U.S. patent application number 11/683192 was filed with the patent office on 2007-06-28 for measuring chip for surface plasmon resonance biosensor and method for producing the same.
This patent application is currently assigned to Isao KARUBE. Invention is credited to Isao Karube, Hitoshi Muguruma, Ryohei Nagata, Hiroyuki Nakamura, Runa NAKAMURA.
Application Number | 20070148702 11/683192 |
Document ID | / |
Family ID | 27279759 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148702 |
Kind Code |
A1 |
NAKAMURA; Runa ; et
al. |
June 28, 2007 |
MEASURING CHIP FOR SURFACE PLASMON RESONANCE BIOSENSOR AND METHOD
FOR PRODUCING THE SAME
Abstract
An objective of the present invention is to provide a measuring
chip for a surface plasmon resonance sensor that can detect a small
amount of target substances in high sensitivity. The present
invention provides a measuring chip for a surface plasmon resonance
sensor comprising a metal layer, one or more plasma polymerization
layers formed on said metal layer, and a biologically active
substance immobilized on the surface of said plasma polymerization
layer.
Inventors: |
NAKAMURA; Runa; (Tokyo-To,
JP) ; Nakamura; Hiroyuki; (Tokyo-To, JP) ;
Nagata; Ryohei; (Tokyo-To, JP) ; Karube; Isao;
(Kanagawa-Ken, JP) ; Muguruma; Hitoshi;
(Kochi-Ken, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
KARUBE; Isao
Kawasaki-Shi
JP
DAI NIPPON PRINTING CO., LTD.
Tokyo-To
JP
|
Family ID: |
27279759 |
Appl. No.: |
11/683192 |
Filed: |
March 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10462747 |
Jun 17, 2003 |
|
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11683192 |
Mar 7, 2007 |
|
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09274321 |
Mar 23, 1999 |
6627397 |
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10462747 |
Jun 17, 2003 |
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Current U.S.
Class: |
435/7.1 ;
435/287.2 |
Current CPC
Class: |
G01N 21/553
20130101 |
Class at
Publication: |
435/007.1 ;
435/287.2 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 24, 1998 |
JP |
10-076144 |
May 18, 1998 |
JP |
10-134780 |
Jan 20, 1999 |
JP |
11-012233 |
Claims
1. A measuring chip for a surface plasmon resonance sensor
comprising a metal layer and one or more plasma polymerization
layers formed on said metal layer.
2. The measuring chip of claim 1, wherein said metal layer
comprises gold, platinum or silver.
3. The measuring chip of claim 1 further comprising a nucleic acid
immobilized on the surface of at least one plasma polymerization
layers.
4. The measuring chip for a surface plasmon resonance sensor of
claim 1 further comprising a non-immune protein immobilized on the
surface of at least one plasma polymerization layer.
5. The measuring chip for a surface plasmon resonance sensor of
claim 1 further comprising a non-immune protein which is avidin,
streptoavidin, biotin or a receptor.
6. The measuring chip for a surface plasmon resonance sensor of
claim 1, further comprising an immunoglobulin-binding protein
immobilized on the surface of at least one plasma polymerization
layer.
7. The measuring chip for a surface plasmon resonance sensor of
claim 1, further comprising an immunoglobulin-binding protein which
is protein A, protein G, or a rheumatoid factor.
8. The measuring chip for a surface plasmon resonance sensor of
claim 1, further comprising a sugar-binding protein immobilized on
the surface of at least one plasma polymerization layer.
9. The measuring chip for a surface plasmon resonance sensor of
claim 1 further comprising a sugar-binding protein which is a
lectin.
10. The measuring chip for a surface plasmon resonance sensor of
claim 1, comprising a metal layer, one or more plasma
polymerization layers formed on said metal layer, and a
sugar-recognizing sugar chain immobilized on the surface of said
plasma polymerization layer.
11. The measuring chip for a surface plasmon resonance sensor of
claim 1, comprising a metal layer, one or more plasma
polymerization layers formed on said metal layer, and a fatty acid
or a fatty acid ester immobilized on the surface of said plasma
polymerization layer.
12. The measuring chip for a surface plasmon resonance sensor of
claim 1 further comprising a fatty acid or fatty acid ester which
is stearic acid, alachidic acid, behenic acid, ethyl stearate,
ethyl arachidate, or ethyl behanate.
13. The measuring chip for a surface plasmon resonance sensor of
claim 1 further comprising a polypeptide or oligopeptide having
ligand-binding activity immobilized on the surface of said at least
one plasma polymerization layer.
14. The measuring chip for a surface plasmon resonance sensor of
claim 1, further comprising a polypeptide or oligopeptide which is
produced by genetic engineering or chemical synthesis.
15. The measuring chip of claim 1 further comprising an optically
transparent substrate on which said metal layer is formed.
16. A measuring chip according to claim 1, wherein said one or more
plasma polymerization layer(s) comprises a compound having one or
more substitutents selected from the group consisting of --COOH,
--CHO, --SH, --NH.sub.2, --OH, .dbd.NH, --CONH.sub.2, --NCO,
--CH.dbd.CH.sub.2, .dbd.C.dbd.O, and ##STR2##
17. A measuring chip of claim 1, comprising two or more plasma
polymerization layers.
18. A measuring chip of claim 1, wherein at least one plasma
polymerization layer is formed from a monomer material containing
nitrogen.
19. The measuring chip for a surface plasmon resonance sensor of
claim 1, wherein said plasma polymerization layer is formed from a
compound containing nitrogen which is
CH.sub.3--(CH.sub.2).sub.n--NH.sub.2 (wherein n is an integer from
1 to 6) and/or NH.sub.2--(CH.sub.2).sub.n--NH.sub.2 (wherein n is
an integer from 1 to 6).
20. The measuring chip for a surface plasmon resonance sensor of
claim 1, comprising a plasma polymerization layer which is produced
from a compound containing nitrogen selected from the group
consisting of pyridine, ethylenediamine, hexamethylenediamine,
n-propylamine, monoethylamine, triethylamine, diethylamine,
allylamine, acrylamide, aniline, acrylonitrile, 1,2,4-triazole,
5-amino-1H-tetrazole, propargylamine and acetonitrile.
21. The measuring chip of claim 1, wherein at least one plasma
polymerization layer is formed from a monomer material containing
sulfur.
22. The measuring chip according to claim 1, wherein at least one
plasma polymerization layer is formed from a compound containing
sulfur selected from the group consisting of dimethyl sulfide,
methyl disulfide, ethanethiol, ethanedithiol, thiophen,
mercaptoethanol and dithreitol.
23. The measuring chip according to claim 1, wherein said plasma
polymerization layer is formed from a monomer compound containing a
halogen.
24. The measuring chip according to claim 1, wherein said at least
one plasma polymerization layer is formed from a monomer material
having one or more groups selected from the group consisting of
--COON, --CHO, --SH, --NH.sub.2, --OH, .dbd.NH, --CONH.sub.2,
--NCO, --CH.dbd.CH.sub.2, .dbd.C.dbd.O and ##STR3##
25. The measuring chip according to claim 1, wherein said monomer
material of a plasma polymerization layer is a compound having
--C.dbd.CCH.sub.2OH.
26. The measuring chip according to claim 1, wherein said plasma
polymerization layer is formed from a monomer material which is a
carbohydrate compound comprising C and H.
27. The measuring chip according to claim 1, wherein said plasma
polymerization layer is formed from a monomer material which is an
organic metal compound.
28. A measuring chip according to claim 27, wherein said organic
metal compound is an organic silicon compound.
29. A measuring chip according to claim 28, wherein said organic
silicon compound is selected from the group consisting of
tetramethylsilane, tetramethyldisiloxane, hexamethyldisiloxane,
hexamethyldisilazane, hexamethylcyclotrisilazane,
dimethylaminotrimethylsilane, trimethylvinylsilane,
tetramethoxysilane, aminopropyltriethoxysilane,
octadecyldiethoxymethylsilane, hexamethyldisilane, and
divinyltetramethyldisiloxane.
30. the measuring chip according to claim 1, wherein plasma
treatment is applied to form said plasma polymerization layer with
a polymeric or non-polymeric monomer.
31. A measuring chip according to claim 30 wherein said
non-polymeric monomer material is selected from the group
consisting of nitrogen, ammonium, hydrazine, hydrogensulfide,
hydrogendisulfide, oxygen, hydrogen, water, halogen gas, and rare
gas.
32. The measuring chip according to claim 1, wherein said plasma
polymerization layer is formed from a mixture of two or more
different types of monomers.
33. The measuring chip for a surface plasmon resonance sensor of
claim 1, further comprising an immune protein or enzyme immobilized
on the surface of said plasma polymerization layer, wherein said
plasma polymerization layer comprises a monomer material selected
from the group consisting of pyridine, triethylamine, diethylamine,
allylamine, acrylamide, aniline, acrylonitrile, 1,2,4-triazole,
5-amino-1H-tetrazole, and acetonitrile.
