METHOD AND APPARATUS FOR DETECTING 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 530 and 570 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), a frequency-doubled Nd:YAG laser emitting at a wavelength of 532 nm is selected for the excitation of the fluorophore.

Description: METHOD AND APPARATUS FOR DETECTING 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 530 and 570 nm by excitation of the fluorophore by means of light from a laser, and detection of fluorescence emitted by the fluorophore; an apparatus for detecting substances which are labelled with a fluorophore having a light absorption maximum at a wavelength between 530 and 570 nm, comprising a laser for excitation of the fluorophore by means of laser light, and a photodetector for detection of fluorescence emitted by the fluorophore; and the use of a specific type of fluorophores for labelling substances to be detected, together with a specific laser for excitation of the fluorophore. BACKGROUND OF THE INVENTION As a fluorophore for labelling substances to be detected, it is known to use a dye marketed under the trademark Cy3 and manufactured by Biological Detection Systems, Inc., Pittsburgh, PA, U.S.A.. The dye Cy3 is a carbocyanine based dye including a reactive succinimidyl ester and having a light absorption maximum at a wavelength of 552 nm and a light emission maximum at a wavelength of 565 nm. To excite the dye Cy3, it is known to use argon lasers and He-Ne lasers. Argon lasers are, however, bulky, expensive and power demanding and, therefore, are not suitable to be built into commercial electrophoresis apparatuses. He-Ne lasers are certainly smaller than argon lasers but their output power is low, normally 0.1-5 m , which causes the excitation efficiency to become low. Another disadvantage of He-Ne lasers and of argon lasers is that their output power is not stable but fluctuates. Moreover, neither He-Ne lasers nor argon lasers can 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 fluorphore of the type mentioned in the introduction, which laser does not exhibit the above disadvantages but is suitable to be built into commercial electrophoresis apparatuses. This is attained by the method according to the invention in that a frequency-doubled Nd.YAG laser emitting at a wavelength of 532 nm, is selected for the excitation of the fluorophore. The object according to the invention is also attained by the apparatus according to the invention in that the laser for the excitation of the fluorophore is a frequency- doubled Nd.YAG laser emitting at a wavelength of 532 nm. The use according to the invention consists of the use of a fluorophore having a light absorption maximum at a wavelength between 530 and 570 nm for labelling substances to be detected, and a frequency-doubled Nd.YAG laser emitting at a wavelength of 532 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 detail, for separating substances which, according to the invention, are labelled with a flurophore, which has a light absorption maximum at a wavelength between 530 and 570 nm. The fluorophore used in a preferred embodiment of the invention, is the dye marketed under the trademark Cy3, mentioned in the introduction, and manufactured by Biological Detection Systems, Inc., Pittsburgh, PA, U.S.A.. The dye Cy3 is a carbocyanine based dye containing a reactive succinimidyl ester and having a light absorption maximum at a wavelength of 552 nm and a light emission maximum at a wavelength of 565 nm. Tetramethylrhodamine having a light absorption maximum at a wavelength of 555 nm and a light emission maximum at a wavelength of 580 nm as well as lissamine rhodamine B having a light absorption maximum at a wavelength of 570 nm and a light emission maximum at a wavelength of 590 nm, are two other examples of fluorophores that can be used. Both these fluorophores are available from the US company Molecular Probes, Inc., Oregon, U.S.A.. When the fluorophore labelled substances migrating along the gel 4 pass through the laser beam 2, the fluorophore is excited to emit fluorescence which is detected by means of a photodetector 7. On the drawing, the excitation of a single fluorophore 8 in a single migration lane is schematically indicated, 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. According to the invention, as laser 1 for excitation of the fluorophore, a commercially available, frequency- doubled Nd:YAG laser emitting at a wavelength of 532 nm, is used, which laser, due to its small size, is well suited to be built into commercial gel electrophoresis apparatuses, particularly for separation of nucleic acid fragments. This laser which can have an output power of 5-100 mW, emits green light with small fluctuations and can furthermore be pulsed. Another advantage of, in accordance with the invention, using a laser which emits green light in comparison to lasers which emit yellow or red light, is that a better geometric resolution is obtained in the applications in question since a green laser beam is narrower than both a red and a yellow laser beam. By the fluorophore-laser combination according to the invention, a combination is obtained which, what concerns size and efficiency, is well suited to be used in commercial electrophoresis apparatuses, particularly for 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 530 and 570 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 a frequency-doubled Nd.YAG laser, emitting at a wavelength of 532 nm, is selected for the excitation of the fluorophore. 2. A method according to claim 2, particularly in a gel electrophoresis apparatus for separating nucleic acid fragments, characterized in that a carbocyanine based dye, marketed under the trademark Cy3, 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 530 and 570 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 a frequency-doubled Nd.YAG laser emitting at a wavelength of 532 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 a carbocyanine based dye marketed under the trademark Cy3. 5. Use of a fluorophore having a light absorption maximum at a wavelength between 530 and 570 nm for labelling substances to be detected, and a frequency-doubled Nd.YAG laser (1) emitting at a wavelength of 532 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 a carbocyanine based dye marketed under the trademark Cy3.

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