GB2078369A - Enzymatic assay method and apparatus - Google Patents
Enzymatic assay method and apparatus Download PDFInfo
- Publication number
- GB2078369A GB2078369A GB8115611A GB8115611A GB2078369A GB 2078369 A GB2078369 A GB 2078369A GB 8115611 A GB8115611 A GB 8115611A GB 8115611 A GB8115611 A GB 8115611A GB 2078369 A GB2078369 A GB 2078369A
- Authority
- GB
- United Kingdom
- Prior art keywords
- substance
- oxidase
- sample
- glucose
- assay method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000007824 enzymatic assay Methods 0.000 title 1
- 239000000126 substance Substances 0.000 claims abstract description 98
- 238000003556 assay Methods 0.000 claims abstract description 54
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 48
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 35
- 239000001301 oxygen Substances 0.000 claims abstract description 35
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 35
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 29
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 29
- 229930006000 Sucrose Natural products 0.000 claims abstract description 22
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 22
- 235000013681 dietary sucrose Nutrition 0.000 claims abstract description 22
- 229960004793 sucrose Drugs 0.000 claims abstract description 22
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 20
- 239000008101 lactose Substances 0.000 claims abstract description 20
- 108091000080 Phosphotransferase Proteins 0.000 claims abstract description 16
- 102000020233 phosphotransferase Human genes 0.000 claims abstract description 16
- 230000003647 oxidation Effects 0.000 claims abstract description 8
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 58
- 102000004190 Enzymes Human genes 0.000 claims description 38
- 108090000790 Enzymes Proteins 0.000 claims description 38
- 229940088598 enzyme Drugs 0.000 claims description 38
- 230000000694 effects Effects 0.000 claims description 25
- 108010015776 Glucose oxidase Proteins 0.000 claims description 21
- 239000004366 Glucose oxidase Substances 0.000 claims description 21
- 102000005548 Hexokinase Human genes 0.000 claims description 21
- 108700040460 Hexokinases Proteins 0.000 claims description 21
- 229940116332 glucose oxidase Drugs 0.000 claims description 21
- 235000019420 glucose oxidase Nutrition 0.000 claims description 21
- 239000004382 Amylase Substances 0.000 claims description 20
- 108010065511 Amylases Proteins 0.000 claims description 14
- 102000013142 Amylases Human genes 0.000 claims description 14
- 235000019418 amylase Nutrition 0.000 claims description 14
- 102000013009 Pyruvate Kinase Human genes 0.000 claims description 9
- 108020005115 Pyruvate Kinase Proteins 0.000 claims description 9
- 102000057621 Glycerol kinases Human genes 0.000 claims description 7
- 108700016170 Glycerol kinases Proteins 0.000 claims description 6
- 239000000460 chlorine Substances 0.000 claims description 4
- 229910052801 chlorine Inorganic materials 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 4
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 3
- 108010000659 Choline oxidase Proteins 0.000 claims description 3
- 102000048120 Galactokinases Human genes 0.000 claims description 3
- 108700023157 Galactokinases Proteins 0.000 claims description 3
- 108010015133 Galactose oxidase Proteins 0.000 claims description 3
- 102000030595 Glucokinase Human genes 0.000 claims description 3
- 108010021582 Glucokinase Proteins 0.000 claims description 3
- 102000001253 Protein Kinase Human genes 0.000 claims description 3
- 108010042687 Pyruvate Oxidase Proteins 0.000 claims description 3
- 108010090622 glycerol oxidase Proteins 0.000 claims description 3
- 108010018734 hexose oxidase Proteins 0.000 claims description 3
- 108060006633 protein kinase Proteins 0.000 claims description 3
- 230000000865 phosphorylative effect Effects 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 12
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 abstract description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 56
- 239000008103 glucose Substances 0.000 description 56
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 24
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 18
- 239000007853 buffer solution Substances 0.000 description 14
- 239000000758 substrate Substances 0.000 description 14
- 108010093096 Immobilized Enzymes Proteins 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 229940107700 pyruvic acid Drugs 0.000 description 9
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 8
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 8
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 8
- 239000001573 invertase Substances 0.000 description 8
- 235000011073 invertase Nutrition 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 102000020006 aldose 1-epimerase Human genes 0.000 description 7
- 108091022872 aldose 1-epimerase Proteins 0.000 description 7
- 229960001231 choline Drugs 0.000 description 7
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 7
- 102100026189 Beta-galactosidase Human genes 0.000 description 6
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 6
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 6
- 229920002472 Starch Polymers 0.000 description 6
- 108010005774 beta-Galactosidase Proteins 0.000 description 6
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- 229930182830 galactose Natural products 0.000 description 6
- 239000008107 starch Substances 0.000 description 6
- 235000019698 starch Nutrition 0.000 description 6
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 4
- 108010082126 Alanine transaminase Proteins 0.000 description 4
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 4
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 4
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 4
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 4
- 108090000553 Phospholipase D Proteins 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 238000003149 assay kit Methods 0.000 description 4
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 150000002402 hexoses Chemical class 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 description 4
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 4
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 4
- 108010066906 Creatininase Proteins 0.000 description 3
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 3
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 3
- 102000004882 Lipase Human genes 0.000 description 3