34. The measuring chip for a surface plasmon resonance sensor of
claim 1, further comprising an immune protein or enzyme immobilized
on the surface of said plasma polymerization layer, wherein said
plasma polymerization layer comprises a monomer material selected
from the group consisting of a compound containing sulfur, an
oxygen containing compound, a carbon containing compound, a
compound containing a halogen, an organic metal compound, an
organic silicon compound, and a carbohydrate compound containing C
and H.
35. A measuring chip according to claim 33, wherein said immune
protein is an antibody.
36. measuring chip according to claim 33, wherein said immune
protein is a Fab fragment of an antibody.
37. A measuring chip according to claim 33, wherein said immune
protein is an F(ab)2 fragment of an antibody.
38. A measuring chip according to claim 3, wherein said nucleic
acid is immobilized on said plasma polymerization layer through a
cross-linking reagent or a condensation reagent.
39. A measuring chip according to claim 4, wherein said non-immune
protein is immobilized on said plasma polymerization layer through
a cross-linking reagent or a condensation reagent.
40. A measuring chip according to claim 6, wherein said
immunoglobulin-binding protein is immobilized on said plasma
polymerization layer through a cross-linking reagent or a
condensation reagent.
41. A measuring chip according to claim 8, wherein said
sugar-binding protein is immobilized on said plasma polymerization
layer through a cross-linking reagent or a condensation
reagent.
42. A measuring chip according to claim 10, wherein said
sugar-recognizing sugar chain is immobilized on said plasma
polymerization layer through a cross-linking reagent or a
condensation reagent.
43. A measuring chip according to claim 11, wherein said fatty acid
or fatty acid ester is immobilized on said plasma polymerization
layer through a cross-linking reagent or a condensation
reagent.
44. A measuring chip according to claim 13, wherein said
polypeptide or oligopeptide is immobilized on said plasma
polymerization layer through a cross-linking reagent or a
condensation reagent.
45. The measuring chip according to claim 38, wherein said
cross-linking reagent is one or more compounds selected from the
group consisting of glutaraldehyde, periodic acid,
N-succinimidyl-2-maleimidoacetic acid,
N-succinimidyl-4-maleimidobutyric acid,
N-succinimidyl-6-maleimidohexanic acid,
N-succinimidyl-4-maleimidomethylcyclohexan-1-carboxylic acid,
N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid,
N-succinimidyl-4-maleimidomethylbanzoic acid,
N-succinimidyl-3-maleimidobenzoic acid, N-sulfosuccinimidyl-3
maleimidobenzoic acid, N-succinimidyl-4-maleimidophenyl-4-butyric
acid, N-sulfosuccinimidyl-4-maleimidophenyl-4-butyric acid,
N,N'-oxydimethylene-dimaleimide, N,N'-o-phenylene-dimaleimide,
N,N'-m-phenylene-dimaleimide, N,N'-p-phenylene-dimaleimide,
N,N'-hexamethylene-dimaleimide, N-succinimidylmaleimidocarboxylic
acid, N-succinimidyl-S-acetylmercaptoacetic acid,
N-succinimidyl-3-(2-pyridyldithio)propionate,
S-acetylmercaptosuccinic anhydride,
methyl-3-(4'-dithiopyridyl)propionimidate,
methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,
iminothiolene, o-carboxymethyl-hydroxylamine,
azodiphenylpilmaleido, bis(sulfosuccinimidyl)sperate,
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene,
4,4'-difluoro-3,3'-dinitrodiphenylsulfon,
1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,
dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,
p-azidophenacylbromide, p-azidophenylglyoxal,
N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,
methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,
N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate,
1,4-benzoquinone, N-succinimidyl-3-(2'-pyridyldithio)propionate,
N-(4-maleimidobutyloxy)sulfosuccinimide sodium salt,
N-(6-maleimidocaproyloxy)sulfosuccinimide sodium salt,
N-(8-maleimidocaproyloxy)sulfosuccinimide sodium salt, N-(
11-maleimidoundecanoyloxy)sulfosuccinimide sodium salt,
N-[2-(1-piperazinyl)ethyl] maleimide bichloric acid,
bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate,
hexamethylenediisothiocyanate, N,N'-ethylenebismaleinimide,
N,N'-polymethylenebisiodoacetamide, 2,4-dinitrobenzenesulfonate
sodium salt, and diazo compounds; or said condensation reagent is
one or more compounds selected from the group consisting of
carbodiimide derivatives represented by RN.dbd.C.dbd.NR (or R'),
N-hydroxysuccinimide, tri-n-butylamine, butyl chloroformate, and
isobutyl isocyanide.
46. A measuring chip according to claim 1, wherein said immune
protein or enzyme is immobilized on said plasma polymerization
layer through a cross-linking reagent or a water-soluble
condensation reagent.
47. A measuring chip according to claim 46, wherein said
cross-linking reagent is one or more compounds selected from the
group consisting of glutaraldehyde,
N-succinimidyl-4-maleimidomethylbanzoic acid,
N-succinimidyl-3-maleimidobenzoic acid,
N-succinimidyl-4-maleimidophenyl-4-butyric acid,
N,N'-oxydimethylene-dimaleimide, N,N'-m-phenylene-dimaleimide,
N,N'-p-phenylene-dimaleimide, N,N'-hexamethylene-dimaleimide,
N-succinimidylmaleimidocarboxylic acid,
N-succinimidyl-S-acetylmercaptoacetic acid,
N-succinimidyl-3-(2-pyridyldithio)propionate,
S-acetylmercaptosuccinic anhydride,
methyl-3-(4'-dithiopyridyl)propionimidate,
methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,
iminothiolene, o-carboxymethyl-hydroxylamine,
azodiphenylpilmaleido, bis(sulfosuccinimidyl)sperate,
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene,
4,4'-difluoro-3,3'-dinitrodiphenylsulfon,
1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,
dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,
p-azidophenacylbromide, p-azidophenylglyoxal,
N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,
methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,
N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate,
1,4-benzoquinone, N-succinimidyl-3-(2'-pyridyldithio)propionate,
bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate,
hexamethylenediisothiocyanate, N,N'-ethylenebismaleinimido,
N,N'-polymethylenebisiodoacetoamide, and diazo compounds; or said
condensation reagent is one or more compounds selected from the
group consisting of carbodiimide derivatives represented by
RN.dbd.C.dbd.NR (or R'), N-hydroxysuccinimide, tri-n-butylamine,
butyl chloroformate, and isobutyl isocyanide.
48. The measuring chip for a surface plasmon resonance sensor of
claim 1, further comprising a substance immobilized on the surface
of said plasma polymerization layer, and an additional plasma
polymerization layer or plasma-treated layer formed on said plasma
polymerization layer.
49. A measuring chip according to claim 48, wherein said substance
to be immobilized is selected from the group consisting of a
non-immune protein, an immunoglobulin-binding protein, a
sugar-binding protein, a sugar-recognizing sugar chain, a fatty
acid or a fatty acid ester, a polypeptide or oligopeptide having
ligand-binding activity, an immune protein, and an enzyme.
50. The measuring chip for a surface plasmon resonance sensor of
claim 1, further comprising a substance immobilized on said plasma
polymerization layer through a hydrophobic bond.
51. The measuring chip according to claim 50, wherein said
substance to be immobilized is selected from the group consisting
of a a non-immune protein, an immunoglobulin-binding protein, a
sugar-binding protein, a sugar-recognizing sugar chain, a fatty
acid or a fatty acid ester, a polypeptide or oligopeptide having
ligand-binding activity, an immune protein, and an enzyme.
52. A method for producing a measuring chip for a surface plasmon
resonance sensor comprising: forming a metal layer on an optically
transparent substrate, forming one or more plasma polymerization
layers on said metal layer, and immobilizing a physiologically
active substance on the surface of said plasma polymerization
layer.
53. The method of claim 52, comprising immobilizing a non-immune
protein on the surface of said plasma polymerization layer.
54. The method of claim 52 comprising immobilizing an
immunoglobulin-binding protein on the surface of said plasma
polymerization layer.
55. The method of claim 52 comprising A immobilizing a
sugar-binding protein on the surface of said plasma polymerization
layer.
56. The method of claim 52 comprising a sugar-recognizing sugar
chain on the surface of said plasma polymerization layer.
57. The method of claim 52 comprising _ immobilizing a fatty acid
or fatty acid ester on the surface of said plasma polymerization
layer.
58. The method of claim 52 comprising immobilizing a polypeptide or
oligopeptide having a ligand binding capability on the surface of
said plasma polymerization layer.
59. A method according to claim 52 wherein the plasma
polymerization layer is formed by a plasma-treatment using a
monomer material.
60. The method of claim 52 comprising immobilizing an immune
protein or enzyme on the surface of said plasma polymerization
layer.
61. A measuring cell for a surface plasmon resonance sensor
comprising a measuring chip according to claim 1.
62. A measuring cell according to claim 61, wherein said chip is
optically analyzed.
63. A surface plasmon resonance biosensor comprising a measuring
chip according to claim 1.
64. A surface plasmon resonance biosensor comprising a measuring
cell according to claim 61.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a surface plasmon resonance
biosensor, specifically, a measuring chip for the same and a method
for producing the measurement chip.
