METHOD AND APPARATUS FOR DETECTING FLUOROPHORE LABELLED SUBSTANCES

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: 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.

Inventors: ANDERSSON BENGT-GOERAN, ANDERSSON, BENGT-GOERAN
Applicants: ANDERSSON BENGT GOERAN, PHARMACIA BIOTECH AB, ANDERSSON, BENGT-GOERAN
Application year: 1996
Main classification: G01N21
IPC codes: G01N21, G01N27, G01N33