WO2005050187A1

WO2005050187A1 - Methods and devices for measuring fluorescence of a sample in a capillary

Info

Publication number: WO2005050187A1
Application number: PCT/EP2004/012300
Authority: WO
Inventors: Lajos NYÁRSIK; Wilfried Nietfeld; Hans Lehrach; Tarso Benigno Ledur Kist
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2003-11-05
Filing date: 2004-10-29
Publication date: 2005-06-02
Also published as: EP1682883A1; BRPI0416120A

Abstract

A method of measuring fluorescence of at least one sample (1) in at least one sample chamber (10, 11) extending along a longitudinal reference direction (x), comprises the steps of positioning the sample (1) in the sample chamber (10, 11), irradiating the sample (1) with excitation light from an excitation light source (20) along an excitation light path, and detecting fluorescence light from the sample (1) with a detector device (30) along a detection light path, wherein one of the excitation and detection light paths extends through the sample chamber (10, 11) parallel to the longitudinal reference direction (x) and the respective other one of the detection and excitation light paths extends in a direction (y) deviating from the longitudinal reference direction (x). Furthermore, a device for measuring fluorescence according to this method is described.

Description

Methods and devices for measuring fluorescence of a sample in a capillary

The present invention relates to methods for measuring fluorescence of at least one sample in at least one sample chamber, in particular to methods for measuring fluorescence of samples in capillaries of a substance separation device like e.g. an isoelectric focusing device. Furthermore, the present inventions relates to fluorescence measuring devices and substance separation devices like e.g. isoelectric focusing devices, particularly being adapted for conducting the above methods .

Isoelectric focusing is used for separating molecules (e.g. proteins) with a net charge depending on the surrounding pH value. The basic of isoelectric focusing devices is the formation of pH gradients along a gel lane or a capillary filled with gel or polymer solution. Methods to generate this pH gradient are generally known. Typically, "Carrier Ampholites" made of bifunctional amphoteric (acid and basic ends) buffers molecules are used which separate out to form a smooth pH gradient in applied electric fields within a matrix (e.g. polyacrilamide, agarose, dextran) . A molecule of a sample to be investigated will migrate within the pH gradient toward the anode or cathode until it arrives at a point in pH gradient equal to it's pi value. At this point the molecule has a zero net charge and is said to focus at the isoelectric point. The pH gradient is generated automatically simple by applying the electrophoresis electric field (see D. F. GarfrLn et al. "Isoelectric Focusing Methods" in "Enzymol." vol. 182, 1990, p. 459-477) . A second commonly known method is the use of the so called "acrylamido buffers for immobilized pH gradients". In this components of the buffer react and are covalently attached to acrylamide derivatives to create immobilized pH gradients (see Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications", Bjellquist B., Ek K. , Righetti P. G., Gianazza E., Georg A., estermeier R. , Postel W.. in "J. Biochem. Biophys . Methods", 1982, 6(4), p. 317-339) . Further methods to generate pH gradients are de- scribed in US 5 047 134, US 5 066 382 and US 5 784 154.

In an isoelectric focusing device, different molecules will be focused at different points along the longitudinal extension of the capillary. The location, quantity and types of separated molecules can be investigated by a fluorescence measurement. The principle of a conventional fluorescence measurement is illustrated in figure 6. The sample 1' in the sample chamber (capillary) 10" is irradiated with excitation light from an excitation light source (laser) 20'. The fluo- rescence emitted from sample 1" is detected with a detector device 30'. This arrangement of the excitation and detector devices has a series of drawbacks. Firstly, both the excitation and fluorescence light pass through the curved capillary walls causing essential reflection losses reducing the sensi- tivity and reproducibility of the measurement. Due to light refraction in the capillary walls, the focussing points of the sample can be detected with restricted precision only. Furthermore, the conventional fluorescence measurement is adapted for detecting fluorescence in a single capillary only. Samples in multi-channel systems can be analyzed with more complex devices or complex procedures only.

