WO1996024042A1 - Method and apparatus for detecting fluorophore labelled substances - Google Patents
Method and apparatus for detecting fluorophore labelled substances Download PDFInfo
- Publication number
- WO1996024042A1 WO1996024042A1 PCT/SE1996/000109 SE9600109W WO9624042A1 WO 1996024042 A1 WO1996024042 A1 WO 1996024042A1 SE 9600109 W SE9600109 W SE 9600109W WO 9624042 A1 WO9624042 A1 WO 9624042A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fluorophore
- wavelength
- laser
- excitation
- emitting
- Prior art date
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.
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- 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 The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
In a method and an apparatus 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 (1), and detection of light emitted by the fluorophore by means of a photodetector (7), either an Nd:YAG-laser emitting at a wavelength of 473 nm or a frequency-doubled laser diode emitting at a wavelength of 490 nm, is selected for the excitation of the fluorophore.
Description
METHOD AND APPARATUS FOR DETECTING FLUOROPHORE LABELLED SUBSTANCES
TECHNICAL FIELD 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.
BACKGROUND OF THE INVENTION
It is known from US 5,062,942 to label a DNA-fragment with fluorescein isothiocyanate to be able to detect the same after an electrophoretic separation in an electrophoresis gel. An argon laser having a wavelength of 488 or 515 nm is used as an excitation light source.
However, 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.
BRIEF DESCRIPTION OF THE INVENTION 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.
This is attained by the method according to the invention in that 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 selected for the excitation of the fluorophore.
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
490 nm.
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.
BRIEF DESCRIPTION OF THE DRAWING
The invention will be described more in detail below with reference to the appended drawing on which the single figure, Fig. 1, schematically shows an embodiment of an apparatus according to the invention.
DESCRIPTION OF PREFERRED EMBODIMENTS
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.
In a preferred embodiment of the invention in a gel electrophoresis apparatus for separating nucleic acid fragments, these are labelled with fluorescein isothiocyanate.
When the fluorophore labelled substances migrating along the gel 4, pass through the laser beam 2 , the fluorophore is excited to emit light which is detected by means of a photodetector 7.
The excitation of a single fluorophore 8 in a single migration lane is schematically indicated on the drawing, but it is to be understood that in a practical embodiment of the apparatus according to the invention, there are several, mutually parallel migration lanes in the gel 4 as well as a corresponding number of photodetectors 7.
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.
By the 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.
Claims
1. 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 (1) , and detection of light emitted by the fluorophore, characterized in that 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 selected for the excitation of the fluorophore.
2. A method according to claim 1, particularly in a gel electrophoresis apparatus for separating nucleic acid fragments, characterized in that fluorescein isothiocyanate is selected as fluorophore.
3. 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 (1) for excitation of the fluorophore by means of laser light, and a photodetector (7) for detection of light emitted by the fluorophore, characterized in that the laser (1) for the excitation of the fluorophore, is 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.
4. An apparatus according to claim 3, particularly in a gel electrophoresis apparatus for separating nucleic acid fragments, characterized in that the fluorophore is fluorescein isothiocyanate.
5. 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.
6. Use according to claim 5, particularly in a gel electrophoresis apparatus for separating nucleic acid fragments, wherein the fluorophore used is fluorescein isothiocyanate.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP96902044A EP0807247A1 (en) | 1995-02-01 | 1996-01-31 | Method and apparatus for detecting fluorophore labelled substances |
| JP8523474A JPH10513555A (en) | 1995-02-01 | 1996-01-31 | Method and apparatus for detecting a phosphor label |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9500361-2 | 1995-02-01 | ||
| SE9500361A SE9500361D0 (en) | 1995-02-01 | 1995-02-01 | Method and apparatus for detecting fluoroformed substances |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1996024042A1 true WO1996024042A1 (en) | 1996-08-08 |
Family
ID=20397051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/SE1996/000109 WO1996024042A1 (en) | 1995-02-01 | 1996-01-31 | Method and apparatus for detecting fluorophore labelled substances |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0807247A1 (en) |
| JP (1) | JPH10513555A (en) |
| SE (1) | SE9500361D0 (en) |
| WO (1) | WO1996024042A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6023071A (en) * | 1996-06-18 | 2000-02-08 | Fuji Photo Film Co., Ltd. | Image reading apparatus |
| US7439522B2 (en) | 2005-03-04 | 2008-10-21 | Hitachi High-Technologies Corporation | Counting system for fluorescent molecules |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0216600A2 (en) * | 1985-09-18 | 1987-04-01 | The Board Of Trustees Of The Leland Stanford Junior University | Laser excitation fluorescence detection electrokinetic separation |
| EP0476792A1 (en) * | 1990-08-21 | 1992-03-25 | The Board of Regents of the University of Nebraska | DNA sequencing |
-
1995
- 1995-02-01 SE SE9500361A patent/SE9500361D0/en unknown
-
1996
- 1996-01-31 WO PCT/SE1996/000109 patent/WO1996024042A1/en not_active Application Discontinuation
- 1996-01-31 JP JP8523474A patent/JPH10513555A/en active Pending
- 1996-01-31 EP EP96902044A patent/EP0807247A1/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0216600A2 (en) * | 1985-09-18 | 1987-04-01 | The Board Of Trustees Of The Leland Stanford Junior University | Laser excitation fluorescence detection electrokinetic separation |
| EP0476792A1 (en) * | 1990-08-21 | 1992-03-25 | The Board of Regents of the University of Nebraska | DNA sequencing |
Non-Patent Citations (1)
| Title |
|---|
| PATENT ABSTRACTS OF JAPAN, Vol. 17, No. 338, (P-1564), 25 June 1993; & JP,A,05 045 332 (HITACHI SOFTWARE ENG CO LTD), 23 February 1993. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6023071A (en) * | 1996-06-18 | 2000-02-08 | Fuji Photo Film Co., Ltd. | Image reading apparatus |
| US7439522B2 (en) | 2005-03-04 | 2008-10-21 | Hitachi High-Technologies Corporation | Counting system for fluorescent molecules |
Also Published As
| Publication number | Publication date |
|---|---|
| SE9500361D0 (en) | 1995-02-01 |
| JPH10513555A (en) | 1998-12-22 |
| EP0807247A1 (en) | 1997-11-19 |
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