[0003] 2. Background Art
[0004] A number of methods using immunological reactions are used
in clinical tests for detecting target substances. Conventional
methods are known to be intricate and require labeling substances.
Thus, immunological sensors using a surface plasmon resonance
biosensor (SPR) is being used, in which no labeling substance is
required and a ligand can be detected with high sensitivity. This
surface plasmon resonance biosensor is based on the phenomenon that
the intensity of a monochromatic light reflected from the interface
between an optically transparent substance such as glass and a
metal thin-film layer is dependent on the refractive index of a
sample placed on the reflecting side of the metal. Accordingly, a
sample can be analyzed by measuring the intensity of the reflected
monochromatic light.
[0005] An optical part of a measuring cell for this surface plasmon
resonance (surface plasmon resonance biosensor) has a structure
shown in FIG. 2. Namely, porous material 5 is formed on metal
thin-film 2 formed on glass substrate 1, and physiologically active
substance 4, such as an enzyme or antibody, is retained or
immobilized on the surface or inside of porous material 5. Examples
of porous material 5 to be used include weaved, knitted or
non-woven cloths made of synthetic fibers, natural fibers,
inorganic fibers or the like, and porous inorganic or organic
materials (see Japanese Patent Laid-open No. 164195/1991).
Furthermore, carboxymethyl dextran is used as a porous material in
a commercial product (BIAcore 2000, Pharmacia Biosensor).
[0006] However, physiologically active substance 4 just exists on
the surface of porous material 5 and interacts with target
substances.
[0007] LB (Langmuir-Blodgett) method is occasionally used to
immobilize physiologically active substance 4 on metal thin-film 2
(see Japanese Patent Laid-open No. 288672/1993). However, this
method has a disadvantage in that LB membrane binds poorly to a
metal thin-film and peels off together with the physiologically
active substance.
[0008] Furthermore, Japanese Patent Laid-open No. 264843/1997
discloses measuring cells for a surface plasmon resonance
biosensor.
SUMMARY OF THE INVENTION
[0009] The present inventors have now found that sensitivity of a
measuring chip for a surface plasmon resonance sensor is extremely
improved when only a small amount of a physiologically active
substance is immobilized on a specific plasma polymerization
layer.
[0010] An objective of the present invention is to provide a
measuring chip for a surface plasmon resonance sensor that can
detect a small amount of target substances in high sensitivity.
[0011] Another objective of the present invention is to provide a
measuring cell for a surface plasmon resonance sensor that can
detect a small amount of target substances in high sensitivity.
[0012] Further objective of the present invention is to provide a
method for producing said measuring chip.
[0013] The present invention provides a measuring chip for a
surface plasmon resonance sensor comprising a metal layer and one
or more plasma polymerization layers formed on said metal
layer.
[0014] The present invention also provides a measuring chip for a
surface plasmon resonance sensor comprising a metal layer, one or
more plasma polymerization layers formed on said metal layer, and a
biologically active substance immobilized on the surface of said
plasma polymerization layer.
[0015] The present invention also provides a measuring cell for a
surface plasmon resonance sensor comprising said measuring
chip.
[0016] The present invention also provides a method for producing a
measuring chip for a surface plasmon resonance sensor comprising
the steps of forming a metal layer on an optically transparent
substrate, forming one or more plasma polymerization layers on said
metal layer, and then immobilizing a biologically active substance
on the surface of said plasma polymerization layer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a schematic sectional view of one embodiment of
the measuring chip for a surface plasmon resonance sensor according
to the present invention.
[0018] FIG. 2 is a schematic sectional view of an optical part of a
measuring chip for a conventional surface plasmon resonance
biosensor. 1: Transparent substrate; 2: Metal thin-film; 3: Plasma
polymerization layer; 4: Physiologically active substance; 5:
Porous material.
[0019] FIGS. 3 (a) and (b) each show a schematic sectional view of
an optical part of a measuring chip for a surface plasmon resonance
biosensor. (a) shows immobilization of an Fab fragment of an
antibody. (b) shows immobilization of an F(ab').sub.2 fragment of
an antibody. 1: Transparent substrate; 2: Metal thin-film; 3:
Plasma polymerization layer; 4: Physiologically active
substance.
[0020] FIG. 4 is a schematic sectional view of an optical part of a
measuring chip for a surface plasmon resonance biosensor.
[0021] FIG. 5 illustrates a surface plasmon resonance biosensor. 7:
Cartridge block; 8: Light source; 9: Detector; 10: Measuring chip;
71: Measuring cell; 72, 73: Flow routes; 80: Incident light; 90:
Reflecting light.
[0022] FIG. 6 illustrates a reflected light intensity curve before
and after plasma polymerization membrane formation.
[0023] FIG. 7 illustrates a schematic view showing the apparatus
used in Example 1.
[0024] FIG. 8 shows the relationship between the concentration of
the complementary DNA and RU in Example 1.
[0025] FIG. 9 shows the relationship between the concentration of
the complementary DNA and RU in Example 2.
[0026] FIG. 10 shows the relationship between the concentration of
the complementary DNA and RU in Example 3.
[0027] FIG. 11 shows the relationship between the concentration of
the complementary DNA and RU in Example 4.
[0028] FIG. 12 shows the relationship between the concentration of
the HSA antigen and RU in Example 5.
[0029] FIG. 13 shows the relationship between the concentration of
the BSA antigen and RU in Example 6.
[0030] FIG. 14 shows the relationship between the concentration of
the sugar and RU in Example 7.
[0031] FIG. 15 shows the relationship between the concentration of
the BSA antigen and RU in Example 8.
[0032] FIG. 16 shows the relationship between the concentration of
the BSA antigen and RU in Example 9.
[0033] FIG. 17 shows the relationship between the concentration of
the BSA antigen and RU in Example 10.
[0034] FIG. 18 shows the relationship between the concentration of
the BSA antigen and RU in Example 11.
[0035] FIG. 19 shows the relationship between the concentration of
the HSA antigen and RU in Example 12.
[0036] FIG. 20 shows the relationship between the concentration of
the HSA antigen and RU in Example 13.
[0037] FIG. 21 shows the relationship between the concentration of
the HSA antigen and RU in Example 14.
[0038] FIG. 22 shows the relationship between the concentration of
the HSA antigen and RU in Example 15.
[0039] FIG. 23 shows the relationship between the concentration of
the HSA antigen and RU in Example 16.
[0040] FIG. 24 shows the relationship between the concentration of
the complementary DNA and RU in Example 17.
[0041] FIG. 25 shows the relationship between the concentration of
the HSA antigen and RU in Example 18.
[0042] FIG. 26 shows the relationship between the concentration of
skatole and RU in Example 19.
[0043] FIG. 27 shows the relationship between the concentration of
the HSA antigen and RU in Example 20.
[0044] FIG. 28 shows the relationship between the concentration of
the HSA antigen and RU in Example 21.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The measuring chip for a surface plasmon resonance sensor
("measuring chip") may have optically transparent substrate
(transparent substrate) 1, metal thin-film 2 formed on transparent
substrate 1, plasma polymerization layer 3 formed on metal
thin-film 2, and physiologically active substance 4 immobilized on
the surface of plasma polymerization layer 3 as shown in FIG.
1.
[0046] Transparent substrate 1 can be any substrate customarily
used in a measuring chip for a surface plasmon resonance sensor.
Generally, substrates made of materials that are transparent to a
laser beam such as glass can be used. The thickness of the
substrate can be about 0.1 to 5 mm.
[0047] Metal thin-film 2 is not particularly restricted, provided
it can induce surface plasmon resonance. Examples of the metal to
be used for metal thin-film 2 include gold, silver and platinum.
They can be used alone or in combination. Furthermore, for better
adhesion to transparent substrate 1, an auxiliary layer made of
chrome or the like may be set between transparent substrate 1 and
the layer made of gold, silver or the like.
[0048] The thickness of metal thin-film 2 is preferably 100 to 2000
angstroms, most preferably 100 to 500 angstroms. If the thickness
exceeds 3,000 angstroms, surface plasmon phenomena of the medium
cannot be sufficiently detected. Furthermore, when an auxiliary
layer made of chrome or the like is formed, the thickness of the
auxiliary layer is preferably 30 to 50 angstroms.
[0049] Plasma polymerization layer 3 can be formed by plasma
polymerization of a monomer material for three-dimensional
cross-linking. A monomer material to be used in the present
invention can be any material that can immobilize a physiologically
active substance by plasma polymerization.
[0050] Examples of a monomer material for a plasma polymerization
layer include compounds of formula (I):
CH.sub.3--(CH.sub.2).sub.n--NH.sub.2 (wherein n is an integer from
1 to 6) (I) and compounds of formula (II):
NH.sub.2--(CH.sub.2).sub.n--NH.sub.2 (wherein n is an integer from
1 to 6) (II) and compounds which comprise carbon (C), hydrogen (H)
and nitrogen (N) and have double bonds or triple bonds, such as
acetonitrile, vinylamine and pyridine.