The problems illustrated above do not occur with isoelectric focusing devices only but rather with all applications of fluorescence measurements in capillaries or other sample chambers extending in a longitudinal direction.

The object of the invention is to provide improved methods of measuring fluorescence in sample chambers having an extended applicability and allowing position selective measurements with increased precision. Another object of the invention is to provide improved devices for conducting the fluorescence measurements in sample chambers and in particular improved isoelectric focussing devices.

The above objects are solved with methods or devices comprising the features of patent claims 1, 19 and 35. Advantageous embodiments and applications of the invention are defined in the dependent claims .

According to a first general aspect of the invention, a method of measuring fluorescence of at least one sample in at least one sample chamber extending along a longitudinal ref- erence direction comprises the steps of irradiating the sample along an excitation light path and detecting fluorescence from the sample along a detection light path, wherein one of the excitation and detection light paths is directed through the sample chamber parallel to the reference direction and the respective other one of the detection and excitation light paths is directed in a direction deviating from the reference direction. One of the advantages of the present invention is given by extending one of the detection and excitation light paths parallel to the longitudinal extension of the sample chamber. This setup allows an essential reflection loss reduction and an improved measurement geometry. In particular, the method of the invention allows simultaneous measurements in a plurality of channels (chambers) providing a high parallelism of measurements, and/or measurements with a plurality of samples in one chamber providing a high ulti- plexation.

According to a preferred embodiment of the invention, the ex- citation light path extends about perpendicularly relative to the detection light path. This may have advantages with regard to a further reflection loss and scattering light reduction. Further advantages with regard to an extended information contents and to the ultiplexation may arise, when a spectrally resolved detection of the fluorescence is provided.

According to a further preferred embodiment of the invention, the detection is associated with a determination of at least one location (position and/or size) of the at least one fluo- rescing sample to be investigated in the sample chamber. This may have advantages with regard to applications of the invention in separation devices for substance analysis.

When according to a further preferred implementation of the invention a plurality of samples are positioned in a plurality of parallel sample chambers, advantages in view of a high-throughput measurement are obtained. In particular, an isoelectric focusing device with axial excitation or detec- tion allows the use of multicapillaries and multiplexation.

The use of many capillaries gives high throughput to this device, as needed by nowadays demands with proteomics and present similar investigation projects in the field of biochemistry and genetic technology. In addition to this the use of multiplexation increases even more the high throughput capability. Moreover, this multiplexation brings the following remarkable advantages over traditional set-ups: i) first, many samples can be analyzed simultaneously in a single channel using internal standards running altogether. So, all sam- pies can be precisely compared to a reference or standard, ii) second, very small differences between samples are more easily perceived. In other words, samples can be compared more precisely as all small fluctuations of temperature, pH_; ionic strength, electric field, etc., that normally occur along the separation channels, will affect all samples and standard in the same way. Therefore, the small differences among proteoms, peptidoms, metabolo e, or the like will be more easily seen and detected by using multiplexation.

If the irradiating and detecting steps are conducted for each of the sample chambers separately, advantages with regard to precision and reproducibility may arise. If the irradiating and detecting steps are conducted for all sample chambers si- multaneously, the measurement speed can be essentially increased.

According to a further advantageous embodiment of the invention, the samples to be investigated are labelled with dif- ferent fluorophores or nanoparticles allowing an improved multiplexation of the measurement.

Preferably, the method of the invention is used for the operation of an isoelectric focusing device with at least one straight sample chamber with axial laser light excitation or detection. Therefore, according to a preferred application of the invention, the sample chamber is a part of an electropho- retic separation device and a sample positioning step is provided with a migration of the at least one sample through the sample chamber under the influence of an electric field. For isoelectric focussing, a pH gradient may be formed in the sample chamber with conventional techniques . According to a particularly preferred embodiment of the invention, the sample chamber comprises a capillary and the excitation light path extends axially along a longitudinal axis of the capillary, while the detection light path extends through a transparent wall portion of the capillary. This embodiment may have particular advantages with regard to a uniform and reproducible sample illumination. Preferably, the excitation light is focussed directly to an end of the capillary allowing a direct in-coupling of excitation light into the sample, sample carrier or matrix material.

