000rpm, 10 min.
Further, the amount of the deionized water used in the step (1.2) is 1.0 mL;
the functionalized modifier solution is glutaraldehyde solution, the concentration is 25% wt, and the dosage is 2 mu L;
the time for the gentle shaking was 1.5h, and the centrifugation conditions were 6000rpm, 10 min.
Further, the serum amyloid A detection antibody solution in the step (1.3) is an anti-serum amyloid A polyclonal antibody solution, the concentration is 9 μ g/mL in 1 × PBS buffer, and the dosage is 1.0 mL;
the centrifugation condition is 6000rpm for 10 min;
the amount of the 1 XPBS buffer was 1.0 mL.
Further, the concentration of the cysteamine solution in step (2.1) is 25mmol/L, and the dosage is 2. mu.L.
Further, the solution of the functional modifier in step (2.2) is glutaraldehyde solution with a concentration of 25% wt, and the amount is 2 μ L.
Further, the serum amyloid A-associated lipocalin capture antibody solution in step (2.3) is a serum amyloid A monoclonal antibody solution at a concentration of 20. mu.g/mL in 1 XPBS buffer in an amount of 2 mL.
Another aspect of the present invention provides a method for detecting serum amyloid a using the kit, the method comprising:
(a) blocking non-specific binding
Firstly, immersing a bottom layer reagent into a BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of serum amyloid A
Dripping 200 mu L of detection sample into the bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water, and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on a Leica microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
Further, the detection sample is one of whole blood, plasma or serum;
when the detection sample is whole blood, an EDTA anticoagulant needs to be added before detection for anticoagulation.
Compared with the prior art, the invention has the following advantages:
the kit provided by the invention adopts a sandwich structure of top layer solution, detection sample and substrate solution, and Raman detection molecules in the top layer solution form strong optical signals through the irradiation of Raman rays, so that the defects of the kit in the prior art are overcome, and the targets of simple operation, low cost, high determination sensitivity and high detection speed can be simultaneously realized.
Drawings
FIG. 1 is a diagram showing the process of forming the structure of the sandwich immunoassay kit of the present invention.
FIG. 2 is a Raman spectrum of serum amyloid according to a first embodiment of the present invention.
FIG. 3 is a Raman spectrum of serum amyloid at various concentration levels according to the first embodiment of the present invention.
FIG. 4 shows a Raman spectrum of ferritin in a second embodiment of the present invention.
FIG. 5 is a Raman spectrum of ferritin at various concentration levels in a second example of the invention.
FIG. 6 shows a Raman spectrum of myeloperoxidase in the third example of the present invention.
FIG. 7 shows Raman spectra of myeloperoxidase at different concentration levels in the third example of the present invention.
FIG. 8 is a Raman spectrum of human lipoprotein-associated phospholipase A2 according to the fourth embodiment of the present invention.
FIG. 9 shows Raman spectra of human lipoprotein-associated phospholipase A2 at different concentration levels according to the fourth embodiment of the present invention.
FIG. 10 is a Raman spectrum of cystatin C in the fifth embodiment of the present invention.
Fig. 11 is a raman spectrum of cystatin C at different concentration levels in the fifth example of the present invention.
FIG. 12 is a Raman spectrum of a D-dimer in a sixth example of the present invention.
FIG. 13 is a Raman spectrum of D-dimer at different concentration levels according to the fifth example of the present invention.
Detailed Description
The objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.
Embodiments of the present invention will hereinafter be described with reference to the accompanying drawings, wherein like reference numerals denote like or similar parts, or like or similar steps.
The first embodiment.
FIG. 1 shows a process diagram of the structure formation of the sandwich immunoassay kit of the present invention. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) 0.6mL of 0.01M NaBH4 solution was rapidly injected into solution one, NaBH4The solution is prepared on site, the mixed solution is stirred by magnetic force at the speed of 1200rpm for 2min, and finally the obtained seed solution is kept stand for 30min at 30 ℃ for standby;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) 250ml of 1.0mM HAuCl was injected into the second solution while magnetically stirring4Stirring the solution at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of serum amyloid A sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2H, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of serum amyloid A polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a serum amyloid A monoclonal antibody solution at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the serum amyloid A sandwich immunoassay kit prepared by the method, and 10mL of 1% BSA solution is provided in the kit.