[0051] Furthermore, when a cross-linking reagent or a condensation
reagent is used as a linking layer, a compound further containing
sulfur (S), oxygen (O) or silicon (Si) can be used as a monomer
material. Generally, a compound appropriately containing any two or
more elements selected from carbon (C), hydrogen (H), nitrogen (N),
sulfur (S), oxygen (O) and silicon (Si) can be used. In addition, a
halogen gas or a rare gas can be used as a monomer material.
[0052] In the present invention, a compound containing nitrogen can
be used as a monomer material. Examples of the compound containing
nitrogen include nitrogen N.sub.2; ammonium; hydrazine; pyridine;
compounds of formulae (I) and (II) such as ethylenediamine
NH.sub.2(CH.sub.2).sub.2NH.sub.2, hexamethylenediamine NH.sub.2
(CH.sub.2).sub.6NH.sub.2, n-propylamine
CH.sub.3(CH.sub.2).sub.2NH.sub.2 and monoethylamine
CH.sub.3(CH.sub.2)NH; compounds of formula
(CH.sub.3).sub.3(CH.sub.2).sub.nN n=0 to 17) such as triethylamine
(C.sub.2H.sub.5).sub.3N; compounds of formula
(CH.sub.3).sub.2(CH.sub.2).sub.nNH (n=0 to 17) such as diethylamine
(C.sub.2H.sub.5).sub.2NH; compounds of formula
CH.sub.2.dbd.CH(CH.sub.2).sub.nNH.sub.2 (n=0 to 17) such as
allylamine CH.sub.2.dbd.CHCH.sub.2NH.sub.2; compounds of formula
CH.sub.3(CH.sub.2).sub.nCN (n=0 to 17) such as acetonitrile
CH.sub.3CN; compounds of formula CH.sub.3(CH.sub.2).sub.nCN;
propargylamine CHCCH.sub.2NH.sub.2; compounds of formula CHC
(CH.sub.2).sub.nNH.sub.2; acrylamide; aniline; acrylonitrile;
1,2,4-triazole; and 5-amino-1H-tetrazole.
[0053] Further examples of of the compound containing nitrogen
include the following:
RaNRb.sub.2:
[0054] Ra is H or CH.sub.3(CH.sub.2).sub.n (n=0 to 17),
[0055] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group,
and
[0056] Rb is H or CH.sub.3(CH.sub.2).sub.n (n=0 to 17),
[0057] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group;
RaNRc:
[0058] Rc is H or CH.sub.3(CH.sub.2).sub.nCH (n=0 to 17), or
CH.sub.2,
[0059] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group;
RdN:
[0060] Rd is CH.sub.3(CH.sub.2).sub.nC (n=0 to 17) or CH,
[0061] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group;
ReNRfNRg.sub.2:
[0062] Re is H or CH.sub.3(CH.sub.2).sub.n (n=0 to 17),
[0063] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group,
[0064] Rf is (CH.sub.2).sub.n (n=0 to 17),
[0065] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group,
[0066] Rg is H or CH.sub.3(CH.sub.2).sub.n (n=0 to 17),
[0067] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group;
RhNRiNRj
[0068] Rh is H or CH.sub.3(CH.sub.2).sub.n (n=0 to 17) or
CH.sub.3(CH.sub.2).sub.n CH (n=0 to 17) or CH).sub.2,
[0069] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group,
[0070] Ri is (CH.sub.2).sub.n (n=0 to 17) or CH (CH.sub.2).sub.n CH
(n=0 to 17),
[0071] and includes a group having a double bond a triple bond or
both in the chain, and further a branched or cyclized group,
[0072] Rj is H or CH.sub.3(CH.sub.2).sub.n CH (n=0 to 17) or
CH.sub.3(CH.sub.2).sub.nCH (n=0 to 17) or CH.sub.2 or
CH.sub.3(CH.sub.2).sub.n C (n=0 to 17) or CH,
[0073] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group;
NRkN:
[0074] Rk is C(CH.sub.2).sub.nC (n=0 to 17),
[0075] and includes a group having a double bond or a triple bond
or both in the chain, and further a branched or cyclized group.
[0076] In the present invention, a compound containing sulfur can
be used as a monomer material. Examples of the compound containing
sulfur include hydrogen sulfide; carbon disulfide thiophene;
compounds of formula CH.sub.3S(CH.sub.2).sub.nCH.sub.3 (n=0 to 17)
such as dimethyl sulfide (CH.sub.3).sub.2S; compounds of formula
CH.sub.3(CH.sub.2).sub.nSS(CH.sub.2).sub.mCH.sub.3 (n=0 to 17, m=0
to 17) such as methyl disulfide CH.sub.3SSCH.sub.3; compounds of
formula CH.sub.3(CH.sub.2).sub.nSH (n=0 to 17) such as ethanethiol
CH.sub.3CH.sub.2SH; compounds of formula SH(CH.sub.2).sub.nSH (n=1
to 17) such as ethanedithiol SH(CH.sub.2).sub.2SH; mercaptoethanol;
and dithreitol.
[0077] Furthermore, compounds having one of or two or more of
groups including --COOH, --CHO, --SH, --NH.sub.2, --OH, .dbd.NH,
CONH.sub.2, --NCO, --CH.dbd.CH.sub.2, .dbd.C.dbd.O and ##STR1## can
be used as a monomer material. Examples of such compounds include
cysteine, glutathione, formyl succinate, aminobenzoate,
aminohexanate, mercaptobenzoate, and compounds having
--C.ident.CCH.sub.2OH.
[0078] In the present invention, a compound containing a halogen
can 10 be used as a monomer material. Examples of the compound
containing a halogen for a plasma polymerization layer include
tetrafluoroethylene, chlorobenzene, hexachlorobenzene,
hexafluorobenzene, and vinyl fluoride.
[0079] In the present invention, an organic metal compound can be
used as a monomer material. Examples of an organic metal compound
for the plasma polymerization layer include an organic silicon
compounds such as tetramethylsilane, tetramethyldisiloxane,
hexamethyldisiloxane, hexamethyldisilazane,
hexamethylcyclotrisilazane, dimethylaminotrimethylsilane,
trimethylvinylsilane, tetramethoxysilane,
aminopropyltriethoxysilane, octadecyldiethoxymethylsilane,
hexamethyldisilane and divinyltetramethyldisiloxane.
[0080] Compounds of formulae (I) and (II) having no double bond or
triple bond are preferably used because a layer is formed slowly so
that the resulting layer is more homogeneous, compared with
compounds having double bonds or triple bonds.
[0081] The thickness of plasma polymerization layer 3 is preferably
100 to 3000 angstroms, most preferably 500 to 1000 angstroms.
[0082] Plasma polymerization layer 3 can be formed by plasma
treatment to a resulting plasma layer with a polymeric or
non-polymeric monomer. Examples of such non-polymeric monomer
material include nitrogen, ammonium, hydrazine, hydrogensulfide,
hydrogendisulfide, oxygen, hydrogen, water, halogen gas, and rare
gas (e.g., argon, neon, helium, krypton, and xenon).
[0083] Furthermore, a mixture of various kinds of monomer materials
can be used as a monomer material. Plasma polymerization layer 3
can also be formed by lamination techniques and optionally using a
mixture as a monomer material.
[0084] Plasma polymerization layer 3 of the present invention has
the following advantages:
[0085] 1) The layer is pinhole-free, amorphous, and dense.
[0086] 2) A thin homogeneous layer down to about 500 angstroms can
be made, which exhibits extremely little fluctuation in its
refractive index.
[0087] 3) By changing the kind of plasma gas, not only a change in
the thickness of the layer but also surface modification and
surface improvement, such as introduction of functional groups, and
control of the density of the functional groups to be introduced
can be attained.
[0088] 4) The layer can be formed in combination with semiconductor
techniques since it is formed under dry conditions.
[0089] 5) The layer has excellent drug tolerance, heat tolerance,
mechanical properties, and stability.
[0090] Furthermore, in the case of a sensor chip for SPR, in which
a metal thin-film is essential, the metal thin-film and the plasma
polymerization layer can be formed in the same chamber. Thus, the
manufacturing process can be simplified.
[0091] It is also advantageous to attain surface improvement, such
as introduction of a functional group, by further exposing a
resulting plasma polymerization layer to plasma treatment with a
non-polymeric or polymeric monomer. The plasma polymerization
treatment is intended to include a treatment with not only a
non-polymeric monomer and an inactive monomer but also a polymeric
monomer.
[0092] Physiologically active substance 4 to be immobilized is not
particularly limited, provided it reacts interacts with a target
substance to be measured. Examples of physiologically active
substance 4 include nucleic acids (e.g., DNA, RNA, and PNA);
non-immune proteins (e.g., avidin (streptoavidin), biotin or a
receptor); immunoglobulin-binding proteins (e.g., protein A,
protein G and a rheumatoid factor (RF)); sugar-binding proteins
(e.g., lectin); sugar-recognizing sugar chains; fatty acids or
fatty acid esters (e.g., stearic acid, alachidic acid, behenic
acid, ethyl stearate, ethyl arachidate, and ethyl behanate);
polypeptides or oligopeptides having ligand binding activity;
immune proteins (e.g., an antibody); and enzyme.
[0093] When an antibody is used as physiologically active substance
4, Fc fragments of the antibody can be immobilized only on the
surface of plasma polymerization layer 3 and the antibody is formed
in a monomolecular layer as shown in FIG. 1. However, since the
sensitivity and the reaction rate decrease as Fab fragments of the
antibody are separated from plasma polymerization layer 3, Fab
fragments (FIG. 3 (a)) or F(ab').sub.2 fragments (FIG. 3 (b)) can
be immobilized directly on plasma polymerization layer 3 as shown
in FIG. 3 to improve the sensitivity and the reaction rate.
[0094] The thickness of physiologically active substance 4 depends
on the size of the physiologically active substance itself, but is
preferably 100 to 3000 angstroms, most preferably 100 to 1000
angstroms.
[0095] In the present invention, the physiologically active
substance can be immobilized on the plasma polymerization layer
through linking agents.
[0096] FIG. 4 is a schematic illustration showing one embodiment of
the measuring chip according to the present invention. The
measuring chip has covalent bond layer 6 between plasma
polymerization layer 3 and physiologically active substance 4.
Substance 4 is immobilized on plasma polymerization layer 3 via
covalent layer 6. The covalent bond can be formed with a
cross-linking reagent or a condensation reagent.
[0097] The cross-linking reagent or a condensation reagent is not
particularly restricted, provided it can covalently and firmly
immobilize substance 4. They can be used alone or in
combination.
[0098] Examples of such cross-linking reagents include
glutaraldehyde, periodic acid, N-succinimidyl-2-maleimidoacetic
acid, N-succinimidyl-4-maleimidobutyric acid,
N-succinimidyl-6-maleimidohexanic acid,
N-succinimidyl-4-maleimidomethylcyclohexan-1-carboxylic acid,
N-sulfosuccinimidyl-4-maleimidomethylcyclohexane-1-carboxylic acid,
N-succinimidyl-4-maleimidomethylbanzoic acid,
N-succinimidyl-3-maleimidobenzoic acid,
N-sulfosuccinimidyl-3-maleimidobenzoic acid,
N-succinimidyl-4-maleimidophenyl-4-butyric acid,
N-sulfosuccinimidyl-4-maleimidophenyl-4-butyric acid,
N,N'-oxydimethylene-dimaleimide, N,N'-o-phenylene-dimaleimide,
N,N'-m-phenylene-dimaleimide, N,N'-p-phenylene-dimaleimide,
N,N'-hexamethylene-dimaleimide, N-succinimidylmaleimidocarboxylic
acid, N-succinimidyl-S-acetylmercaptoacetic acid,
N-succinimidyl-3-(2-pyridyldithio)propionate,
S-acetylmercaptosuccinic anhydride,
methyl-3-(4'-dithiopyridyl)propionimidate,
methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,
iminothiolene, o-carboxymethyl-hydroxylamine,
azodiphenylpilmaleido, bis(sulfosuccinimidyl)sperate,
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene,
4,4'-difluoro-3,3'-dinitrodiphenylsulfon,
1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,
dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,
p-azidophenacylbromide, p-azidophenylglyoxal,
N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,
methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,
N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate,
1,4-benzoquinone, N-succinimidyl-3-(2'-pyridyldithio)propionate,
N-(4-maleimidobutyloxy)sulfosuccinimide sodium salt,
N-(6-maleimidocaproyloxy)sulfosuccinimide sodium salt,
N-(8-maleimidocaproyloxy)sulfosuccinimide sodium salt,
N-(11-maleimidoundecanoyloxy)sulfosuccinimide sodium salt,
N-[2-(1-piperazinyl)ethyl]maleimide bichloric acid,
bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate,
hexamethylenediisothiocyanate, N,N'-ethylenebismaleinimide,
N,N'-polymethylenebisiodoacetamide, 2,4-dinitrobenzenesulfonate
sodium salt, and diazo compounds. Glutaraldehyde is preferable as a
cross-linking reagent.
[0099] Examples of such condensation reagents include carbodiimide
derivatives represented by formula RN.dbd.C.dbd.NR (or R'),
N-hydroxysuccinimide, tri-n-butylamine, butyl chloroformate, and
isobutyl isocyanide.
[0100] By introducing covalent layer 6 to the measuring cell to
firmly mobilize physiologically active substance 4 via covalent
bonds, substance 4 can be maintained immobilized when the measuring
cell is washed, which enables the cell to be used for repetitive
measurements for another advantageous feature. The thickness of
covalent layer 6 is preferably 10 to 100 angstroms, most preferably
10 to 20 angstroms.
[0101] The physiologically active substance can also be immobilized
by hydrophobic bond, by integrating substance 4 into a plasma
polymerization layer or by an additional plasma treatment.
[0102] A preferred group of the measuring chip according to the
present invention is a measuring chip comprising a metal layer, one
or more plasma polymerization layers formed on said metal layer,
and an immune protein or enzyme immobilized on the surface of said
plasma polymerization layer, wherein said plasma polymerization
layer comprises a monomer material selected from the group
consisting of pyridine, triethylamine, diethylamine, allylamine,
acrylamide, aniline, acrylonitrile, 1,2,4-triazole,
5-amino-1H-tetrazole, and acetonitrile,
[0103] Another preferred group of the measuring chip according to
the present invention is a measuring chip comprising a metal layer,
one or more plasma polymerization layers formed on said metal
layer, and an immune protein or enzyme immobilized on the surface
of said plasma polymerization layer, wherein said plasma
polymerization layer comprises a monomer material selected from the
group consisting of pyridine, triethylamine, diethylamine,
allylamine, acrylamide, aniline, acrylonitrile, 1,2,4-triazole,
5-amino-1H-tetrazole, and acetonitrile and wherein said immune
protein or enzyme is immobilized on said plasma polymerization
layer through a cross-linking reagent or a water-soluble
condensation reagent.
[0104] A cross-linking reagent for the preferred group above can be
selected from the group consisting of glutaraldehyde,
N-succinimidyl-4-maleimidomethylbanzoic acid,
N-succinimidyl-3-maleimidobenzoic acid,
N-succinimidyl-4-maleimidophenyl-4-butyric acid,
N,N'-oxydimethylene-dimaleimide, N,N'-m-phenylene-dimaleimide,
N,N'-p-phenylene-dimaleimide, N,N'-hexamethylene-dimaleimide,
N-succinimidylmaleimidocarboxylic acid,
N-succinimidyl-S-acetylmercaptoacetic acid,
N-succinimidyl-3-(2-pyridyldithio)propionate,
S-acetylmercaptosuccinic anhydride,
methyl-3-(4'-dithiopyridyl)propionimidate,
methyl-4-mercaptobutylimidate, methyl-3-mercaptopropionimidate,
iminothiolene, o-carboxymethyl-hydroxylamine,
azodiphenylpilmaleido, bis(sulfosuccinimidyl)sperate,
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene,
4,4'-difluoro-3,3'-dinitrodiphenylsulfon,
1,5-difluoro-2,4-dinitrobenzene, p-phenylenediisothiocyanate,
dimethyladipimidate, dimethylpimelimidate, dimethylsuberimidate,
p-azidophenacylbromide, p-azidophenylglyoxal,
N-hydroxysuccinimidyl-4-azidobenzoate, 4-fluoro-3-nitrophenylazide,
methyl-4-azidobenzoimidate, N-5-azido-2-nitrobenzoyloxysuccinimide,
N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate,
1,4-benzoquinone, N-succinimidyl-3-(2'-pyridyldithio)propionate,
bisdiazobenzidine, hexamethylenediisocyanate, toluenediisocyanate,
hexamethylenediisothiocyanate, N,N'-ethylenebismaleinimido,
N,N'-polymethylenebisiodoacetoamide, and diazo compounds; or said
condensation reagent is one or more compounds selected from the
group consisting of carbodiimide derivatives represented by
RN.dbd.C.dbd.NR (or R'), N-hydroxysuccinimide, tri-n-butylamine,
butyl chloroformate, and isobutyl isocyanide.
[0105] The measuring chip according to the present invention can be
formed as follows:
[0106] First, metal thin-film 2 is formed on transparent substrate
1. Metal thin-film 2 can be formed by conventional methods such as
sputtering, CVD, PVD, or vacuum evaporation.
[0107] Second, plasma polymerization layer 3 is formed on metal
thin-film 2. Plasma polymerization layer 3 can be formed by plasma
polymerization using a plasma polymerization apparatus. The rate of
plasma formation is preferably 100 to 3000 angstroms/min, most
preferably 500 to 1000 angstroms/min. If the rate exceeds 3000
angstroms/min, it becomes difficult to obtain a smooth plasma
polymerization layer. More specifically, the plasma 35
polymerization can be preferably carried out at a monomer material
flow rate of 0.05 to 100 sccm at a room temperature or at a
temperature of 10 to 20.degree. C. at a pressure between
1.0.times.10.sup.-2 and 1.0.times.10.sup.2 Pa using a discharge
power of 20 to 300 W at a discharge frequency of 10 MHz or 13.56
MHz. However, polymerization conditions are not restricted to the
conditions above.
[0108] After formation of plasma polymerization layer 3,
physiologically active substance 4 is finally immobilized on plasma
polymerization layer 3. Immobilization can be done by conventional
methods. For example, a specified amount of physiologically active
substance 4 can be immobilized by contacting it with plasma
polymerization layer 3 for a specified period of time. If the
measuring cell is a flow-cell type, a specified volume of the
physiologically active substance 4 can be immobilized by contacting
it with plasma polymerization layer 3 by pouring a specified volume
for a specified period of time.
[0109] When an antibody is used as a physiologically active
substance and its Fab fragment is immobilized directly on plasma
polymerization layer 3, the same treatment can be done after the
antibody is partly digested with papain. On the other hand, when
the F(ab').sub.2 fragment is immobilized directly on plasma
polymerization layer 3, the same treatment can be done after the
antibody is partly digested with pepsin.
[0110] When covalent bond layer 6 is formed, a cross-linking
reagent or a condensation reagent is allowed to be in contact with
plasma polymerization layer 3 in the same manner as with active
substance 4, after which substance 4 can be immobilized.
[0111] The measuring cell for a surface plasmon resonance sensor
according to the present invention comprises the measuring chip.
The measuring chip can be mounted on an optical part to be
optically analyzed. The term "optical part" as used herein refers
to a part where a light is projected and an evanescent wave and a
surface plasmon can be induced.
[0112] The surface plasmon resonance biosensor according to the
present invention comprises the measuring cell.
[0113] FIG. 5 is a schematic view of one embodiment of the surface
plasmon resonance biosensor according to the present invention. The
surface plasmon resonance biosensor has cartridge block 7, light
source 8, and detector 9 and measuring chip 10 is mounted on
cartridge block 7. The upper side of cartridge block 7 has a hollow
and this hollow and measuring chip 10 construct measuring cell
71.
[0114] The body of measuring chip 10 comprises a transparent
substrate, and a layer comprising a metal thin-film, a plasma
polymerization layer formed under said metal film. A
physiologically active substance is immobilized on the surface of
said plasma polymerization layer facing the hollow of cartridge
block 7. Measuring cell 71 is constructed from the hollow of
cartridge block 7 and measuring chip 10; and cartridge block 7 has
flow routes 72 and 73 providing passages to the outside of
measuring cell 71 and cartridge block 7, which makes measuring cell
71 a flow-cell type. However, the present invention is not
restricted to this type and a batch type cell can also be used.
Using measuring cell 71 of this flow-cell type, a sample can be
measured either continuously or intermittently. In this sensor, the
sample flows into measuring cell 71 via flow route 72 and is
discharged after measurement via flow route 73. The flow rate of
the sample is preferably 0. 5 to 5 .mu.l/min. The flow rate is
controlled, for example, using a computer-operated pump.
[0115] Monochromatic light (incident light 80) is irradiated from
light source 8 toward the optical part of measuring chip 10 and its
reflected light 90, which is reflected by metal thin-film 2 set on
the reverse side of measuring chip 10, reaches detector 9. Detector
9 can detect the intensity of reflected light 90. Light source 8
and detector 9 are not particularly restricted, and can be any
types customarily used for a surface plasmon resonance biosensor.
In the sensor according to the present invention, the incident
light is wedge-shaped and the light reflected in different
directions can be measured simultaneously. However, the present
invention is not restricted to this type of sensor. The
configuration of this type does not require a mobile part, thereby
producing excellent stability and durability, and enabling real
time measurement of samples as well.
[0116] The configuration as described above yields a reflected
light intensity curve that forms a trough relative to a given angle
of incidence (see FIG. 6). The trough in the reflected light
intensity curve is due to surface plasmon resonance. Namely, when
light is totally reflected at the interface between the transparent
substrate and the exterior of measuring chip 10, a surface wave
known as an evanescent wave is generated at the interface and a
surface wave known as a surface plasmon is also generated on the
metal thin-film. Resonance occurs when the wave number of these two
surface waves coincides and a part of light energy is consumed to
excite the surface plasmon, resulting in a decrease in the
intensity of the reflected light. The wave number of the surface
plasmon is affected by the refractive index of the medium proximate
to the surface of the metal thin-film. Therefore, when the
refractive index of the medium changes due to an interaction
between the substance to be measured and the physiologically active
substance, a surface plasmon resonance is induced to change the
angle of incidence. Thus, a change in the concentration of the
substance to be measured can be perceived by a shift of the trough
in the reflected light intensity curve. The change in the angle of
incidence is called a resonance signal and a change of 10.sup.-4
degree is expressed as 1 RU. In the surface plasmon resonance
biosensor of this example, highly effective and reliable
measurement can be done if measuring chip 10 is made to be freely
attachable and detachable and disposable. Furthermore, if a
covalent bond layer is provided between the plasma polymerization
layer and the physiologically active substance, measuring chip 10
can be used repeatedly by washing the inside of measuring cell 71,
resulting in a decrease in the cost.
[0117] The surface plasmon resonance biosensor of the present
invention can be used for quantitative or qualitative analysis,
identification of a target substance present in a sample.
EXAMPLE
[0118] The present invention is further illustrated by the
following Examples that are not intended as a limitation of the
invention.
Example 1
[0119] A measuring chip having layers shown in FIG. 1 on an optical
recognition part was constructed.
[0120] A glass plate with a thickness of 0.15 mm (18 mm.times.18
mm) was used for a transparent substrate. A chrome layer and then a
gold layer were deposited on this transparent substrate by
sputtering. The sputtering was carried out at 100 W for 40 seconds
for the chrome layer and at 100 W for 2 minutes and 30 seconds for
the gold layer. The resulting chrome layer was 40 angstroms thick
and the resulting gold layer was 500 angstroms thick.
[0121] A plasma polymerization layer was formed on the metal
layers. An apparatus as shown in FIG. 7 was used for plasma
polymerization. Ethanedithiol was used as a monomer material for
the plasma polymerization layer to introduce a thiol group.
Conditions for plasma polymerization were as follows:
[0122] Flow volume of monomer material: 15 sccm
[0123] Temperature: 15.degree. C.
[0124] Pressure: 4.7 Pa
[0125] Discharge electric power: 20 W
[0126] Discharge frequency: 10 MHz, FM modulation
[0127] Duration of discharge: 60 seconds.
[0128] Under the conditions described above, a thiol group was
introduced on the surface of plasma polymerization layer. The
sensor chip with the introduced thiol group was mounted on the
cartridge block of the surface plasmon resonance biosensor and
maleimidized avidin (see "Ultrahigh Sensitivity Enzyme Immunoassay"
by Eiji Ishikawa) was poured through a flow route into the
measuring cell at a flow rate of 5 .mu.l/min for immobilization on
the thiol group on the plasma polymerization layer for 60 minutes.
50 .mu.l of 10 pM-biotinized DNA were then poured and the probe DNA
was immobilized via the avidin for 10 minutes. A DNA
(7.5.times.10.sup.-7 M) having a DNA sequence complementary to this
probe DNA was introduced and after the reaction, a signal of about
500 RU was obtained. TABLE-US-00001 Concentration of Complementary
DNA (.mu.M) 0.00075 0.0075 0.075 0.75 7.5 75 RU 10 25 100 500 1000
1100
[0129] It was confirmed by an XPS analysis that the resulting
membrane has a mercapto group.
[0130] FIG. 6 shows the reflected light intensity curve before and
after the formation of the plasma polymerization layer, which show
the intensity of reflected light corresponding to the angle of
incidence .theta.) FIG. 6 shows that the plasma polymerization
layer is formed on the surface of the gold layer. The thickness of
the plasma polymerization layer can be estimated from
.DELTA..theta..
Example 2
[0131] The same apparatus and method as in Example 1 were used.
[0132] Acetonitrile was used as a monomer material for the plasma
polymerization layer. Conditions for plasma polymerization were as
follows:
[0133] Flow volume of monomer material: 1.5 sccm+Ar dilution 15
(sccm)
[0134] Temperature: room temperature
[0135] Pressure: 4.7 Pa
[0136] Discharge electric power: 80 W
[0137] Discharge frequency: 13.56 MHz
[0138] Duration of discharge: 15 seconds.
[0139] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and avidin
(concentration: 20 .mu.g/ml) was also poured at a flow rate of 5
.mu.l/min to immobilize for 60 minutes. 10 .mu.M biotin-labeled
probe RNA were then poured at a flow rate of 1 .mu.l/min to
immobilize the probe RNA for 10 minutes. DNA (7.5.times.10.sup.-7
M) having a DNA sequence complementary to this probe RNA was
introduced and after the reaction, a signal of about 500 RU was
obtained. TABLE-US-00002 Concentration of Complementary DNA (.mu.M)
0.00075 0.0075 0.075 0.75 7.5 75 RU 8 20 80 400 800 880
[0140] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 3
[0141] The same apparatus and method as in Example 1 were used.
[0142] Conditions for plasma polymerization layer formation were
the same as in Example 2.
[0143] Under the conditions described above, a plasma
polymerization layer was formed.
[0144] The sensor chip was mounted on the cartridge block of the
surface plasmon resonance biosensor, 5% glutaraldehyde was poured
through a flow route into the measuring cell at a flow rate of 5
.mu.l/min for 10 minutes and streptoavidin (concentration: 20 82
g/ml) was also poured at a flow rate of 5 .mu.l/min to immobilize
for 60 minutes. 10 .mu.M biotin-labeled probe RNA was then poured
at a flow rate of 1 .mu.l/min for 10 minutes to immobilize the
probe RNA. DNA (7.5.times.10.sup.-7 M) having a DNA sequence
complementary to this probe RNA was introduced and after the
reaction, a signal of about 375 RU was obtained. TABLE-US-00003
Concentration of Complementary DNA (.mu.M) 0.00075 0.0075 0.075
0.75 7.5 75 RU 7.5 18.75 75 375 750 825
[0145] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 4
[0146] The same apparatus and method as in Example 1 were used.
[0147] Conditions for plasma polymerization layer formation were
the same as in Example 2 except that propargylamine was used as a
monomer material.
[0148] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 0.4 M
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide was poured through a
flow route into the measuring cell at a flow rate of 5 .mu.l/min
for 10 minutes and avidin (concentration: 20 .mu.g/ml) was also
poured at a flow rate of 5 .mu.l/min to immobilize for 60 minutes.
10 .mu.M biotin-labeled probe RNA was then poured at a flow rate of
1 .mu.l/min for 10 minutes to immobilize the probe RNA. DNA
(7.5.times.10.sup.-7 M) having a DNA sequence complementary to this
probe RNA was introduced and after the reaction, a signal of about
450 RU was obtained. TABLE-US-00004 Concentration of Complementary
DNA (.mu.M) 0.00075 0.0075 0.075 0.75 7.5 75 RU 0.9 22.5 90 450 900
990
[0149] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 5
[0150] The same apparatus and method as in Example 1 were used.
[0151] Conditions for plasma polymerization layer formation were
the same as in Example 4.
[0152] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and protein A
(concentration: 400 .mu.g/ml) was also poured at a flow rate of 5
.mu.l/min to immobilize for 60 minutes. An anti-HSA antibody
(concentration: 400 .mu.l/ml) was then poured at a flow rate of 1
.mu.l/min for 10 minutes to immobilize the antibody. An HSA antigen
(10 .mu.g/ml) complementary to this anti-HSA antibody was
introduced and after the reaction, a signal of about 250 RU was
obtained. TABLE-US-00005 Concentration of HSA antigen (.mu.g/ml)
0.01 0.1 1 10 100 1000 RU 5 12.5 50 250 500 550
[0153] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 6
[0154] The same apparatus and method as in Example 1 were used.
[0155] Conditions for plasma polymerization layer formation were
the same as in Example 4.
[0156] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and protein G
(concentration: 400 .mu.g/ml) minutes. An anti-BSA antibody
(concentration: 400 .mu.l/ml) was then poured at a flow rate of 1
.mu.l/min for 10 minutes to immobilize the antibody. A BSA antigen
(10 .mu.g/ml) complementary to this anti-BSA antibody was
introduced and after the reaction, a signal of about 225 RU was
obtained. TABLE-US-00006 Concentration of BSA antigen (.mu.g/ml)
0.01 0.1 1 10 100 1000 RU 4.5 11.25 45 225 450 495
[0157] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 7
[0158] The same apparatus and method as in Example 1 were used.
[0159] Conditions for plasma polymerization layer formation were
the same as in Example 4.
[0160] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and
mannose-binding lectin (concentration: 200 .mu.g/ml) was also
poured at a flow rate of 5 .mu.l/min to immobilize for 60
minutes.
[0161] A sugar (10 .mu.g/ml) complementary to this mannose-binding
lectin was introduced and after the reaction, a signal of about 200
RU was obtained.
[0162] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 8
[0163] TABLE-US-00007 Concentration of sugar (.mu.g/ml) 0.01 0.1 1
10 100 1000 RU 4 10 40 200 400 440
[0164] The same apparatus and method as in Example 1 were used.
[0165] Conditions for plasma polymerization layer formation were
the same as in Example 4 except that pyridine was used as a monomer
material.
[0166] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and an anti-BSA
antibody (concentration: 400 .mu.g/ml) was also poured at a flow
rate of 5 .mu.l/min to immobilize for 60 minutes. A BSA antigen (10
.mu.g/ml) complementary to this anti-BSA antibody was introduced
and after the reaction, a signal of about 187.5 RU was obtained.
TABLE-US-00008 Concentration of BSA antigen (.mu.g/ml) 0.01 0.1 1
10 100 1000 RU 3.75 9.375 37.5 187.5 375 412.5
[0167] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 9
[0168] The same apparatus and method as in Example 1 were used.
[0169] Conditions for plasma polymerization layer formation were
the same as in Example 8 except that acrylonitrile was used as a
monomer material.
[0170] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and an anti-BSA
antibody (concentration: 400 .mu.g/ml) was also poured at a flow
rate of 5 .mu.l/min to immobilize for 60 minutes. A BSA antigen (10
.mu.g/ml) complementary to this anti-BSA antibody was introduced
and after the reaction, a signal of about 200 RU was obtained.
TABLE-US-00009 Concentration of BSA antigen (.mu.g/ml) 0.01 0.1 1
10 100 1000 RU 4 10 40 200 400 440
[0171] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 10
[0172] The same apparatus and method as in Example 1 were used.
[0173] Conditions for plasma polymerization layer formation were
the same as in Example 9 except that ethanethiol was used as a
monomer material.
[0174] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor and
maleimidized anti-BSA antibody was poured through a flow route at a
flow rate of 5 .mu.l/min to immobilize for 60 minutes. A BSA
antigen (10 .mu.g/ml) complementary to this anti-BSA antibody was
introduced and after the reaction, a signal of about 200 RU was
obtained. TABLE-US-00010 Concentration of BSA antigen (.mu.g/ml)
0.01 0.1 1 10 100 1000 RU 4 10 40 200 400 440
[0175] It was confirmed by the XPS analysis that the resulting
membrane has a mercapto group.
Example 11
[0176] The same apparatus and method as in Example 1 were used.
[0177] Conditions for plasma polymerization layer formation were
the same as in Example 10 except that thiophene was used as a
monomer material.
[0178] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor and
maleimidized anti-BSA antibody was poured through a flow route at a
flow rate of 5 .mu.l/min to immobilize for 60 minutes. A BSA
antigen (10 .mu.g/ml) complementary to this anti-BSA antibody was
introduced and after the reaction a signal of about 187.5 RU was
obtained. TABLE-US-00011 Concentration of BSA antigen (.mu.g/ml)
0.01 0.1 1 10 100 1000 RU 3.75 9.375 37.5 187.5 375 412.5
[0179] It was confirmed by the XPS analysis that the resulting
membrane has a mercapto group.
Example 12
[0180] The same apparatus and method as in Example 1 were used.
[0181] Conditions for plasma polymerization layer formation were
the same as in Example 11 except that acetonitrile was used as a
monomer material.
[0182] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and an anti-HSA
antibody (concentration: 400 .mu.g/ml) was also poured at a flow
rate of 5 .mu.l/min to immobilize for 60 minutes. HSA antigen (10
.mu.g/ml) complementary to this anti-HSA antibody was introduced
and after the reaction, a signal of about 250 RU was obtained.
TABLE-US-00012 Concentration of HSA antigen (.mu.g/ml) 0.01 0.1 1
10 100 1000 RU 5 10 50 250 500 550
[0183] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 13
[0184] The same apparatus and method as in Example 1 were used.
[0185] Conditions for plasma polymerization layer formation were
the same as in Example 12.
[0186] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and the Fab
fragment of an anti-HSA antibody (concentration: 400 .mu.g/ml) was
also poured at a flow rate of 5 .mu.l/min to immobilize for 60
minutes.
[0187] A HSA antigen (10 .mu.g/ml) complementary to this Fab
fragment of the anti-HSA antibody was introduced and after the
reaction, a signal of about 275 RU was obtained. TABLE-US-00013
Concentration of HSA antigen (.mu.g/ml) 0.01 0.1 1 10 100 1000 RU
5.5 11 55 275 550 605
[0188] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 14
[0189] The same apparatus and method as in Example 1 were used.
[0190] Conditions for plasma polymerization layer formation were
the same as in Example 13.
[0191] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and the
F(ab).sub.2 fragment of an anti-HSA antibody (concentration: 400
.mu.g/ml) was also poured at a flow rate of 5 .mu.l/min to
immobilize for 60 minutes.
[0192] A HSA antigen (10 .mu.g/ml) complementary to this
F(ab).sub.2 fragment of the anti-HSA antibody was introduced and
after the reaction, a signal of about 300 RU was obtained.
TABLE-US-00014 Concentration of HSA antigen (.mu.g/ml) 0.01 0.1 1
10 100 1000 RU 6 12 60 300 600 660
[0193] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 15
[0194] The same apparatus and method as in Example 1 were used.
[0195] Conditions for plasma polymerization layer formation were as
follows:
(1) Monomer: hexadiene Flow volume of monomer material: 1.5 sccm
+Ar dilution 15 (sccm)
[0196] Temperature: room temperature
[0197] Pressure: 1.6 Pa
[0198] Discharge electric power: 80 W
[0199] Discharge frequency: 13.56 MHz
[0200] Duration of discharge: 15 seconds;
(2) Monomer: ethylenediamine
[0201] Flow volume of monomer material: 1.5 sccm
[0202] Temperature: room temperature
[0203] Pressure: 1.6 Pa
[0204] Discharge electric power: 80 W
[0205] Discharge frequency: 13.56 MHz
[0206] Duration of discharge: 5 seconds.
The targeted surface was obtained by the two-step process
above.
[0207] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and an anti-HSA
antibody (concentration: 400 .mu.g/ml) was also poured at a flow
rate of 5 .mu.l/min to immobilize for 60 minutes. A HSA antigen (10
.mu.g/ml) complementary to this anti-HSA antibody was introduced
and after the reaction, a signal of about 250 RU was obtained.
TABLE-US-00015 Concentration of HSA antigen (.mu.g/ml) 0.01 0.1 1
10 100 1000 RU 5 10 50 250 500 550
[0208] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 16
[0209] The same apparatus and method as in Example 1 were used.
[0210] Conditions for plasma polymerization layer formation were as
follows:
(1) Monomer: hexamethyldisiloxane
[0211] Flow volume of monomer material: 1.5 sccm+Ar dilution 15
(sccm)
[0212] Temperature: room temperature
[0213] Pressure: 1.6 Pa
[0214] Discharge electric power: 80 W
[0215] Discharge frequency: 13.56 MHz
[0216] Duration of discharge: 15 seconds;
(2) Monomer: ethylenediamine
[0217] Flow volume of monomer material: 1.5 sccm
[0218] Temperature: room temperature
[0219] Pressure: 1.6 Pa
[0220] Discharge electric power: 80 W
[0221] Discharge frequency: 13.56 MHz
[0222] Duration of discharge: 5 seconds.
The targeted surface was obtained by the two-step process
above.
[0223] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and an anti-HSA
antibody (concentration: 400 .mu.g/ml) was also poured at a flow
rate of 5 .mu.l/min to immobilize for 60 minutes. A HSA antigen (10
.mu.g/ml) complementary to this anti-HSA antibody was introduced
and after the reaction, a signal of about 225RU was obtained.
TABLE-US-00016 Concentration of HSA antigen (.mu.g/ml) 0.01 0.1 1
10 100 1000 RU 4.5 9 45 225 450 495
[0224] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 17
[0225] The same apparatus and method as in Example 1 were used.
[0226] Conditions for plasma polymerization layer formation were
the same as in Example 2 except that propylamine was used as a
monomer material.
[0227] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 5%
glutaraldehyde was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and avidin
(concentration: 20 .mu.g/ml) was also poured at a flow rate of 5
.mu.l/min to immobilize for 60 minutes. 10 .mu.M biotin-labeled
probe RNA was poured at a flow rate of 1 .mu.l/min to immobilize
the probe RNA for 10 minutes. DNA (7.5.times.10.sup.-7 M) having a
DNA sequence complementary to this probe RNA was introduced and
after the reaction, a signal of about 400 RU was obtained.
TABLE-US-00017 Concentration of Complementary DNA (.mu.M) 0.00075
0.0075 0.075 0.75 7.5 75 RU 8 20 80 400 800 880
[0228] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 18
[0229] The same apparatus and method as in Example 1 were used.
[0230] Conditions for plasma polymerization layer formation were as
follows:
(1) Monomer: propargyl alcohol
[0231] Flow volume of monomer material: 1.5 sccm
[0232] Temperature: room temperature
[0233] Pressure: 1.6 Pa
[0234] Discharge electric power: 20 W
[0235] Discharge frequency: 13.56 MHz
[0236] Duration of discharge: 15 seconds;
(2) Monomer: oxygen (plasma treatment)
[0237] Flow volume of monomer material: 1.5 sccm
[0238] Temperature: room temperature
[0239] Pressure: 1.6 Pa
[0240] Discharge electric power: 20 W
[0241] Discharge frequency: 13.56 MHz
[0242] Duration of discharge: 5 seconds.
The targeted surface was obtained by the two-step process
above.
[0243] Under the conditions described above a plasma polymerization
layer was formed. The sensor chip was mounted on the cartridge
block of the surface plasmon resonance biosensor, a 0.5 M
carbodiimide solution was poured through a flow route into the
measuring cell at a flow rate of 5 .mu.l/min for 10 minutes and an
anti-HSA antibody (concentration: 400 .mu.g/ml) was also poured at
a flow rate of 5 .mu.l/min to immobilize for 60 minutes. A HSA
antigen (10 .mu.g/ml) complementary to this anti-HSA antibody was
introduced and after the reaction, a signal of about 250 RU was
obtained. TABLE-US-00018 Concentration of HSA antigen (.mu.g/ml)
0.01 0.1 1 10 100 1000 RU 5 10 50 250 500 550
[0244] It was confirmed by the XPS analysis that the resulting
membrane has a carboxyl group.
Example 19
[0245] The same apparatus and method as in Example 1 were used.
[0246] Conditions for plasma polymerization layer formation were
the same as in Example 2 except that propagylamine was used as a
monomer material.
[0247] Under the conditions described above, a plasma
polymerization layer was formed. The sensor chip was mounted on the
cartridge block of the surface plasmon resonance biosensor, 0.5 M
carbodiimide was poured through a flow route into the measuring
cell at a flow rate of 5 .mu.l/min for 10 minutes and behenic acid
(concentration: 400 .mu.g/ml) was also poured at a flow rate of 5
.mu.l/min to immobilize for 60 minutes. Skatole (10 .mu.g/ml)
complementary to this behenic acid was introduced and after the
reaction, a signal of about 225 RU was obtained. TABLE-US-00019
Concentration of skatole (.mu.g/ml) 0.01 0.1 1 10 100 1000 RU 4.5 9
45 225 450 495
[0248] It was confirmed by the XPS analysis that the resulting
membrane has a primary amine.
Example 20
[0249] Example 20 shows a formation of hydrophobic bond.
[0250] A layer comprising chrome and gold was formed on a
transparent substrate (glass plate) by sputtering. A plasma
polymerization layer in which trifluoroethylene was used as a
monomer was then formed on the resulting metal layer under the
following conditions
[0251] Flow volume: 1.5 sccm
[0252] Temperature: room temperature
[0253] Pressure: 5 Pa
[0254] Discharge electric power: 50 W
[0255] Discharge frequency: 13.56 MHz
[0256] Duration of discharge: 30 seconds.
[0257] The plasma polymerization layer obtained under the
conditions described above was hydrophobic. An anti-HSA antibody
(concentration: 100 .mu.g/ml) was allowed to flow at a flow rate of
5 .mu.l/min for 60 minutes to immobilize the antibody via
hydrophobic bond. HSA at a specified concentration was further
reacted with this antibody-immobilized plasma polymerization layer.
The following results were obtained. TABLE-US-00020 Concentration
of HSA antigen (.mu.g/ml) 0.01 0.1 1 10 100 1000 RU 3 6 30 150 300
330
Example 21
[0258] Example 21 shows an inclusion of an antibody by plasma
polymerization.
[0259] A layer comprising chrome and gold was formed on a
transparent substrate (glass plate) by sputtering. A plasma
polymerization layer in which propargyl alcohol was used as a
monomer was then formed on the resulting metal layer under the
following conditions:
[0260] Flow volume: 1.5 sccm
[0261] Temperature: room temperature
[0262] Pressure: 1.6 Pa
[0263] Discharge electric power: 20 W
[0264] Discharge frequency: 13.56 MHz
[0265] Duration of discharge: 15 seconds.
[0266] The plasma polymerization layer obtained under the
conditions described above was highly hydrophilic. An antibody
solution (concentration: 100 .mu.g/ml) was spread evenly on this
propargyl alcohol plasma polymerization layer and after drying,
plasma treatment was further carried out on this surface under the
following conditions:
[0267] Flow volume: 1.5 sccm
[0268] Temperature: room temperature
[0269] Pressure: 1.6 Pa
[0270] Discharge electric power: 20 W
[0271] Discharge frequency: 13.56 MHz
[0272] Duration of discharge: 8 seconds.
[0273] HSA at a specified concentration was reacted with the
membrane in which the antibody was thus integrated and immobilized
by plasma treatment. The following results were obtained, from
which a calibration curve could be drawn. TABLE-US-00021
Concentration of HSA antigen (.mu.g/ml) 0.01 0.1 1 10 100 1000 RU 5
10 50 250 500 550 *Flow rate for HSA: 5 .mu.l/min.
* * * * *