If a multi-channel detector extending along the capillary is used as the detector device, a position-selective signal of the multi-channel detector may be used directly for a paral- lei determination of the location, size and/or distribution of one ore more samples. Alternatively, the detector device (in particular a single-channel detector) can be moved along the capillary for sequentially obtaining the above geometry- related information. This variant may have advantages for special measurement purposes (e.g. high resolution measurements at certain locations) .

According to an alternative embodiment of the invention, the sample chamber comprises a capillary and the detection light path extends along a longitudinal axis of the capillary, while the excitation light path extends through a transparent wall (or transparent wall portion) of the capillary. In this case, a detector (e.g. CCD, PMT, or APD) can be placed at the end of the capillary and a laser beam is focused preferably orthogonal to the capillary and put to scan the whole capillary along its whole extension. In this case part of the fluorescent light will propagate by internal total reflection to the end where the detectors are placed to detect the fluorescent light. According to a preferred modification of this embodiment, the excitation light source can be moved along the capillary for allowing an evaluation the above geometry-related information from a position of the excitation light source.

If the at least one sample is irradiated with at least two different wavelengths, different fluorophores can be excited.

Accordingly different fluorescence properties can be meas- ured. In this case, advantages with regard to multiplexation of the measurement may arise.

According to a further embodiment of the invention, the at least one sample chamber is placed in contact with a heat dissipating material. E.g. the capillaries of an isoelectric focusing device are placed between dielectric plates, i.e., sandwiched between dielectric solid materials, in order to have a more efficient heat dissipation, which in turn allows more elevated voltages to be applied and correspondingly im- proved separation results.

According to a second general aspect of the invention, a device for measuring fluorescence of at least one sample is provided, comprising at least one sample chamber for accommo- dating the at least one sample and extending along a longitudinal reference direction, wherein an excitation light source for irradiating the sample along an excitation light path and a detector device for detecting fluorescence along a detection light path are arranged such that one of the excitation and detection light paths extends through the sample chamber parallel to the reference direction and the respective other one of the detection and excitation light paths extends in another direction. According to a particularly preferred embodiment of the invention, the device for measuring fluorescence comprises a plurality of sample chambers each being adapted for accommodating at least one sample. Preferably, all sample chambers extend in parallel along the same reference direction, so that the adaptation of the excitation light source and the detector to the chamber arrangement can be facilitated.

For a preferred application of the invention, each sample chamber comprises one capillary for accommodating a sample carrier or matrix material containing or carrying the sample to be investigated. Preferably, the capillary has at least one transparent wall portion.

According to a further preferred embodiment of the invention, the excitation light source comprises at least one laser. This may have advantages with regard to intensity and wavelength control depending on the particular sample to be investigated. If the excitation light from each laser is sub- mitted to the sample chamber via at least one light guiding fibre, further advantages with regard to the use of a single laser for a plurality of capillaries and a flexible adaptation of the excitation light source to the geometry of the measuring device are obtained.

If the detector device comprises a multi-channel detector extending along the capillary, parallel provision of position selective information can be facilitated. If the detector device is movable along the capillary, different portions of the capillary can be investigated with different resolution.

According to a third general aspect of the invention, a separation device, in particular for isoelectric focusing of sam- pies is provided comprising a measuring device having in particular the features characterized above.

An essential advantage of the invention is given by the pro- vision of isoelectric focusing devices which are capable to use simultaneously multichannels and/or multiplexation. Moreover, they are able to work with multiplexation in a capillary. The use of multichannels (multi-capillaries) and multiplexation (many samples per capillary) brings advantages in terms of high-throughput measurements and new applications.