- 108090001060 Lipase Proteins 0.000 description 3
- 239000004367 Lipase Substances 0.000 description 3
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 3
- 102000011420 Phospholipase D Human genes 0.000 description 3
- -1 a-glucosidase Proteins 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940109239 creatinine Drugs 0.000 description 3
- 239000000174 gluconic acid Substances 0.000 description 3
- 235000012208 gluconic acid Nutrition 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 235000019421 lipase Nutrition 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 150000003904 phospholipids Chemical class 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- 108010017192 4-hydroxy-4-methyl-2-oxoglutarate aldolase Proteins 0.000 description 2
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 2
- 102100029589 Acylpyruvase FAHD1, mitochondrial Human genes 0.000 description 2
- 102000004420 Creatine Kinase Human genes 0.000 description 2
- 108010042126 Creatine kinase Proteins 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- 102000002704 Leucyl aminopeptidase Human genes 0.000 description 2
- 108010004098 Leucyl aminopeptidase Proteins 0.000 description 2
- 102000017055 Lipoprotein Lipase Human genes 0.000 description 2
- 108010069823 Oxaloacetate decarboxylase Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- XTDYIOOONNVFMA-UHFFFAOYSA-N dimethyl pentanedioate Chemical compound COC(=O)CCCC(=O)OC XTDYIOOONNVFMA-UHFFFAOYSA-N 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 229960003082 galactose Drugs 0.000 description 2
- 229960001031 glucose Drugs 0.000 description 2
- 229960005150 glycerol Drugs 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000013379 molasses Nutrition 0.000 description 2
- 229950004354 phosphorylcholine Drugs 0.000 description 2
- PYJNAPOPMIJKJZ-UHFFFAOYSA-N phosphorylcholine chloride Chemical compound [Cl-].C[N+](C)(C)CCOP(O)(O)=O PYJNAPOPMIJKJZ-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- WRWGAMITZOWMJJ-UHFFFAOYSA-N 2-hydroxypropanoic acid 2-oxopropanoic acid Chemical compound C(C(=O)C)(=O)O.C(C(=O)C)(=O)O.C(C(O)C)(=O)O WRWGAMITZOWMJJ-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 108090000489 Carboxy-Lyases Proteins 0.000 description 1
- 102000002745 Choline Kinase Human genes 0.000 description 1
- 108010018888 Choline kinase Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 108010022355 Fibroins Proteins 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 102000043296 Lipoprotein lipases Human genes 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010067035 Pancrelipase Proteins 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 101710163410 Probable glycerol kinase Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229960002737 fructose Drugs 0.000 description 1
- 150000002303 glucose derivatives Chemical class 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 235000011147 magnesium chloride Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- RLOWWWKZYUNIDI-UHFFFAOYSA-N phosphinic chloride Chemical compound ClP=O RLOWWWKZYUNIDI-UHFFFAOYSA-N 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Emergency Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
In assay methods involving converting a first substance in a sample to a second substance, oxidising the thus-formed second substance using an oxidase, and measuring the oxygen consumed or hydrogen peroxide generated during the oxidation, errors which occur due to the presence of amounts of the second substance in the original sample can be eliminated by contacting the sample, prior to the step involving the conversion of the first substance to the second substance, with an immobilised kinase in the presence of ATP to phosphorylate any second substance which may be present in the original sample and which would otherwise by oxidised by the oxidase. The method can be used for the assay of, for example, lactose or saccharose.
Description
SPECIFICATION
Assay method and apparatus
This invention relates to an assay method and apparatus.
Enzymatic analysis has been commonly used for the quantitative determination and analysis of a component in sample. Enzymatic analysis is, however, uneconomical due to the use of a large amount of expensive enzyme. Also it is disadvantageous because it can require the use of complicated procedures, the assay can take a long time and problems can occur due to the lability of enzyme.
The use of an immobilized enzyme has been introduced to improve enzymatic analysis, especially in combination with an electro-chemical measurement means to form a so-called enzyme electrode. The assay method using an enzyme electrode has number of advantages. For example, it is simple to operate, requires less amount of reagents, shortens the assay time, and less enzyme is consumed. Many enzyme electrodes have been reported, especially in the field of clinical biochemistry. Further this technique has become popular for analysis of starting materials, products and compositions in the field of fermentation, foods and others. The application of the enzyme electrode for fermentation process measurement and quality control process measurement in the fermentation and food industries has been much noticed.
In the fermentation industry, for the purpose of proper cultivation of fungi or yeast and higher productivity, accurate and rapid measurement for saccharose in a medium is required. In the food industry, quality control for dairy products containing saccharose and lactose requires rapid and accurate analysis of these sugars. In clinical biochemistry, for example an assay for amylase activity is performed by hydrolysing a starch substrate to maltose and the maltose to hexose. Hence it is necessary to measure maltose and hexose rapidly and accurately.
Examples of the assay of saccharose, lactose, maltose and amylase activity, and related invertase, a-glucosidase or B-galactosidase activity are as follows:
invertase mutarotase Saccharose aD-fructose + a-glucose ,P-D-glucose O2 > glucose oxidase H gluconic acid -galactosidase Lactose D-galactose + ss-D-glucose 2 > glucose oxidase H202 oxidase gluconic acid amylase a-glucosidase Starch 3 maltose * ss-D-glucose 2 - glucose oxidase H2O2 gluconic acid In these reaction systems, glucose is formed by the action of mutarotase, ss-galactosidase or a-glucosidase. The glucose is oxidised by glucose oxidase and the amount of the decrease in oxygen or of the hydrogen peroxide generated measured, thereby determining the amount of each objective component.