FIG. 2 shows a Raman spectrum of serum amyloid in a first embodiment of the present invention. The serum amyloid a sandwich immunoassay kit of the examples was used to detect patient serum.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of serum amyloid A sandwich immunoassay kit in the embodiment is taken according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of serum amyloid A
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Collecting Raman spectrum of the 2 samples on a Leica microscope by using 785nm laser as excitation light source and using a 20-time objective lens, wherein the spectrum is 1800cm in 800--1In the range, the exposure is 10 s.
The obtained Raman spectrum is shown in FIG. 2, and the result shows that 1013cm is in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, can be determined preliminarily that the kit can detect the presence of serum amyloid a, and in order to further prove the accuracy thereof, and the detection limit of the kit, we performed the following assay.
FIG. 3 shows Raman spectra of serum amyloid at different concentration levels in the first embodiment of the present invention. Serum amyloid a was detected at different concentrations using the serum amyloid a sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and serum amyloid A was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL, respectively, to obtain samples Nos. 1 to 6.
Then, 6 parts of the serum amyloid A sandwich immunoassay kit in the examples are taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of serum amyloid A
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
And (3) taking 785nm laser as an excitation light source, and carrying out Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800 cm.
The obtained raman spectrum is shown in fig. 2, and the result shows that the raman intensity shows a monotonous rising trend along with the enhancement of the concentration of the serum amyloid A, so that the serum amyloid A sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human serum amyloid A, and the detection limit is in the fg level.
Example two.
This example utilizes a sandwich immunoassay kit to detect ferritin. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6 mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of ferritin sandwich immunoassay kit
(1) Preparation of detection reagent
(1.1) adding 2 mu L of 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2H, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of ferritin polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a detection reagent, and storing at 4 ℃;
(2) preparation of Capture reagent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a ferritin monoclonal antibody solution at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
The detection reagent 10mL and the capture reagent 10mL of the ferritin sandwich immunoassay kit prepared by the method are provided, and in addition, 10mL of BSA solution with the concentration of 1% is provided in the kit.
FIG. 4 shows a Raman spectrum of ferritin in a second embodiment of the present invention. The patient sera were tested using the ferritin sandwich immunoassay kit of the example.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of the ferritin sandwich immunoassay kit in the embodiment is taken to be operated according to the following steps:
a) blocking non-specific binding
Immersing the capture reagent in BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilising ferritin
Respectively dripping 200 mu L of a test sample and a blank sample into 2 parts of the capture reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a detection reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 4, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The presence of a distinct peak, but the absence of such a peak in the blank, allows a preliminary determination that the kit can detect the presence of ferritin, and to further demonstrate its accuracy, and the detection limits of the kit, we proceed as follows.
FIG. 5 shows Raman spectra of ferritin at different concentration levels in a second example of the invention. Ferritin was detected at different concentrations using the ferritin sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and ferritin was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL, and 0fg/mL, respectively, to give samples Nos. 1-6.
Then, 6 parts of the ferritin sandwich immunoassay kit in the embodiment is taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the capture reagent in BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilising ferritin
Dripping 200 mu L of No. 1-6 sample into 6 parts of capture reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a detection reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 4, and the result shows that the Raman intensity shows a monotonous rising trend along with the increase of the concentration of the ferritin, so that the ferritin sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human ferritin, and the detection limit is in the fg level.
Example three.
This example utilizes a sandwich immunoassay kit for detecting Myeloperoxidase (MPO). The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6 mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of Myeloperoxidase (MPO) sandwich immunoassay kit
(1) Preparation of reagent one
(1.1) adding 2 mu L of 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2H, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture II into 1.0mL of Myeloperoxidase (MPO) polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a reagent I, and storing at 4 ℃;
(2) preparation of reagent two
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a Myeloperoxidase (MPO) monoclonal antibody solution at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
The first 10mL and the second 10mL of reagents of the Myeloperoxidase (MPO) sandwich immunoassay kit prepared by the method are provided, and in addition, 10mL of BSA solution with the concentration of 1% is provided in the kit.
FIG. 6 shows a Raman spectrum of myeloperoxidase in the third embodiment of the present invention. Patient sera were tested using the Myeloperoxidase (MPO) sandwich immunoassay kit of the examples.