According to a preferred embodiment of the invention, the separation device comprises a first sample chamber holding block for carrying a first end of the at least one sample chamber. Advantageously, the first sample chamber holding block is further adapted for accommodating samples to be introduced into the respective sample chamber.

Preferably, the second end of each sample chamber is con- nected with a second sample chamber holding block which is capable of collecting samples from the at least one sample chamber. If the first or second sample chamber holding block being made of a transparent material, the transmission of excitation or fluorescence light can be facilitated.

Further details and advantages of the invention are described in the following with reference to the attached drawings, which show: Figures 1 to 3 schematic representations of various embodiments of the invention; Figure 4 a perspective view of a multichannel isoelectric focusing device according to the invention;

Figure 5 a schematic representation of an embodiment adapted for spectrally resolved fluorescence measurements; and

Figure 6 an illustration of a conventional fluorescence measurement set-up.

The invention is described in the following with reference to an isoelectric focusing device with electrophoretic molecule separation in capillaries. It is emphasized that the invention can be implemented in an analogous way with modified isoelectric focusing devices or other applications of fluorescence measurements. As an example, capillaries with other sizes or shapes can be used, or straight open channels or gel lanes can be used instead of the capillaries. Although straight sample chambers are preferred, the invention can be implemented with bend or curved chamber shapes.

Figure 1 shows an embodiment of a fluorescence measurement device 100 according to the present invention, comprising a sample chamber 10, an excitation light source 20, a detector device 30, a first holding block 40, and a voltage source 60.

The sample chamber 10 is a hollow compartment or channel hav- ing a main extension parallel to the x-direction (longitudinal reference direction) . In the example, the sample chamber 10 is a capillary 11, made of a plastic or quartz, e.g. with the following dimensions: length: 10 mm, outer diameter: 200 μm, inner diameter: 75 μm. The fluorescence measurement de- vice 100 comprises at least one capillary. Preferably, the number of capillaries is adapted to a typical format used for parallel sample processing, e.g. in micro- or nanotiter plates, like 8, 16 or higher multiples. In this case, the in- troduction of samples into the capillaries e.g. with conventional multichannel-pipettes is facilitated.

A first end 12 of the capillary 11 is fixed to the first holding block 40, while the second end 13 is connected with a second holding block (50, see figure 4). Both ends have an open connection with sample reservoirs in the blocks (see below) .

Capillary 11 is filled with a gel or solution 2 serving as a carrier material for a sample 1 migrating through the capillary under the influence of a driving voltage. The carrier material 2 is introduced into the capillary in conventional manner with a pumping device (not shown) .

The excitation light source 20 comprises a laser, like e.g. a Ar ion laser or a semiconductor laser (preferred due to small size) being adapted for fluorescence excitation and emitting e.g. in the blue or green wavelength range (e.g. 488 nm, power: 50-150 mW) . Laser 20 may be equipped with a light guiding fibre (not shown) .

Excitation light from laser 20 is focused axially and parallel to the x-direction to the second end 13 of the capillary 11. Excitation light travels along an excitation path through the capillary. If the device 100 comprises a plurality of parallel capillaries, a corresponding number of light guiding fibers can be fed from one laser to the ends of the capillaries. The focus at the end 13 of each capillary can be im- proved by a focusing optic (focusing lens, reference numeral

21, see figure 4) .

The detector device 30 comprises generally a light sensitive fluorescence light detector like known in the art, e.g. a photodiode, an APD or a photomultiplier . Figure 1 schematically shows a CCD line detector 30 extending adjacent to the outer side of the capillary 11. The CCD line detector 30 comprises a plurality of detector elements (pixel) collecting light from fluorescing molecules within the capillary along a detection path. The signals of the detector elements represent the position of a fluorescing molecule in x-direction. The CCD line detector 30 is e.g. the spectroscopic camera DV401 with 1024 x 128 pixels from Andor Technology . (distribu- tion in Germany LOT Oriel Gruppe Europe) .