In general, the assay of a component in a sample proceeds as follows.
enzyme second substance Component to be assayed in sampler 2 corresponding oxidase H2O2 oxidized second substance By this reaction scheme, the enzyme activity or component in the sample is assayed by using the oxidase of the second substance (corresponding oxidase for the second substance). The examples of this reaction scheme are not limited to the above analysis of sugar related substances. For example:
(1) To be assayed: phospholipid, phosphoryl chlorine, activity of phospholipase D, phosphatase.
the second substance: chlorine.
oxidase: chlorine oxidase.
measure: oxygen consumed or hydrogen peroxide generated.
(2) To be assayed: neutral fat
activity of lipase, lipoprotein lipase.
the second substance: glycerol.
oxidase: glycerol oxidase.
measure: oxygen consumed or hydrogen peroxide generated.
(3) To be assayed: activity of leucine-aminopeptidase,
synthetic substrate containing amino acid.
the second substance: amino acid.
oxidase: amino acid oxidase.
measure: oxygen consumed or hydrogen peroxide generated.
(4) To be assayed: lactic acid,
lactate dehydrogenase (LDH) activity.
the second substance: pyruvic acid.
oxidase: pyruvate oxidase.
measure: oxygen consumed or hydrogen peroxide generated.
(5) Others: [pyruvic acid formed as final (second) substance, refer to Japanese Patent Laid-Open
Publication No. 55-13068]
(a) ADP, phosphoenolpyruvate and pyruvate kinase.
(b) Alanine, c:-ketoglutarate and glutamic-pyruvic-transaminase (GPT).
(c) aspartate, -ketoglutarate and glutamic-oxaloacetic-transaminase (GOT) and oxaloacetate
decarboxylase.
(d) glycerol, ATP, glycerol kinase and pyruvate kinase.
(e) creatinine, creatininase, ATP, creatinphosphokinase and pyruvate kinase.
In orderto obtain good and accurate results, the second substance such as glucose, galactose, fructose, choline, glycerol, amino acid and pyruvic acid should be completely formed, and additional amounts of the second substance should be not present with the component to be assayed in the original assay sample. The presence of these additional amounts of the second substance can cause errors in the analysis. Generally a sample for assay, excepting standard samples for use in producing standard curves, will be contaminated by amounts ofthe second substance such as glucose and chlorine.
To avoid errors, the contamination of the original sample for assay by a second substance such as glucose and galactose should be determined first. Subsequently, after finishing the assay of the sample, the first value obtained for the contamination of the sample must be deleted from the total value to obtain the correct value. This is a disadvantage. Further, the contaminating second substance such as glucose is oxidised as a pretreatment by an oxidase such as glucose oxidase prior to the assay ofthe sample. This pretreatment using an oxidase can have disadvantages also. For example, dissolved oxygen in a sample is consumed by the action of the oxidase and hydrogen peroxide is generated. These phenomena affect the final determination.
We have investigated the determination of saccharose, lactose, maltose and amylase activity and have found that the problems caused by the presence of glucose in the original sample to be assayed can be overcome by treating the sample first with hexokinase in the presence of ATP to phosphorylate the glucose to glucose-6-phosphate. When the assay is subsequently effected there is no decrease in the level of dissolved oxygen and no hydrogen peroxide is generated attributable to the original amounts of glucose in the sample. However, as a result of this pretreatment, hexokinase remains in the reaction mixture, which causes phosphorylation of the glucose formed in the actual assay. To avoid this effect, denaturation or removal of the remaining hexokinase is required.However, heat or pH denaturation will cause the denaturation of the substance to be assayed, and removal of hexokinase is quite difficult.
We have found that the difficulties caused by the hexokinase remaining after the phosphorylation of the glucose in the original assay sample can be overcome using an immobilized hexokinase. The thus treated sample can then be assayed without errors caused by the original glucose. This technique has general application to assays conducted by converting a first substance to a second substance, oxidising this second substance using an oxidase and measuring the oxygen consumed or hydrogen peroxide generated during the oxidation.
Accordingly, the present invention provides an assay method which comprises:
(i) converting a first substance in a sample to a second substance,
(ii) oxidising the thus formed second substance using an oxidase, and
(iii) measuring the oxygen consumed or hydrogen peroxide generated in the oxidation step (ii), wherein, prior to step (i), the sample is contacted with an immobilised kinase in the presence of ATP to phosphorylate any second substance which may be present in the sample and which would otherwise be oxidised in step (ii).
The present invention also provides apparatus for use in assaying a sample which comprises a first substance and which may also contain a second substance, which apparatus comprises: an immobilised kinase for phosphorylating any second substance in the original sample in the presence of ATP, means for converting the first substance to the second substance, an oxidase for oxidising the thus-formed second substance and means for measuring either the oxygen consumed or the hydrogen peroxide generated during the oxidation.
For ease of reference, any second substance present in the sample to be assayed prior to step (i) will be referred to herein as admixed second substance.
In an embodiment of the present invention, a sample to be assayed is treated in step (i) by one or more enzymes to form the second substance in one or more reactions. This second substance is oxidised by the corresponding oxidase in step (ii) and the oxygen consumed or hydrogen peroxide generated is measured.
The enzymatic reaction system are not limited. Similarly, examples of samples to be analysed which contain an admixed second substance which can cause an error on assay are not limited. Clinical diagnostic samples such as serum and urine, fermentation media, culture filtrates, food raw materials and food products can be analysed.