First, 10mL of patient serum was taken as a test sample, then 10mL of calf serum was taken as a blank sample, and then 2 parts of Myeloperoxidase (MPO) sandwich immunoassay kit in the example were taken according to the following procedure:
a) blocking non-specific binding
Soaking the reagent II in BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized Myeloperoxidase (MPO)
Respectively dropwise adding 200 mu L of test sample and blank sample into the 2 parts of reagent II treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding the reagent I, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 6, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The presence of a distinct peak, but not the absence of such a peak in the blank sample, can be a preliminary determination that the kit can detect the presence of Myeloperoxidase (MPO), and to further demonstrate its accuracy, and the detection limits of the kit, we performed the following assays.
As shown in FIG. 7, Raman spectra of myeloperoxidase at different concentration levels in the third example of the present invention are shown. Myeloperoxidase (MPO) was detected at various concentrations using the Myeloperoxidase (MPO) sandwich immunoassay kit of the example.
First, 6 samples were prepared, and Myeloperoxidase (MPO) was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL, respectively, to obtain samples Nos. 1-6.
Then, 6 parts of Myeloperoxidase (MPO) sandwich immunoassay kit in the examples were prepared according to the following procedure:
(a) blocking non-specific binding
Soaking the reagent II in BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized Myeloperoxidase (MPO)
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of reagent II treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding the reagent I, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 6, and the result shows that the raman intensity is in a monotonous rising trend along with the increase of the concentration of the Myeloperoxidase (MPO), so that the Myeloperoxidase (MPO) sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human Myeloperoxidase (MPO) and the detection limit is in the fg level.
Example four
This example utilizes a sandwich immunoassay kit to detect human lipoprotein-associated phospholipase A2(LP-PLA 2). The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6 mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of human lipoprotein-associated phospholipase A2(LP-PLA2) sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2H, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of human lipoprotein-associated phospholipase A2(LP-PLA2) polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing supernatant, dispersing the precipitate into 1mL of 1 XPBS buffer to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a solution of human lipoprotein-associated phospholipase A2(LP-PLA2) monoclonal antibody at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then rinsed with purified water and dried under nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the human lipoprotein-associated phospholipase A2(LP-PLA2) sandwich immunoassay kit prepared by the method, and 10mL of BSA solution with the concentration of 1% is provided in the kit.
FIG. 8 shows a Raman spectrum of human lipoprotein-associated phospholipase A2 in the fourth embodiment of the present invention. Patient sera were tested using the human lipoprotein-associated phospholipase A2(LP-PLA2) sandwich immunoassay kit of the examples.
First, 10mL of patient serum was taken as a test sample, then 10mL of calf serum was taken as a blank sample, and then 2 parts of the human lipoprotein-associated phospholipase a2(LP-PLA2) sandwich immunoassay kit in the example were taken according to the following procedure:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human lipoprotein-associated phospholipase A2(LP-PLA2)
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 8, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, can be determined preliminarily that the kit can detect the existence of human lipoprotein-associated phospholipase A2(LP-PLA2), and in order to further prove the accuracy and detection limit of the kit, we carried out the following determination.
FIG. 9 shows Raman spectra of human lipoprotein-associated phospholipase A2 at different concentration levels according to the fourth embodiment of the present invention. In the embodiment, a sandwich immunoassay kit of human lipoprotein-related phospholipase A2(LP-PLA2) is used for detecting different concentrations of human lipoprotein-related phospholipase A2(LP-PLA 2).
First, 6 samples were prepared, and human lipoprotein-associated phospholipase A2(LP-PLA2) was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL, and 0fg/mL, respectively, to give samples Nos. 1-6.
Then, 6 parts of the human lipoprotein-associated phospholipase A2(LP-PLA2) sandwich immunoassay kit in the example was prepared according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human lipoprotein-associated phospholipase A2(LP-PLA2)
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The raman spectrum obtained is shown in fig. 8, and the result shows that the raman intensity is in a monotonous rising trend along with the increase of the concentration of the human lipoprotein-associated phospholipase a2(LP-PLA2), so that the human lipoprotein-associated phospholipase a2(LP-PLA2) sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human lipoprotein-associated phospholipase a2(LP-PLA2), and the detection limit is in the fg level.