The first holding block 40 is adapted for accommodating samples to be introduced into capillary as described below with reference to figure 4. Block 40 is preferably made of an electrically conducting material, e.g. platin covered inox or plastic with platin electrodes.

The voltage source 60 is a high voltage power source as known in the art of electrophoretic separation techniques, which is connected via an electrically conducting connection 61 with the carrier material 2 at the end 13 of the capillary 11.

Figure 2 shows an alternative embodiment of the measuring device 100 being similar like the embodiment of figure 1. The only difference concerns the detector device 30 which is a movable single- or multi-channel detector. The x-position of the detector can be changed with a driving device (not shown) . Control signals of the driving device or the position of the detector 30 represent the position of a fluorescing molecule in x-direction. As a single-channel detector, a PMT or APD can be used. As a multi-channel detector, a CCD camera can be used.

With the embodiment of the measuring device 100 shown in figure 3, the excitation and detection paths are reversed compared with figures 1 and 2. The excitation source, e.g. laser 20 is arranged for illuminating the samples within the capil- lary according to the y-direction. Laser 20 is movable in x- direction with a driving device (not shown) . Control signals of the driving device or the position of laser 20 represent the position of a fluorescing molecule in x-direction. On the other hand, the detector device 30 is arranged at the end 13 of the capillary 11.

Figure 4 illustrates a multi-channel arrangement with a plurality of capillaries 11 between the first and second holding blocks 40, 50. The first holding block 40 contains conic holes 41 for accommodating samples to be introduced into the respective capillaries. The first holding block 40 is made by a metal and works as the ground electrode or, alternatively, it may be made by any dielectric material and in this case electrodes must be inserted in order to keep this end at the right electrostatic potential. The second holding block 50 is made of transparent material like plastic, e.g. PMMA, or quartz. It contains a central channel 51 for collecting carrier material from the capillaries. The excitation light source 20 is movable along the second holding block 50, i. e. parallel to a z-reference direction. The laser 20 scans all capillaries, once at a time. The illumination is done in the axial x-direction, allowing a lot of light to be used to excite all bands at once. The detector 30 is a movable CCD- camera as shown in figure 2. Alternatively, the fluorescent light might be detected by a PMT or PDA.

The measuring device may comprise a heat dissipating compo- nent 70 (schematically shown) placed in contact with the at least one or all capillaries 11. The heat dissipating component 70 can be formed as a monolithic plastics block into which all capillaries are molded.

Reference numeral 80 indicates a control device being adapted for controlling the function of the components of the measuring device 100. The control device 80 contains a circuit for determining at least one location, size or distribution of the at least one sample along the longitudinal extension of the sample chamber (location determining device) .

In operation of the device 100, samples are introduced into the conic holes 41 in the first sample holding block 40. Using a multi-channel pipette or any automated system all sam- pies and/buffer can be manipulated at once. In the next step a high voltage is applied to the carrier material (buffer) that fills the hole of the block that holds the opposite end of the capillaries or channels 11. The application of this high voltage will make the molecules to migrate into the channels or capillaries 11. An alternative way to introduce the samples is simple applying a vacuum to the hole on the exit end 13 of the capillaries. Afterwards, the sample left over in the conic holder is removed and one of the IEF running buffers is placed in. Then the high voltage is applied again until the end of the run, which takes about 10 to 30 min. Then the detection systems are activated in order to get the spatial profile of the sample bands (e.g. reference numeral 1 in figures 1 and 2) . The detection starts with the movement of the laser as shown in figure 4. The laser light propagates along the capillary by internal total reflection causing the excitation of the labeled or unlabeled bands that are focused around their isoelectric points. A CDD camera 30 takes a picture of the whole capillary or only pieces of them. These photos are then later mounted to get a panoramic photo.

For multiplexed experiments a transmission diffraction grating 31 is positioned in the detection light path, as shown in figure 5. Between the diffraction grating 31, optical lenses 32 and a mirror 33 are arranged before the CCD camera 30. An alternative to the diffraction grating is an optical filter transmitting fluorescence light only.