Examples of the component in a sample to be analysed, the first substance, include saccharose, lactose, starch, maltose, phospholipid, phosphorylcholine, neutral fat, glycerol, lactic acid and creatinine. The enzyme used in the conversion step (i) may be amylase, invertase, ss-galactosidase, a-glucosidase, phospholipase-D, phosphatase, lipase, lipoprotein lipase, leucine aminopeptidase, LDH, GPT, GOT, pyruvate kinase, glycerokinase, oxaloacetic acid decarboxylase, creatininase or creatinephosphokinase.
The present invention can be used for the quantitative determination of the first substance, and for assaying the activity of an enzyme employed in the conversion step (i). The reaction system can be selected and multiplied.
The second substance is the substance produced from the first substance in step (i) and should not be identical to the first substance. Examples of the second substance are substances which are produced by enzymatic action on the first substance and can be oxidised by a corresponding oxidase. The second substance can be glucose, galactose, choline, glycerol or pyruvic acid.The second substances, enzymes and the appropriate first substances are illustrated as follows:
Second substance: enzyme: first substance:
gluxose invertase saccharose
galactose, glucose ss-galactosidase lactose
glucose amylase, a-glucosidase starch
choline phospholipase D phospholipid
choline phosphatase phosphorylcholine
choline choline esterase benzoylcholine
glycerol lipase, lipoproteinlipase neutral fat
pyruvic acid LDH lactic acid
pyruvic acid pyruvate kinase phosphoenol pyruvate
and ADP
pyruvic acid GPT alanine and
a-ketoglutarate
pyruvic acid GOT and oxaloacetate
decarboxylase a-ketoglutarate and aspartate
pyruvic acid glycerol kinase, glycerol and
pyruvate kinase phosphoenolpyruvate
and ATP
pyruvic acid creatininase,
creatinephosphokinase creatinine, ATP and
and pyruvate kinase phosphoenolpyruvate
The admixed second substance therefore refers to a substance in the original assay sample which is identical with the second substance produced from the first substance in step (i) of the process of this invention.
The kinase for the admixed second substance is an enzyme which phosphorylates the said admixed second substance in the presence of ATP. The kinase must correspond to whatever the admixed second substance is. Examples are glucokinase or hexokinase when glucose is the admixed second substance, galactokinase when galactose is the admixed second substance and similarly hexokinase for the other hexoses, cholinekinase for choline, glycerolkinase for glycerol and pyruvate kinase for pyruvate. The immobilized kinase can be prepared by conventional methods for immobilizing enzymes. Preferable techniques are immobilization by acrylamide, protein cross-linking after mixing the enzyme with a protein such as albumin, entrapping with collagen and fibroin or covalently linking therewith, adsorption on polyporous organic polymer or covalent linking therewith, and entrapping by photopolymerization.The immobilized kinase, or any immobilized enzyme used in the present invention, may be in the form of a membrane, fiber, pellet or tube. After contact with the immobilized kinase the sample to be assayed is fed to step (i) of the process of the invention.
The oxidase employed in step (ii) of the process of the invention is an enzyme which at least catalyzes the oxidation of the second substance in the presence of oxygen, generating hydrogen peroxide. Examples are glucose oxidase for glucose, galactose oxidase or hexose oxidase for galactose, hexose oxidase for the other hexoses, glycerol oxidase for glycerol, choline oxidase for choline and pyruvate oxidase for pyruvic acid. The oxidase is preferably immobilized and by this immobilization automatic analysis by electrochemical means can be used, such as an oxygen electrode, hydrogen peroxide electrode or enzyme electrode.
Electrochemical measuring means for an oxygen decrease or for hydrogen peroxide formation involve electrical detection of variations in the amount of oxygen or hydrogen peroxide. A Clark or Galvanic oxygen electrode or hydrogen peroxide electrode are preferably used. Immobilization of the enzyme can save the amount of expensive enzyme. Also an electrode connecting the immobilized enzyme with an electric detector, for example an enzyme electrode such as an enzyme electrode for an oxygen electrode and an enzyme electrode for a hydrogen peroxide electrode is quite preferable because of the rapid detection that results. Also, there is no requirement for reagents, the electrode can be repeatedly used and there is no inhibitory effects caused by colored substances in a sample.
An embodiment of the apparatus of the invention will now be described, by way of example only, with reference to Figure 1 of the accompanying drawings. In Figure 1,the numberals represent various components as follows:
1: buffer solution vessel, 2: pump, 3: sample injector, 4: kinase column, 5: enzyme column for conversion of the first substance to the second substance, 6: immobilized oxidase column, 7: electrode for electrochemical detection means, 8: flow cell, 9: amplifier for electric current change from electrode, 10: recorder, 11: constant temperature vessel, 12: the second injector and 13: exhaust. The second injector is not needed when an immobilised enzyme column 5 is used.The second injector is used for injection of a non-immobilized enzyme solution employed for conversion of the first substance to the second substance, or for injection of a substrate for assay of enzyme activity.