Example five.
This example utilizes a sandwich immunoassay kit to detect cystatin C (Cys-C). The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6 mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of cystatin C (Cys-C) sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2H, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture II into 1.0mL of cystatin C (Cys-C) polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of cystatin C (Cys-C) monoclonal antibody solution at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then rinsed with purified water and dried under nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the cystatin C (Cys-C) sandwich immunoassay kit prepared by the method, and 10mL of 1% BSA solution is provided in the kit.
Fig. 10 shows a raman spectrum of cystatin C in the fifth example of the present invention. The cystatin C (Cys-C) sandwich immunoassay kit of the examples was used to detect patient sera.
Firstly, 10mL of patient serum is obtained as a test sample, then 10mL of calf serum is used as a blank sample, and then 2 parts of cystatin C (Cys-C) sandwich immunoassay kit in the embodiment is taken to operate according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) stationary cystatin C (Cys-C)
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 570nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 10, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, can be determined preliminarily that the kit can detect the presence of cystatin C (Cys-C), and in order to further prove the accuracy thereof, and the detection limit of the kit, we performed the following determination.
Fig. 11 shows raman spectra of cystatin C at different concentration levels in the fifth example of the present invention. Cystatin C (Cys-C) was detected at different concentrations using the cystatin C (Cys-C) sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and cystatin C (Cys-C) was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL, and 0fg/mL, respectively, to give samples Nos. 1-6.
Then, 6 parts of cystatin C (Cys-C) sandwich immunoassay kit in the embodiment is taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) stationary cystatin C (Cys-C)
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 570nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 10, and the result shows that the raman intensity shows a monotonous rising trend along with the increase of the concentration of cystatin C (Cys-C), so that the cystatin C (Cys-C) sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of human cystatin C (Cys-C), and the detection limit is in the fg level.
Example six.
This example utilizes a sandwich immunoassay kit for the detection of D-dimer. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6 mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of two, D-dimer sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2H, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of D-dimer polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a 20. mu.g/mL in 1 XPBS buffer solution of the D-dimer monoclonal antibody, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the D-dimer sandwich immunoassay kit prepared by the method, and 10mL of BSA solution with the concentration of 1% is provided in the kit.
FIG. 12 shows a Raman spectrum of a D-dimer in the sixth embodiment of the present invention. Patient sera were tested using the D-dimer sandwich immunoassay kit of the example.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of the D-dimer sandwich immunoassay kit in the embodiment are taken according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized D-dimers
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 570nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 12, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, can be determined primarily as the presence of D-dimer which can be detected by the kit, and in order to further demonstrate its accuracy, and the detection limit of the kit, we proceed as follows.
As shown in FIG. 13, Raman spectra of D-dimer at different concentration levels in the fifth example of the present invention are shown. Different concentrations of D-dimer were detected using the D-dimer sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and D-dimer was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL, respectively, to give samples Nos. 1-6.
Then, 6 parts of the D-dimer sandwich immunoassay kit in the example were taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized D-dimers
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Surface enhanced Raman spectroscopy
And (3) performing surface enhanced Raman spectroscopy detection by using 570nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is in the range of 800-1800cm < -1 > and is exposed for 10 s.
The obtained raman spectrum is shown in fig. 12, and the result shows that the raman intensity shows a monotonous rising trend along with the increase of the concentration of the D-dimer, so that the D-dimer sandwich immunoassay kit prepared in the embodiment of the present invention can detect the presence of human D-dimer according to the results of test example 1 and test example 2, and the detection limit is at the fg level.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (3)
1. A kit, comprising: 10ml of top layer reagent, 10ml of bottom layer reagent, 10ml of BSA solution and 10ml of anticoagulant LEDTA;
the top layer reagent comprises: the method comprises the following steps of (1) preparing a gold-core silver-shell nanorod substrate, a cysteamine modified molecule, a glutaraldehyde modified molecule and a 1 XPBS buffer solution;
the bottom layer reagent comprises: a gold core silver shell nanorod substrate, 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole Raman detection molecules and glutaraldehyde functional modification molecules;
the concentration of the BSA solution was 1%.