For the purpose of multiplexation, different fluorophores (e.g. FITC, NDA, succini idyl ester, nanodots) are used to label different samples, which in turn allows many samples to be analyzed simultaneously in a single channel or capillary. Alternatively, samples are labeled with different nanodots, that allow the simultaneous analysis of multiple samples in a single channel (multiplexation with quantum nanodots) . Nanodots are described e.g. in "High quantum yield blue emission form water-soluble Au nanodots" Zheng J. , Petty J. T., Dickson R. M. in "J. Am. Chem. Soc." 2003, 125(26), p. 7780- 7781.

An alternative to CCD camera detection is the use of a pho- tomultiplier tube (PMT) or an avalanche photodiode (APD) . In this case the whole capillary must be scanned, as most of these devices don't have spatial resolution. In this case the spectrum is also scanned at each position in order to carry out multiplexed experiments. There are various approaches to do that which include the use of a rotating disk with many filters between the capillary and the microscope objective or between the microscope objective and the detector (PMT or

APD) . Also a moving transmission diffraction grating can be used. This oscillatory movement would be synchronized with the rate of data acquisition, so that spatial position and spectral interval of the detected photons are precisely known.

Claims

1. Method of measuring fluorescence of at least one sample

(1) in at least one sample chamber (10, 11) extending along a longitudinal reference direction (x) , comprising the steps of:

- positioning the sample (1) in the sample chamber (10, 11), - irradiating the sample (1) with excitation light from an excitation light source (20) along an excitation light path, and

- detecting fluorescence light from the sample (1) with^" a detector device (30) along a detection light path, characterized in that one of the excitation and detection light paths extends through the sample chamber (10, 11) parallel to the longitudinal reference direction (x) and the respective other one of the detection and excitation light paths extends in a direc- tion (y) deviating from the longitudinal reference direction (x) •

2. Method according to claim 1, wherein the excitation light path extends about perpendicularly relative to the detection light path.

3. Method according to claim 1 or 2, wherein the detecting step comprises a spectrally resolved detection of the fluorescence light.

4. Method according to at least one of the claims 1 to 3, wherein the detecting step comprises a step of determining at least one position, size or distribution of the at least one sample along the longitudinal extension of the sample chamber.

5. Method according to at least one of the claims 1 to 4, wherein a plurality of samples are positioned in a plurality of sample chambers (11) .

6. Method according to claim 5, wherein the irradiating and detecting steps are conducted for each of the sample chambers separately or for all sample chambers simultaneously.

7. Method according to claim 5 or 6, wherein the samples are labelled with different fluorophores or nanoparticles.

8. Method according to at least one of the claims 1 to 7 , wherein the sample chamber (10, 11) is a part of an electro- phoretic separation device (100) and the sample positioning step comprises migrating of the at least one sample through the sample chamber under the influence of an electric field.

9. Method according to claim 8, further comprising the step of forming a pH gradient in the sample chamber (10, 11).

10. Method to according at least one of the claims 1 to 9, wherein the sample chamber (10) comprises a capillary (11), the excitation light path extending along a longitudinal axis of the capillary (11) and the detection light path extending through a transparent wall of the capillary (11) .

11. Method according to claim 10, wherein the excitation light is focussed to an end (13) of the capillary (11) .

12. Method according to claim 10 or 11, wherein a multichannel detector (30) extending along the capillary is used as the detector device and the location determining step comprises an evaluation of a signal of the multi-channel detector.

13. Method according to claim 10 or 11, wherein the detector device (30) is moved along the capillary (11) and the location determining step comprises an evaluation of at least one position of the detector device (30) during the detecting step.

14. Method according at least one of the claims 1 to 9, wherein the sample chamber (10) comprises a capillary (11) , the detection light path extending along a longitudinal axis of the capillary (11) and the excitation light path extending through a transparent wall of the capillary (11) .

15. Method according to claim 14, wherein the detector device is arranged at an end of the capillary (11) .

16. Method according to claim 14, wherein the excitation light source (20) is moved along the capillary (11) and the location determining step comprises an evaluation of a position of the excitation light source (20) during the detecting step.

17. Method according at least one of the claims 1 to 16, wherein the irradiating step comprises irradiating the sample with at least two different wavelengths.

18. Method according at least one of the claims 1 to 17, wherein the at least one sample chamber (10, 11) is placed in contact with a heat dissipating component (70) .

19. Device for measuring fluorescence of at least one sample (1) , comprising:

- at least one sample chamber (10, 11) being adapted for accommodating the at least one sample and extending along a longitudinal reference direction (x) ,

- an excitation light source (20) for irradiating the sample in the sample chamber (10, 11) with excitation light along an excitation light path, and

- a detector device (30) for detecting fluorescence light from the sample (1) along a detection light path, characterized in that the excitation light source (20) and the detector device (30) are arranged such that one of the excitation and detection light paths extends through the sample chamber (10, 11) par- allel to the longitudinal reference direction and the respective other one of the detection and excitation light paths extends in a direction (y) deviating from the longitudinal reference direction (x) .

20. Measuring device according to claim 19, wherein the excitation light path extends about perpendicularly relative to the detection light path.

21. Measuring device according to claim 19 or 20, wherein the detector device (30) comprises a spectrally resolving component (31) .

22. Measuring device according to at least one of the claims 19 to 21, further comprising a location determining device for determining at least one location of the at least one sample along the longitudinal extension of the sample chamber (10, 11) .

23. Measuring device according to at least one of the claims 19 to 22, comprising a plurality of sample chambers (10, 11) each being adapted for accommodating at least one sample.

24. Measuring device to according at least one of the claims 19 to 23, wherein each sample chamber (10) comprises one capillary (11) having at least one transparent wall portion.

25. Measuring device according to claim 24, wherein the exci- tation light source (20) being arranged for directing the excitation light along a longitudinal axis of the capillary (11) and the detector device (30) being arranged for collecting detection light passing through the wall of the capillary (11).

26. Measuring device according to claim 25, wherein the excitation light source comprises at least one laser (20) .

27. Measuring device according to claim 26, wherein the exci- tation light source (20) further comprises at least one light guiding fibre.

28. Measuring device according to at least one of the claims 25 to 27, wherein the detector device comprises a mult±- channel detector (30) extending along the capillary (11) .

29. Measuring device according to at least one of the claims 25 to 28, wherein the detector device is movable along the capillary (11) .

30. Measuring device according to claim 24, wherein the excitation light source (20) being arranged for directing the excitation light through the wall of the capillary (11) and the detector device (30) being arranged for collecting detection light along a longitudinal axis of the capillary (11) .

31. Measuring device according to claim 30, wherein the de— tector device (30) being arranged at an end of the capillaxy

(11) •

32. Measuring device according to claim 31, wherein the excitation light source (20) is movable along the capillary (11).

33. Measuring device according at least one of the claims 1 to 32, wherein the excitation light source (20) being adapted for irradiating the sample with at least two different wavelengths .

34. Measuring device according at least one of the claims 1 to 33, further comprising a heat dissipating component (70) placed in contact with the at least one sample chamber (10) .

35. Separation device, in particular for isoelectric focusing of samples, comprising a measuring device according to at least one of the claims 19 to 34.

36. Separation device according to claim 35, comprising a first sample chamber holding block (40) being further adapted for accommodating samples to be introduced into the at least one sample chamber.

37. Separation device according to claim 35 or 36, comprising a second sample chamber holding block (50) being further adapted for collecting samples to be drained from the at least one sample chamber.

38. Separation device according to claim 37, wherein the first or second sample chamber holding block being made of a transparent material.

39. Separation device according to at least one of the claims 35 to 38, wherein the detector device comprising a transmission diffraction grating (31) .