In an embodiment for lactose assay, column 4 is an immobilized hexokinase or galactokinase, column 5 is an immobilized B-galactosidase, column 6 is an immobilized glucose oxidase or galactose oxidase, and electrode 7 is an oxygen electrode or hydrogen peroxide electrode which is connected to flow cell 8. In an embodiment for saccharose assay, column 4 is an immobilised hexokinase, column 5 is a double linked column of immobilized invertase and mutarotase and column 6 is an immobilized glucose oxidase, or column 5 is an immobilized invertase and column 6 is an immobilized mutarotase and glucose oxidase. In an embodiment for assay or amylase activity, column 4 is an immobilized glucokinase or hexokinase, column 5 is an immobilized a-glucosidase, and colum 6 is an immobilized glucose oxidase.
The amount of immobilized enzyme depends on the flow rate of the reaction medium, the Km value of enzyme, the amount of admixed second substance and the dilution of the sample and is not limited.
Preferably the amount is 20 - 200 mg (wet wt.) of an immobilized enzyme containing hexokinase [above 100
U/g (wet wt.)], glucose oxidase [above 30 U/g (wet wt.)i, invertase [above 2000 U/g (wet wt.)], mutarotase [above 800 U/g (wetwt.)], P-galactosidase [above 30 U/g (wetwt.)] ora-glucosidase [above 140 Ulg (wetwt.)j As explained below in the Examples 13.8 U (total activity) of hexokinase can remove 400 mg/dl of glucose in a sample.
The buffer solution in the buffer solution vessel should be a stable pH buffer for enzymes, such as dimethylglutarate buffer or phosphate bluffer. ATP can be contained in the buffer solution for use in phosphorylation by the kinase. The amount of ATP is preferably slightly larger than content of the admixed second substance in the sample. Salts which keep the enzymes stable may be added. Examples are magnesium chloride, potassium chloride or manganese chloride.
In operation, a constant volume of buffer is supplied at 0.1 - 2 ml/min. and the device is stabilized. After stabilization a sample to be assayed is injected at an aliquot of 5 - 50 yl by using micro-syringe or a micro-pipette. The injected sample is transferred to the immobilized kinase column and the admixed second substance is phosphorylated. The reaction temperature is preferably kept at 25 - 40"C, more preferably at 37"C. Admixed glucose, for example, is removed by conversion to glucose-6-phosphate. The sample is passed through the next column and the first substance in the sample is converted to the second substance.
For example, saccharose or lactose (to be assayed) is enzymatically changed to glucose (as the second substance) by an enzyme, preferably an immobilized enzyme.
In the case where an immobilized enzyme is not used in step (i) of the process of the invention, an enzyme solution is injected into the system via the second injector 12. For assaying enzyme activity, operation differs depending on the number of the enzymatic reaction steps, i.e. one step or two or more steps. In a one step enzymatic reaction, if a substrate is injected into the system via the sample injector 3, this substrate passes through the kinase column and hence is affected by the kinase which can cause unexpected results.
Therefore, the substrate should be injected in via the second injector 12. In a two or more step enzymatic reaction, the substrate is treated as a first step in a column for assaying enzymatic acitivity, and the treated substrate is subjected to further enzymatic action or other reagents in order to convert it to the second substance. In this case at the first step the substrate is not reacted with the kinase, and hence the substrate can be injected into the buffer solution or injected into the second injector. The substrate is converted to the second substance in the second or further step of the column.
The second substance is preferably oxidized by an immobilized oxidase column. The reactant solution is introduced into the flow cell, preferably 0.05 - 0.5 my in volume, and the oxygen consumed or hydrogen peroxide generated is detected by an electrode as a change in electric current change, which is recorded through an amplifier.
In the above reactor system, the column 6 and electrode 7 can be replaced by an enzyme electrode of the same immobilized oxidase and set up at the detector part of electrode. A multiple detecting system set up using a plurality of enzyme electrodes in one system can be employed. In this system, in a part of column 5 a corresponding immobilized enzyme column is connected.
Further, a computer controlled or a computerized automatic device can be set up. For example, sample injection can be preformed by an auto-sampler, each valve set up with an electro-magnetic valve. Other possible attachments include a micro-computer with channels for automatic input and control, etc.
connected to a display panel and an operation key board which can respectively display and input, for example, reaction-times and conditions. The device can be automatically controlled by the computer throughtthis interface.
The following Examples illustrate the present invention:
EXAMPLE 1 (1) Removal of the admixed second substance, glucose, by immobilized hexokinase:
Referring to Figure 1 of the accompanying drawings, a buffer solution containing 3 mM ATP and 2 mM magnesium chloride in 0.1 M dimethylglutarate buffer (pH 6.5) was prepared in buffer solution vessel 1. The flow rate in pump 2 was set up at 1 ml/min. Other components of the apparatus of Figure 1 were as follows.
Sample injector: 3. Column 4: (2.8 x 30 mm) immobilized hexokinase (138 U/g, wet. wt., 100 mg). Column 5: (2.8 x 30 mm) immobilized B-galactosidase (35 U/g, wet wt., 100 mg). Column 6: (2.8 x 15 mm) immobilized glucose oxidase (70 U/g, wet wt., 50 mg). Electrode 7: oxygen electrode connected to the flow cell 8 (vol. 0.1 ml). A constant temperature of 37"C was maintained. The oxygen electrode was connected to digital recorder 11 through amplifier 9. The apparatus was to be used for lactose assay.