2. The kit according to claim 1, wherein the method for preparing the kit comprises:
(1) preparation of reagent one
(1.1) adding 2 mu L of 4-amino-3-mercapto-5-pyrazine-4 (H) -1,2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2H, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture II into 1.0mL of Myeloperoxidase (MPO) polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a reagent I, and storing at 4 ℃;
(2) preparation of reagent two
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a Myeloperoxidase (MPO) monoclonal antibody solution at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
3. A method for detecting Myeloperoxidase (MPO) using the kit of claim 1 or 2, which comprises:
(a) blocking non-specific binding
Soaking the reagent II in BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized Myeloperoxidase (MPO)
Respectively dropwise adding 200 mu L of test sample and blank sample into the 2 parts of reagent II treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
After drying by nitrogen in the step (b), adding a reagent I, placing in a greenhouse for 20min, washing by purified water after finishing cultivation, and drying by nitrogen to obtain a sandwich structure;
(d) spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011202912.9A CN113820505A (en) | 2020-06-19 | 2020-06-19 | Kit |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010564029.8A CN113820503A (en) | 2020-06-19 | 2020-06-19 | Kit |
| CN202011202912.9A CN113820505A (en) | 2020-06-19 | 2020-06-19 | Kit |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010564029.8A Division CN113820503A (en) | 2020-06-19 | 2020-06-19 | Kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113820505A true CN113820505A (en) | 2021-12-21 |
Family
ID=78911958
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010564029.8A Pending CN113820503A (en) | 2020-06-19 | 2020-06-19 | Kit |
| CN202011202248.8A Pending CN113820504A (en) | 2020-06-19 | 2020-06-19 | Kit |
| CN202011202912.9A Pending CN113820505A (en) | 2020-06-19 | 2020-06-19 | Kit |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010564029.8A Pending CN113820503A (en) | 2020-06-19 | 2020-06-19 | Kit |
| CN202011202248.8A Pending CN113820504A (en) | 2020-06-19 | 2020-06-19 | Kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN113820503A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100107648A (en) * | 2009-03-26 | 2010-10-06 | 한양대학교 산학협력단 | Combinational surface-enhanced raman scattering probe and method for detecting target substance by using the same |
| US20180024147A1 (en) * | 2015-01-09 | 2018-01-25 | Indutry-University Cooperation Foundation Hanyang University Erica Campus | Reagent kit for detecting sex hormone and method for detecting sex hormone using same |
| WO2019179386A1 (en) * | 2018-03-19 | 2019-09-26 | 上海交通大学 | Raman probe, and manufacturing method and application thereof |
| CN110749585A (en) * | 2019-09-11 | 2020-02-04 | 亳州市新健康科技有限公司 | Biosensor based on surface enhanced Raman technology and preparation method thereof |
| CN111239395A (en) * | 2020-01-15 | 2020-06-05 | 亳州市新健康科技有限公司 | Preparation method of HPV16 type E7 protein sandwich immunodetection kit |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2040075A1 (en) * | 2007-09-24 | 2009-03-25 | Julius-Maximilians-Universität Würzburg | Compounds and markers for surface-enhanced raman scattering |
| WO2011116402A2 (en) * | 2010-03-19 | 2011-09-22 | University Of Wyoming, An Institution Of Higher Education Of The State Of Wyoming | Surface enhanced raman spectroscopy |
| US20130052665A1 (en) * | 2011-08-25 | 2013-02-28 | Bruce Xuefeng Ling | Methods for diagnosis of systemic juvenile idiopathic arthritis |
| CN105548571A (en) * | 2016-02-02 | 2016-05-04 | 潍坊三维生物工程集团有限公司 | Kit and method for detecting content of serum amyloid protein A and application |
| CN110618123B (en) * | 2019-09-11 | 2022-01-28 | 亳州市新健康科技有限公司 | Efficient surface-enhanced Raman scattering substrate material and preparation method thereof |
| CN110646399B (en) * | 2019-09-29 | 2021-08-13 | 吉林大学 | SERS-based high-throughput protein detection chip |
| CN111175517B (en) * | 2020-01-07 | 2023-08-08 | 中国人民解放军军事科学院军事医学研究院 | Immunochromatography test paper for detecting radiation markers, preparation method and application thereof and kit |
-
2020
- 2020-06-19 CN CN202010564029.8A patent/CN113820503A/en active Pending
- 2020-06-19 CN CN202011202248.8A patent/CN113820504A/en active Pending
- 2020-06-19 CN CN202011202912.9A patent/CN113820505A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100107648A (en) * | 2009-03-26 | 2010-10-06 | 한양대학교 산학협력단 | Combinational surface-enhanced raman scattering probe and method for detecting target substance by using the same |
| US20180024147A1 (en) * | 2015-01-09 | 2018-01-25 | Indutry-University Cooperation Foundation Hanyang University Erica Campus | Reagent kit for detecting sex hormone and method for detecting sex hormone using same |
| WO2019179386A1 (en) * | 2018-03-19 | 2019-09-26 | 上海交通大学 | Raman probe, and manufacturing method and application thereof |
| CN110749585A (en) * | 2019-09-11 | 2020-02-04 | 亳州市新健康科技有限公司 | Biosensor based on surface enhanced Raman technology and preparation method thereof |
| CN111239395A (en) * | 2020-01-15 | 2020-06-05 | 亳州市新健康科技有限公司 | Preparation method of HPV16 type E7 protein sandwich immunodetection kit |
Non-Patent Citations (1)
| Title |
|---|
| BEATA WRZOSEK ET AL: "Surface-Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection", ANAL.CHEM., vol. 79, no. 11, pages 2 - 8 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113820504A (en) | 2021-12-21 |
| CN113820503A (en) | 2021-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Campello et al. | Microparticles as biomarkers of venous thromboembolic events | |
| CA2575475C (en) | Method for diagnosing liver fibrosis | |
| JP3370334B2 (en) | Method for measuring oxidized lipoprotein and its use | |
| Chiriacò et al. | On-chip screening for prostate cancer: an EIS microfluidic platform for contemporary detection of free and total PSA | |
| JP2019060880A (en) | In-vitro early detection method for potential inflammation especially related to implantation rejection reaction, neurodegenerative disease, or depression | |
| CA2509063A1 (en) | Scd40l and placental growth factor (plgf) as biochemical marker combinations in cardiovascular diseases | |
| CN111929443A (en) | Heparin binding protein determination kit and preparation and determination method thereof | |
| WO2006015873A1 (en) | Method for diagnosing liver fibrosis | |
| Chuang et al. | Smartphone and home-based liquid crystal sensor for rapid screening of acute myocardial infarction by naked-eye observation and image analysis | |
| CN106093411A (en) | An a kind of step homogeneous CK MB detection kit and application thereof | |
| CN110554197A (en) | Fluorescence immunochromatography joint detection kit and preparation method thereof | |
| CN113820505A (en) | Kit | |
| Eriksson et al. | Present and future biochemical markers for detection of acute coronary syndrome | |
| JP6578119B2 (en) | Prostate-specific antigen measurement method and measurement kit | |
| Liu et al. | Detection of biomarkers of acute myocardial infarction by high-throughput suspension array technology in serum sample | |
| CN113820501A (en) | Sandwich immunoassay kit | |
| AU2014210241A1 (en) | Novel disease-marker | |
| CN111487222B (en) | Preparation method of SPR chip for detecting BNP | |
| Nechaeva et al. | Rapid Automatic Determination of Four Cardiomarkers in the Blood Plasma of Patients with Cardiopathologies | |
| KR100921237B1 (en) | Quartz crystal microbalance immunosensor Apparatus for C-reactive protein detection and High-sensitivity detection method for C-reactive protein using quartz crystal microbalance immunosensor | |
| António et al. | Gold nanoparticle probes for colorimetric detection of plasma galectin-3: a simple and rapid approach | |
| JP5048207B2 (en) | Apparatus and method for determining the onset and presence of septic conditions | |
| WO2022186318A1 (en) | Cancer detection method, cancer test method and kits to be used therein | |
| Prylutskyi et al. | Determination of the concentration of polyamines with SPR-based immune biosensor for early diagnostics of breast cancer | |
| Marlęga-Linert et al. | Lipoprotein apheresis affects the concentration of extracellular vesicles in patients with elevated lipoprotein (a) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |