EP0807247A1 - Verfahren und vorrichtung zum nachweis von mit einem fluorophor markierten substanzen - Google Patents
Verfahren und vorrichtung zum nachweis von mit einem fluorophor markierten substanzenInfo
- Publication number
- EP0807247A1 EP0807247A1 EP96902044A EP96902044A EP0807247A1 EP 0807247 A1 EP0807247 A1 EP 0807247A1 EP 96902044 A EP96902044 A EP 96902044A EP 96902044 A EP96902044 A EP 96902044A EP 0807247 A1 EP0807247 A1 EP 0807247A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- fluorophore
- wavelength
- laser
- excitation
- emitting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 239000000126 substance Substances 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims abstract description 7
- 230000005284 excitation Effects 0.000 claims abstract description 16
- 230000031700 light absorption Effects 0.000 claims abstract description 8
- 230000023077 detection of light stimulus Effects 0.000 claims abstract description 5
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 7
- 150000007523 nucleic acids Chemical group 0.000 claims description 6
- 238000001502 gel electrophoresis Methods 0.000 claims description 5
- 238000002372 labelling Methods 0.000 claims description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 8
- 229910052786 argon Inorganic materials 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 3
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- -1 BODIPY-FL Chemical compound 0.000 description 1
- MZZINWWGSYUHGU-UHFFFAOYSA-J ToTo-1 Chemical compound [I-].[I-].[I-].[I-].C12=CC=CC=C2C(C=C2N(C3=CC=CC=C3S2)C)=CC=[N+]1CCC[N+](C)(C)CCC[N+](C)(C)CCC[N+](C1=CC=CC=C11)=CC=C1C=C1N(C)C2=CC=CC=C2S1 MZZINWWGSYUHGU-UHFFFAOYSA-J 0.000 description 1
- GRRMZXFOOGQMFA-UHFFFAOYSA-J YoYo-1 Chemical compound [I-].[I-].[I-].[I-].C12=CC=CC=C2C(C=C2N(C3=CC=CC=C3O2)C)=CC=[N+]1CCC[N+](C)(C)CCC[N+](C)(C)CCC[N+](C1=CC=CC=C11)=CC=C1C=C1N(C)C2=CC=CC=C2O1 GRRMZXFOOGQMFA-UHFFFAOYSA-J 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Definitions
- the present invention relates to a method for detecting substances which are labelled with a fluorophore having a light absorption maximum at a wavelength between 480 and 520 nm by excitation of the fluorophore by means of light from a laser, and detection of light emitted by the fluorophore; an apparatus for detecting substances which are labelled with a fluorophore having a light absorption maximum at a wavelength between 480 and 520 nm, comprising a laser for excitation of the fluorophore by means of laser light, and a photodetector for detection of light emitted by the flurophore; and the use of a specific type of flurophores for labelling substances which are to be detected, together with a specific type of lasers for excitation of the fluorophores.
- argon lasers are bulky, expensive and power demanding and, therefore, are not suitable to be built into commercial electrophoresis apparatuses.
- Argon lasers have another disadvantage in that their output power is not stable but fluctuates. Moreover, argon lasers can not be pulsed which can be desirable in certain applications, e.g. time resolved fluorescence measurement.
- the object of the invention is therefore to define a laser for excitation of a fluorophore of the type mentioned in the introduction, which laser does not have the disadvantages mentioned above and which therefore is suitable to be built into commercial electrophoresis apparatuses.
- the object according to the invention is also attained by means of the apparatus according to the invention in that the laser for the excitation of the fluorophore, is either a Nd.YAG-laser emitting a a wavelength of 473 nm or a frequency-doubled laser diode emitting at a wavelength of
- the use according to the invention consists of the use of a fluorophore having a light absorption maximum at a wavelength between 480 and 520 nm for labelling substances to be detected, and either a Nd.YAG-laser emitting at a wavelength of 473 nm or a frequency-doubled laser diode emitting at a wavelength of 490 nm for excitation of the fluorophore.
- FIG. 1 schematically shows an embodiment of an apparatus according to the invention.
- 1 generally denotes a laser which emits a laser beam 2, which via a mirror 3 is directed into a gel 4 through one of its lateral edges, which gel 4 is provided between two glass plates 5 and 6.
- the gel 4 is included in an electrophoretic separation system, not shown in any greater detail, for separating substances which, according to the invention, are labelled with a fluorophore which has a light absorption maximum at a wavelength between 480 and 520 nm.
- Fluorescein isothiocyanate (FITC) , BODIPY-FL, YOYO-1 and TOTO-1, all available from the US company Molecular Probes, Inc., Oregon, U.S.A., are examples of such fluorophores.
- Either a Nd.YAG-laser emitting at a wavelength of 473 nm or a frequency-doubled laser diode emitting at a wavelength of 490 nm is preferably used as laser 1 according to the invention, both of which being well suited, due to their size, to be built into commercial gel electrophoresis apparatuses, particularly for separating nucleic acid fragments. These lasers emit blue light with small fluctuations and can, moreover, be pulsed.
- Another advantage of using lasers emitting blue light in comparison to lasers emitting green, yellow or red light, is that a better geometric resolution is obtained in the applications in question in that a blue laser beam is narrower than a red as well as a yellow and a green laser beam.
- fluorophore-laser combination defined in accordance with the invention, a combination is obtained which, what concerns size and efficiency, is extremely well suited for use in commercial electrophoresis apparatuses, particularly for the separation of nucleic acid fragments.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Electrochemistry (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9500361A SE9500361D0 (sv) | 1995-02-01 | 1995-02-01 | Förfarande och anordning för detektering av fluoroformärkta substanser |
| SE9500361 | 1995-04-04 | ||
| PCT/SE1996/000109 WO1996024042A1 (en) | 1995-02-01 | 1996-01-31 | Method and apparatus for detecting fluorophore labelled substances |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0807247A1 true EP0807247A1 (de) | 1997-11-19 |
Family
ID=20397051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP96902044A Withdrawn EP0807247A1 (de) | 1995-02-01 | 1996-01-31 | Verfahren und vorrichtung zum nachweis von mit einem fluorophor markierten substanzen |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0807247A1 (de) |
| JP (1) | JPH10513555A (de) |
| SE (1) | SE9500361D0 (de) |
| WO (1) | WO1996024042A1 (de) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0814594B1 (de) * | 1996-06-18 | 2004-09-08 | Fuji Photo Film Co., Ltd. | Bildlesegerät |
| JP4812393B2 (ja) | 2005-03-04 | 2011-11-09 | 株式会社日立ハイテクノロジーズ | 蛍光分子計測システム |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5207880A (en) * | 1984-03-29 | 1993-05-04 | The Board Of Regents Of The University Of Nebraska | DNA sequencing |
| US4675300A (en) * | 1985-09-18 | 1987-06-23 | The Board Of Trustees Of The Leland Stanford Junior University | Laser-excitation fluorescence detection electrokinetic separation |
-
1995
- 1995-02-01 SE SE9500361A patent/SE9500361D0/xx unknown
-
1996
- 1996-01-31 JP JP8523474A patent/JPH10513555A/ja active Pending
- 1996-01-31 WO PCT/SE1996/000109 patent/WO1996024042A1/en not_active Ceased
- 1996-01-31 EP EP96902044A patent/EP0807247A1/de not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| See references of WO9624042A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1996024042A1 (en) | 1996-08-08 |
| SE9500361D0 (sv) | 1995-02-01 |
| JPH10513555A (ja) | 1998-12-22 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 19970718 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): DE FR GB |
|
| RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: APBIOTECH AKTIEBOLAG |
|
| RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: AMERSHAM BIOSCIENCES AB |
|
| 17Q | First examination report despatched |
Effective date: 20030603 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 20031014 |