The buffer solution was supplied by the pump at a flow rate of 1 ml/min. After confirming the stabilization of the level of dissolved oxygen by the oxygen electrode set in the flow cell, a 5 ll1 aliquot of each standard glucose solution (concentration 100,200,300,400,500,600 and 700 mg/dl.) was injected into the system via the sample injector. Each standard glucose solution passed into the immobilized hexokinase column where the glucose was converted to glucose-6-phosphate by ATP. If glucose remained, the amount of dissolved oxygen in the standard solution decreased as a result of oxidation of the glucose by the immobilized glucose oxidase column. The oxygen decrease was recorded by the oxygen electrode as a current change through the amplifier.As shown in Figure 2 of the accompanying drawings hexokinase (total activity 13.9 U) can remove at least 400 mg/dl. of admixed glucose. Therefore, at least 800 mg/dl. of admixed glucose can be removed by 30 U of hexokinase. The same result was obtained by replacing the immobilized ss-galactosidase column 5 by a column without P-galactosidase.
(2) Lactose assay in sample containing lactose admixed with glucose:
The apparatus and same conditions in (1 ) were used. The flow rate of the buffer was 1 ml/min. After the amount of dissolved oxygen had stabilised, a sample (5 Fi) was injected. The sample was an aliquot of lactose of concentration 100, 200, 300, 400 or 500 mg/dl. containing glucose (300 mg/dl.). Controls having the same lactose concentration but containing no glucose were also tested. The glucose in the sample was converted to glucose-6-phosphate by the action of the immobilized hexokinase column, and the lactose was hydrolysed by the immobilized frgalactosidase column to glucose. The glucose was oxidized by the immobilized glucose oxidase column and the electric current change caused by the decrease in the amount of dissolved oxygen was recorded.
The results are shown in Figure 3 of the accompanying drawings, in which - represents the results obtained for the glucose-containing samples and 0-0 represents the results obtained for the control samples without glucose. As can be seen, lactose was assayed with good accuracy even in the presence of glucose.
(3) Saccharose array in sample containing saccharose admixed with glucose:
In the apparatus employed for the lactose assay (2) the immobilized ss-galactosidase column was replaced by an immobilized invertase column (2950 Ulg, wetwt., 100 mg) (2.8 x 30 mm) and the immobilized glucose oxidase column was replaced by a column on which both glucose oxidase and mutarotase had been immobilised (glucoseoxidase 43 Ulg, wetwt., mutarotase 1200 Ulg, wetwt.,50 mg) (2.8 x 15 mm). A buffer solution consisting of 3 mM ATP and 2 mM magnesium chloride containing 0.1 M phosphate buffer (pH 7.0) was used. The samples to be tested were aliquots of saccharose (concentration 100, 200, 300, 400 or 500 mg/dl.) containing 300 mg/dl: glucose.Saccharose solutions without glucose were used as controls. The assay was effected in the same way as in the lactose assay (2) above. The results are shown in Figure 4 of the accompanying drawings. In this Figure, - represents the results obtained for samples with glucose and 0-0 represents the results obtained for the controls without glucose. As can be seen, saccharose can be assayed even in the presence of glucose.
(4) Amylase assay in sample containing amylase admixed with glucose:
In the apparatus employed for the lactose assay (2), the immobilized ss-galactosidase column was replaced by an immobilized a-glucosidase column (160 U/g, wetwt., 100 mg) (2.8 x 30 mm). A buffer solution consisting of 3mM ATP and 2 mM magnesium chloride containing 0.1 M phosphate buffer (pH 7.0) was used.
The substrate was soluble starch in 0.1 M phosphate buffer (pH 7.0) (500 mgldl.). Aliquots of amylase (product of Boehringer G.m.b.H., porcine pancrease a-amylase, 100 U/ml: 1,2,3,4,5,6 ml/lit.) containing glucose 200 mg/dl were employed.
The a-amylase solution (10 iil) was added to the substrate solution (100 yl) and incubated at 38"C for 10 minutes. The buffer solution was supplied at a flow rate 1 ml/min. After stabilizing the level of dissolved oxygen, the above incubated sample (5 yl) was injected into the system via the sample injector. Maltose produced from the starch by the a-amylase was decomposed into glucose through the immobilized a-glucosidase column. This glucose was oxidized by the immobilized glucose oxidase column and the electric current change caused by a decrease of the level of dissolved oxygen was recorded. The admixed glucose was converted into glucose-6-phosphate through the immobilized hexokinase column. If completely converted, no effect on the change in electric current will be observed.The results are shown in Figure 5 of the accompanying drawings.
EXAMPLE 2
In the amylase activity assay apparatus of Example 1-(4), the a-amylase activity in an irregular control serum sample (trade name, seraclea NA: Nihon Shoji Co., a-amylase: 1360 lUll, glucose: 230 mg/dl.) was assayed. The incubation time of the substrate and sample was 10 min., 20 min. and 30 min. Each 20 yl sample was injected into the system via the injector. Operation was in the same way as in Example 1-(4).
Buffer solutions without ATP were employed as controls.
The results are shown in Figure 6 of the accompanying drawings in which - represents the results for the buffer solution with ATP and 0-0 represents the results for the controls without ATP. In the case of the controls without ATP, no conversation of glucose to glucose-6-phosphate occurred, resulting in a higher change of electric current. In the case of the buffer solution with ATP, no effect of glucose was observed and the changes in electric current were proportional to the reaction time. amylase activity in the serum can be assayed with good accuracy.
a-Amylase activities in unknown human blood sera were assayed, with the control of the above, as compared with a commercially available kit (trade name: amylase test Daiichi). The results of both assays are identical (assay by the present invention 185 lU/lit., commercially available kit: 181 lU/lit.)
EXAMPLE 3
Saccharose in a culture filtrate was assayed by using the saccharose assay apparatus of Example 1-(3).
Culture liquid (at starting time of cultivation: molasses 25%, yeast cultivation medium) was collected at certain times after the beginning of cultivation of the culture. The supernatant of each culture liquid was diluted 10 times by adding water. A 5 ul sample was injected into the system via the sample injector.
Operation was the same as in Example 1-(3). The saccharose concentration, shown by electric current change, was calculated by the standard curve in Figure 4. Also, a commercially available saccharose assay kit (Boehringer G.m.b.H., F-kit, saccharose/glucose, lot. 139041, UV absorption method) was used to assay the same sample. The results areshown in Figure 7 of the accompanying drawings in which represents the result of the assay according to the present invention and 0-0 represents the result of the assay using the commercially available assay kit.
In spite of the presence of glucose- in molasses, the saccharose in the culture liquid can be assayed. In the
UV absorption assay method in a commercially available assay kit, previously admixed glucose is oxidized by glucose oxidase to remove glucose. Removal of glucose by the present invention requires only 2 minutes, whereas the commercially available assay kit requires 75 minutes for the removal of glucose and thereafter 15 minutes for denaturation of the glucose oxidase. Further constant dissolved oxygen should be supplied and pH control is required if necessary. In addition to these complicated operations, the effect of colored substances in the sample causes error on assay. The present invention, on the other hand, is simple to operate, effects an assay rapidly, does not cause errors due to the presence of colored substances and accurate assays can be achieved.
Claims (15)
1. An assay method which comprises:
(i) converting a first substance in a sample to a second substance.
(ii) oxidising the thus-formed second substance using an oxidase, and
(iii) measuring the oxygen consumed or hydrogen peroxide operated in the oxidation step (ii),
wherein, prior to step (i), the sample is contacted with an immobilised kinase in the presence of ATP to phosphorylate any second substance which may be present in the sample and which would otherwise be oxidised in step (ii).
2. An assay method according to claim 1 wherein an immobilised oxidase is used in step (ii).
3. An assay method according to claim 1 or 2 wherein measurement is effected in step (iii) by electrochemical means.
4. An assay method according to claim 3 wherein an oxygen electrode, a hydrogen peroxide electrode or an enzyme electrode is employed.
5. An assay method according to any one of the preceding claims wherein the kinase is glycerol kinase, pyruvate kinase, chlorine kinase, glucokinase, galactokinase or hexokinase.
6. An assay method according to any one of the preceding claims wherein the oxidase in step (ii) is glycerol oxidase, pyruvate oxidase, choline oxidase, glucose oxidase, galactose oxidase or hexose oxidase.
7. An assay method according to any one of the preceding claims wherein an enzyme is employed to convert the first substance to the second substance instep (i) and it is the activity of this enzyme which is assayed.
8. An assay method according to any one of claims 1 to 6 which is employed for the quantitative determination of the first substance.
9. An assay method according to any of the preceding claims wherein, prior to step (i), the sample is contacted with the immobilised kinase in the presence of ATP substantially as hereinbefore described in
Example 1(1).
10. A method of assaying lactose substantially as herein before described in Example 1(2).
11. A method of assaying saccharose substantially as hereinbefore described in Example 1(3) of Example 3.
12. A method of assaying amylase substantially as hereinbefore described in Example 1(4) or Example 2.
13. An assay method substantially as hereinbefore described with reference to Figure 1 of the accompanying drawings.
14. Apparatus for use in assaying a sample which comprises a first substance and which may also contain a second substance, which apparatus comprises: an immobilised kinase for phosphorylating any second substance in the original sample in the presence of ATP, means for converting the first substance to the second substance, an oxidase for oxidising the thus-formed second substance and means for measuring eitherthe oxygen consumed or the hydrogen peroxide generated during the oxidation.
15. Apparatus for use in assaying sample which comprises a first substance and which may also contain a second substance, said apparatus being substantially as hereinbefore described with reference to and as illustrated by Figure 1 of the accompanying drawings.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6824580A JPS56164797A (en) | 1980-05-21 | 1980-05-21 | Improved method of determining components in samples |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB2078369A true GB2078369A (en) | 1982-01-06 |
| GB2078369B GB2078369B (en) | 1983-06-22 |
Family
ID=13368181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8115611A Expired GB2078369B (en) | 1980-05-21 | 1981-05-21 | Enzymatic assay method and apparatus |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPS56164797A (en) |
| FR (1) | FR2483081A1 (en) |
| GB (1) | GB2078369B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2145815A (en) * | 1983-07-28 | 1985-04-03 | Vysoka Skola Chem Tech | Method for performing and tracing enzymatic reactions and a device for carrying out the method |
| WO1987001133A1 (en) * | 1985-08-22 | 1987-02-26 | Max-Planck-Gesellschaft Zur Förderung Der Wissensc | Process for identifying compounds which can absorb or release elections after catalysis by elections |
| US4725539A (en) * | 1983-03-08 | 1988-02-16 | Oriental Yeast Co. Ltd. | Method for analyzing plural oxidizable components in a liquid |
| WO1989009280A1 (en) * | 1988-03-31 | 1989-10-05 | Biometra Biomedizinische Analytik Gmbh | Process for detecting compounds which can take up or give off electrons when catalysed by oxidases |
| US5175088A (en) * | 1983-03-08 | 1992-12-29 | Oriental Yeast Co. Ltd. | Rapid analysis of plural components |
| WO2002055730A3 (en) * | 2001-01-11 | 2003-05-15 | Hygiena Llc | Hygiene monitoring |
| EP0881301A4 (en) * | 1995-12-27 | 2004-12-08 | Asahi Kasei Pharma Corp | Method for assaying vital sample |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5562098B2 (en) * | 2010-03-31 | 2014-07-30 | シーシーアイ株式会社 | Biosensor |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AT339505B (en) * | 1974-03-14 | 1977-10-25 | Boehringer Mannheim Gmbh | ENZYMATIC ANALYSIS PROCEDURE |
| DE2938737A1 (en) * | 1978-09-26 | 1980-04-03 | Toyo Jozo Kk | DEVICE AND METHOD FOR SIMULTANEOUSLY DETERMINING MULTIPLE COMPONENTS IN A SAMPLE BY MEANS OF ENZYMATIC ANALYSIS |
| DE2913553C2 (en) * | 1979-04-04 | 1981-09-17 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method and reagent for the enzymatic determination of enzyme substrates |
-
1980
- 1980-05-21 JP JP6824580A patent/JPS56164797A/en active Granted
-
1981
- 1981-05-21 GB GB8115611A patent/GB2078369B/en not_active Expired
- 1981-05-21 FR FR8110136A patent/FR2483081A1/en active Granted
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4725539A (en) * | 1983-03-08 | 1988-02-16 | Oriental Yeast Co. Ltd. | Method for analyzing plural oxidizable components in a liquid |
| US5175088A (en) * | 1983-03-08 | 1992-12-29 | Oriental Yeast Co. Ltd. | Rapid analysis of plural components |
| GB2145815A (en) * | 1983-07-28 | 1985-04-03 | Vysoka Skola Chem Tech | Method for performing and tracing enzymatic reactions and a device for carrying out the method |
| WO1987001133A1 (en) * | 1985-08-22 | 1987-02-26 | Max-Planck-Gesellschaft Zur Förderung Der Wissensc | Process for identifying compounds which can absorb or release elections after catalysis by elections |
| WO1989009280A1 (en) * | 1988-03-31 | 1989-10-05 | Biometra Biomedizinische Analytik Gmbh | Process for detecting compounds which can take up or give off electrons when catalysed by oxidases |
| EP0881301A4 (en) * | 1995-12-27 | 2004-12-08 | Asahi Kasei Pharma Corp | Method for assaying vital sample |
| WO2002055730A3 (en) * | 2001-01-11 | 2003-05-15 | Hygiena Llc | Hygiene monitoring |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6258719B2 (en) | 1987-12-07 |
| FR2483081A1 (en) | 1981-11-27 |
| JPS56164797A (en) | 1981-12-17 |
| FR2483081B1 (en) | 1985-03-15 |
| GB2078369B (en) | 1983-06-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Scheller et al. | Enzyme electrodes and their application | |
| US4353983A (en) | Analytical process and means for measuring the amount of hydrogen peroxide in aqueous media and of organic substrates generating hydrogen peroxide by enzymatic oxidation | |
| Vadgama | Enzyme electrodes as practical biosensors | |
| US4374013A (en) | Oxygen stabilized enzyme electrode | |
| EP0794260B1 (en) | Bioluminescence reagent and method for quantitative determination of adenosine phosphate ester using the reagent | |
| EP0125136B1 (en) | Assay systems utilising more than one enzyme | |
| US4019961A (en) | Analytical enzymatic determination | |
| US6309852B1 (en) | Method and reagent for quantitative determination of 1,5-anhydroglucitol | |
| GB2078369A (en) | Enzymatic assay method and apparatus | |
| Montagné et al. | Simultaneous use of dehydrogenases and hexacyanoferrate (III) ion in electrochemical biosensors for l-lactate, d-lactate and l-glutamate ions | |
| US5962248A (en) | Quantitative determination method for chloride ions | |
| US4042462A (en) | Creatine phosphokinase determination method | |
| Murachi | Use of immobilized enzyme reactors in automated clinical analysis | |
| EP0089210B1 (en) | Reagent for assaying cholinsterase | |
| EP1213357A2 (en) | Method and reagent for quantitative determination of 1, 5-Anhydroglucitol | |
| Ngo | Single-enzyme-based amperometric assay for phosphate ion | |
| US4725539A (en) | Method for analyzing plural oxidizable components in a liquid | |
| Yao et al. | An electroanalytical method for estimation of fish freshness using a flow‐injection system with some immobilized enzyme reactors | |
| TABATA et al. | Use of phospholipase D and choline oxidase for the enzymatic determination of calcium ion in serum | |
| Boyacı et al. | Measurement of glucose, sucrose and lactose in food samples with enzyme‐immobilised packed‐bed column reactors integrated to an amperometric enzyme electrode | |
| US5175088A (en) | Rapid analysis of plural components | |
| JP2000300293A (en) | Determination of 1,5-anhydroglucitol and deterioration reagent | |
| Guilbault | Symposium on bioelectrochemistry of microorganisms. 3. Electrochemical analysis of enzymatic reactions | |
| JPH08228760A (en) | Sensor for measuring fructose | |
| KR100715924B1 (en) | Method and reagent for quantitative determination of 1,5-anhydroglucitol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |