CA2284195A1 - Device and method for capillary electrophoresis - Google Patents

Abstract

The invention relates to an electrophoresis device comprising a plurality of capillary columns (10) each having a detection area (10a), and a detection device (40) having an imaging device (41) and a detecting camera (42). The capillary columns are mounted on a shared fixing device (50) in such a way that the detection areas (10a) form a straight row (13) which is imaged on the detecting camera by means of the imaging device.

Description

Apparatus and Method for Capillary Electrophoresis The invention relates to an apparatus for capillary electrophoresis with a large number of separation capillaries and an optical detection system as well as a method for using an apparatus of this kind.
Electrophoretic separation of substances and mixed substances is an analytic procedure that is widespread especially in biochemistry and molecular biology. The substances to be separated are separated in a separation medium subjected to an electric field and separately detected. In capillary electrophoresis the separation medium is in a capillary (typical inner diameter of < 150 um). The separation procedure is performed in the capillary;
detection can be performed both inside and at the end of the capillary. This is a special advantage in terms of speed, resolving power and minimizing the amount of sample. For the analysis of complex biochemical reactions or molecular biological processes (eg for the analysis of complex genomes or proteins) it is necessary to use an extremely large number of different samples (eg 105 to 10') .
Consequently there is interest in apparatus for multiple capillary electrophoresis with high sample throughput and high-grade parallel analyses. For this purpose the multichannel or multiplex arrangements, named as examples in what follows, are known, which, although they allow highly parallel processing, are usually so complicated in their structure that routine use is restricted. US-A-5 498 324, for instance, describes a multiplex fluorescence detector system for capillary electrophoresis in which the capillaries are connected to optical fibers through which the excitation light is conducted separately to the capillaries. Fluorescence detection is performed by a microscope with a CCD camera. This structure is complicated and subject to interference because of the coupling of optical fibers to the capillaries. The amount of light that can be coupled in is limited, so the sensitivity of fluorescence detection is also restricted. The system is unsuitable for routine use in particular because of the substantial maintenance effort required by the capillary array (difficult changing of capillaries) with the optical fibers.
US-A-5 582 705 discloses of a multiplex capillary electrophoresis system in which a CCD detector is optically connected to the capillaries in such a way that the inside of a capillary is imaged on a pixel of the CCD detector. The disadvantage of this system is that the detector arrangement is complicated and highly specialized, calls for the use of specially matched optical components and is thus less compatible with existing laboratory systems for fluorescence detection. Furthermore, there is increased risk of crosstalk from one capillary to another in the event that the concentration differences of the analytes are very large.
US-A-5 584 982 and US-A-5 567 294 describe multiple capillary systems with a socalled sheath flow cuvette that, although it achieves an increase in detection sensitivity, is disadvantageous because of the complicated structure lacking the ruggedness required for routine laboratory operation. The use of replaceable separation media is especially difficult with such a cuvette. The cuvette can be soiled when replacing the medium and there is the risk of the separation medium slowly flowing out during separation.
Finally, US-A-5 675 155 discloses of a raster or scanning system in which the fluorescence signals of a coplanar capillary group are detected by a scanner detector. With this detector the excitation or measurement light is consecutively directed at the individual separation capillaries by a moving mirror. The disadvantage here is the susceptibility to disturbance because of the use of moving parts and the restricted reading speed. In capillary electrophoresis it is possible that the samples to be detected separately will be so fast that reliable detection is not possible during one raster scan. The capillaries at the edge of the capillary array, in particular, are not scanned at even intervals. What is more, the scanning systems are not rugged enough for routine use.
In multiple capillary electrophoresis there is not only interest in stability and parallelism of processing but also in automation of the entire analytical procedure, starting with the loading of a front-end reservoir through the actual separation operations to cleaning of the separation capillaries. Because of the disadvantages mentioned, automation of capillary separation arrangements has not been achieved to date with multiple capillary systems but only with single capillary systems.
The object of the invention is to provide an improved apparatus for capillary electrophoresis that is characterized by a simplified and stable structure and allows automation of the parallel separation of a large = number of samples. It is also the object of the invention to provide methods for the use of such an apparatus.
These objects are solved by an electrophoresis apparatus with the features of patent claim 1 and a method with the features of patent claim 17 respectively. Advantageous embodiments are defined in the dependend claims.
The invention is based on the idea of arranging a large number of separation capillaries, each having a detection range, so that the samples in all detection ranges are exposed to simultaneous and uniform illumination or excitation and a detector device simultaneously detects the images of all detection ranges. For this purpose the following measures are implemented, singly or together, on a generic multiple capillary separation apparatus with a front-end reservoir with a large number of samples, a correspondingly large number of separation capillaries (each with a detection range), attached to a common holding device, a collector device and a measurement system with an illuminating device and a detector device.
The holding device is a support for the separation capillaries on which the separation capillaries are arranged so that the detection ranges form a straight row. The detection ranges are, for example, detection windows on each of the separation capillaries, which are also provided with protective or shielding layers. The holder can also offer "optical isolation" between the capillaries to prevent crosstalk. In addition, the holder is modular (eg six holders for 16 capillaries each), ie it allows replacement of smaller capillary arrays without having to dismantle the w entire arrangement. The illuminating device preferably forms a line-type, uniform illumination field whose shape is matched to the row of detection ranges. A special advantage of the invention is that the illumination or excitation of the samples in the capillaries is direct from the outside by illumination of the capillary wall in the region of the particular detection range. No additional devices are necessary for input coupling, and adjustment is implemented by the fixed but detachable location on the holding device.
The detector device is based on detection of the light emitted in the detection ranges through the capillary wall.
All detection ranges are simultaneously imaged on a detector camera by a suitable imaging device. Depending on the analysis requirements, the detector device comprises imaging on a single detector row or on a large number of detector rows, forming a two-dimensional matrix of detector elements.
In the latter case at least one dispersion element can be provided in the detector device allowing, in addition to simultaneous detection of the detection ranges, analysis of the spectral properties of the light emitted from the detection ranges.
The separation capillaries exit into a common collector device that fulfills a dual function. Firstly, the collector device contains the carrier medium for loading the separation capillaries. Secondly, the separated substances are jointly collected on the collector device. For this purpose the collector device will preferably contain a means of collection for the molecules of the samples to be separated. This means of collection, or molecule trap, is a semi-permeable wall element that separates the ends of the separation capillaries from the high-voltage power supply for generating molecular movement in the separation capillaries.
During electrophoretic separation the molecules are drawn through the porous wall element to the electrode and thus collected in the molecule trap. Passive back-diffusion through the wall element is hindered to a large extent because the pores are very small. After completion of analysis a pressure of up to 5 bar is applied to the collector device (reservoir), but in the region outside the molecule trap. This prevents ready analyzed molecules from being pressed back into the capillaries and disturbing subsequent separation.
An important feature of the method according to the invention is that both illumination or excitation of the samples to be separated in the detection ranges and detection of the light from the samples through the capillary wall is from outside into the capillary or vice versa. Thin-walled capillaries of approx. 35 to 50 um are preferably used to reduce background signals. But larger designs - with thicker walled capillaries - are also possible. Furthermore, other forms of the detection range are possible, eg by coupling the capillaries into a cuvette or into a microstructure with channels. The design of the detector unit with lenses and objectives allows simple alteration of the imaging scale (for optimum imaging of the detection range on the detector elements) and thus greater flexibility in terms of the form of the detection range. The separation device according to the invention is best operated with a low-viscosity separation medium. In this way - the loading pressure to be applied to the collector device (or outlet vessel) is reduced and the loading speed increased. Simple replacement of the separation medium allows adequate flushing of the capillaries (either with the separation medium itself or beforehand with a cleansing agent) and thus extends the service life of the capillaries.
The invention possesses the following advantages. The separation device is compact and without moving parts.
Illumination and detection are compatible with available laboratory setups and with currently used dye markers. This means advantages on the one hand in routine operation by personnel without highly specialized training and on the other hand in maintenance. The invention allows, for the first time, an entirely automated analysis, details of which are explained below. Some 15000 different samples can be analyzed, for example, before an operator has to intervene for the first time. The system possesses high multiplex capability. Both the sample feed (preferably with common formats, eg from microtiter plates) and the illumination and detection are simultaneous in all channels formed by a separation capillary. Special detection structures like a sheath flow cuvette are unnecessary. The holding device for the separation capillaries is of rugged design, prevents stray light between the capillaries and allows bundled attachment of the capillaries to simplify maintenance. The loading pressure of the carrier medium can be reduced from approx. 70 bar for conventional carrier media (eg 2~
hydroxyethylcellulose, viscosity approx. 1000 centiStokes) to approx. 5 bar if carrier media with viscosity of 100 centiStokes (eg 10-15% dextran or 4-8$
- polydimethylacrylamide) are used.
Further advantages and details of the invention are described in what follows with reference to the attached drawings, which show:
Fig. 1: a schematic overview of the setup of an electrophoresis apparatus according to the invention, Fig. 2: a partial view of a holding device that is part of a separation device according to Fig. l, Fig. 3: an overview to illustrate spectrally resolved detection according to the invention, Fig. 4: a further overview to illustrate spectrally resolved detection according to the invention, Fig. 5: an illustration of the detection of detector signals, Fig. 6: a schematic side view of a collector device that is part of a separation device according to Fig. 1, Fig. 7: a special capillary form used for electrophoretic separation according to the invention, whereby the capillary is metallically coated at the end and - serves simultaneously as an electrode, Fig. 8: a curve illustrating uniform illumination by the line Generator, Fig. 9: a curve illustrating detector signals of three adjacent separation capillaries, Fig. 10: curves illustrating the dependence on concentration of the separation medium viscosity, and Fig. 11: curves illustrating experimental results with a separation device according to the invention.
The invention is described in what follows with reference to a preferred embodiment in which samples in microtiter plates are electrophoretically separated by detecting the migration of probe components through separation capillaries with a carrier medium influenced by high voltage. But the invention is not restricted to alignment of the capillary entrances with reference to a microtiter plate or certain carrier media or a certain separation effect. Instead it can be implemented in all electrophoresis capillary systems with a large number of separation capillaries.
Fig. 1 shows an electrophoresis apparatus according to the invention in which a large number of separation capillaries are attached to a common holding device 50, the details of which are explained below with reference to Fig. 2. The separation capillaries 10 are aligned so that detection - ranges, in the form of detection windows for example, form a straight row 13. An illuminating device 60 with a light source 61 and an imaging optical system 62 forms a line-type illuminating field 63 that coincides with the row 13 of detection windows of the capillaries 10. There is also a detector device 40 containing an imaging device 41, a detector camera 42 and a dispersion element 43. The dispersion element 43 is an optional assembly that can be dispensed with for certain applications, as explained below.
The detector camera 42 is connected to a control and data storage device 44, for example in the form of a computer and electronic controller 45.
The separation capillaries 10 lead from an inlet reservoir 20 (samples (21) or storage (24) reservoir) to a collector device 70. The electrophoretic separation circuits are formed by applying a high voltage between the inlet reservoir 20 and the collector device 70 through electrodes 11 or 71. For this purpose the inlet reservoir 20 is connected to ground potential, for example, and the collector device 70 to a high-voltage power supply device 72. This high-voltage power supply device can produce DC
voltage or - for special purposes - a modulated voltage (eg pulse or sinusoidal) and is controlled by the unit 44, 45.
The inlet reservoir 20 is a samples reservoir 21 (during injection) or a storage reservoir 24 (during separation).
The samples reservoir 21 is preferably a flat substrate with a large number of samples arranged in predetermined fashion, which are to be subjected to electrophoretic separation in parallel (or simultaneously). This substrate is preferably a microtiter plate with a common format (eg 96-hole, 384-hole - or 1536-hole plate). For complete automation of separation starting with the feeding of samples, the samples reservoir is arranged on a transport device 22 by means of which a required samples reservoir 21 can be moved from a storage device 23 to the operating position at the injection ends of the separation capillaries by control signals from the unit 44, 45. For this purpose the transport device 22 and/or storage device 23 are equipped with appropriate means of actuation and positioners. The transport device 22 is also intended, after loading the injection ends of the separation capillaries, for replacing the samples reservoir 21 by a storage reservoir 24 so that the circuit is again completed.
The injection ends 11 of the separation capillaries 10 are aligned so that they match the positions of the samples to be separated on the substrate or samples reservoir 21. The separation capillaries 10 have an outer diameter of 100 to 400 um for example. Preferred forms are capillaries with an outer diameter of 375 um and an inner diameter of 100 um, as well as capillaries with an outer diameter of 200 um and an inner diameter between 50 and 100 um, or capillaries with an outer diameter of 150 um and an inner diameter of 75 um. The capillary wall thickness may be of any other figure, however, that allows reproducible detection with the illuminating and detection devices described below. The total length of the separation capillaries is approx. 40 to 50 cm in the shown implementation, although this length can be modified as a function of application.
At the injection end the separation capillaries form what is essentially a flat, two-dimensional "brush-like" fan, the dimensions of which correspond more or less to those of the - samples reservoir 21. Towards the holding device 50 the separation capillaries are brought together in such a way that they, at least for the length where the detection ranges are arranged as a row 13, are closely adjacent (see Fig. 2) .
In the region where the separation capillaries are brought together before the holding device 50, it is possible to provide a tempering device. This tempering device can be designed to circulate a tempered medium (eg air) around the separation capillaries and to regulate the heat. Preferably a temperature between 10 and 60°C will be set.
The illuminating device 60 is intended to illuminate the row of detection ranges of the separation capillaries on the holding device 50 as uniformly as possible so that the sample components, passing the detection ranges in the separation process, are exposed to the same amount of light.
Seeing as the row 13 of detection ranges extends over the approx. 5 cm width of the capillary group for example, a laser light source 61 is preferably combined with an optical system 62 forming a line-type illumination field (socalled line generator) in order to produce sufficient illumination or excitation intensity. The laser 61 is selected as a function of the spectral properties of the fluorescence marker that is used (eg Ar laser of approx. 50 to 200 mW) and can also be driven through the control unit 44, 45.
The optical system 62 forms the line-type illumination field. The optical system can take the form of a mirror swinging or rotating at high speed for example, which may be disadvantageous for the stability of the arrangement - however. The use of a cylindrical lens is also possible, preferably avoiding moving parts, but scaled so that, despite the Gaussian distributed intensity of the illumination field, sufficiently homogeneous illumination of the capillary row is enabled. Consequently, in a preferred implementation of the invention, the optical system 62 is created by a socalled Powell lens (producer: OZ Optics, Canada), allowing homogeneous illumination and optimum utilization of laser power (see Fig. 8). The Powell lens can also be connected to the laser 61 direct by an optical fiber 64, producing a rugged and transportable setup in which no moving parts like rotating mirrors and the like are contained, and ensuring that, apart from the line-type illumination field, no coherent or highly focused light escapes (user safety).
The optical fiber 64 also allows simple replacement of the laser (eg for adaptation to other fluorescence markers) or -combined with special couplers - conduction of the light from two different lasers into one illumination field.
The light intensity of the illumination field of the Powell lens is uniformly distributed in the direction of the line and Gaussian distributed perpendicular to it (ie in a direction parallel to the orientation of the separation capillaries). The parameters of the Powell lens and the configuration referred to the detection windows are selected to focus into a line with a width of approx. 1 mm or less.
The narrower the illumination field, the higher is the resolution achieved by the separation apparatus.

Details of the detection device 40 and the collector device - 70 are explained below with reference to Fig. 3, 4 and 5.
Fig. 2 shows a section through a holding device 50 as a schematic perspective to illustrate the planar, parallel attachment of the separation capillaries on the top of the holding device (Fig. 2 bottom) and a magnified perspective with a capillary section as a phantom view (Fig. 2 top). The holding device comprises at least the shown capillary holder 51, offering the separation capillaries 10 mechanical support in the focus plane of the illuminating device and ensuring optical isolation between the separation capillaries. Several capillary holders of 16 capillaries each, for example, can be provided, whereby the overall holder 50 is then modular and single capillary holders can be replaced as modules. Except for the detection ranges, the separation capillaries are provided with protective layers that may be impervious to light. In the detection ranges l0a the separation capillaries are free of any coating. The capillary holder 51 supports the separation capillaries at least on a lengthwise section where the detection ranges are located. In terms of the total length of the separation capillaries, this is in the rear quarter or rear third of the length of the separation capillaries looking from the injection end. Thus the detection ranges or detection windows of the separation capillaries, given a separation capillary length of approx. 50 cm, form a straight row with a perpendicular spacing of approx. 5 to 20 cm, preferably 10 cm, from the exit ends of the capillaries. Generally, to improve resolving power, the detection windows should be as far as possible downstream referred to the direction of motion of the samples to be separated.

The capillary holder is a block with grooves 52 in which the separation capillaries are inserted. The dividing walls 53 serve for optical isolation between the detection ranges of the individual separation capillaries. The dividing walls are formed of segments that are as thin as possible to allow tight packing of the capillaries and to prevent shadowing of the illuminating or excitation light from above.
Alternatively it is possible to create the capillary holder 51 without grooves and to lay the capillaries next to one another flat on top of the capillary holder. But this requires, instead of the dividing walls 53 for optical isolation, the provision of coatings impervious to light in the detection ranges on the sides of the separation capillaries facing adjacent separation capillaries, or exact optical imaging.
On the holding device there are also (not illustrated) releasable means of attachment, eg clamps, for the separation capillaries 10 in the grooves 52. It is possible for the holding device to support the separation capillaries in lengthwise sections outside the detection ranges. But as a rule it is sufficient for the separation capillaries to be conducted through the air from the inlet reservoir 20 to the holding device 50 (see Fig. 1). But tempering devices may also be provided in this region so that the separation operation can be performed in predetermined temperature conditions.
The principle of spectrally resolved multiplex detection is explained in what follows with reference to Fig. 3 and 4. In simple analysis, where only one fluorescence marker has to be detected, it is sufficient to attach suitable filters to the imaging device 41 of the detector device 40 (see Fig. 1) to shield the excitation light in fluorescence measurement, but more complicated analysis requires spectral separation of the light from the detection ranges. This is the case in DNA sequencing, for example, when nucleotides are specifically provided with fluorescence tags and are to be selectively detected as four separate fluorescence bands for instance.
The invention simultaneously produces excellent spectral and local resolution by mapping the detection window row on a two-dimensional detector matrix with resolution corresponding to the two matrix dimensions. This is illustrated schematically in Fig. 3. The vertical array of separation capillaries 10 with the detection window row 13, differing from the operating position, is mapped by an imaged device (not shown) and the dispersion element 43 on a CCD matrix 42. The dispersion element 43 is symbolized by a prism but can be formed of any wavelength-dispersive structure with high local resolution. The imaging on the CCD
matrix produces the local resolution on the Y axis and the spectral resolution on the X axis. Thus the matrix contains illuminated pixel rows, the number of which corresponds to the number of separation capillaries. Each pixel delivers a detector signal as a function of the amount of incident light, so that the detector signal response of each X row corresponds to the spectral response of the light from a separation capillary. It is possible, depending on the fluorescence dye or tag that is used, for only a subrange of one or more X rows to be read out, corresponding to the expected wavelength emission band of the fluorescence dye or tag. The spectral resolution is designed for measuring in at - least three spectral bands.
Details of spectrally and locally resolved detection are shown in Fig. 4. In a preferred embodiment the detection window row 13 is imaged by a first objective 411 onto a slit 412. A second, inverted objective 413 sends the light from the slit through the dispersion element 43. One or more filters 414 for masking the excitation light may be located either in front of the objective 411 (as shown) or between the inverted objective 413 and the dispersion element 43.
The dispersion element 43 is either a classic spectral device with prisms and/or gratings (drawback: alteration of the imaging direction, reduced local resolution) or preferably a direct-vision prism 431 (socalled Amici prism, Fig. 4A) or a prism/grating/prism combination 432 (Fig. 4B) or an L-shaped arrangement with a holographic transmission grating 433 (Fig. 4C). In the last mentioned example of Fig.
4C the light is reflected at a certain angle by a mirror 434 onto the transmission grating 433. The direct-vision prism 431 or the combination 432 or the L-shaped arrangement with transmission grating 433 offers the advantage of compactness and extra ruggedness. Interference through aberration and astigmatism is excluded for the most part. Furthermore, high light throughput is guaranteed plus small focal length. In the first two designs a straight optical axis from the detection windows to the detector camera is maintained. The entire structure of objectives, slit, filters and dispersion element can be configured in a tube shielded against stray light, even functioning as a portable spectrometer. Suitable selection of the imaging parameters of the objectives and the dispersion element makes the imaging scale variable over a wide range, resulting in high flexibility in terms of the number of separation capillaries and their diameter.
The light through the dispersion element 43 is imaged on the camera 42 by an objective 415. The CCD chip 421 of the camera 42 is 500 * 500 pixels, for example, from which pixel groups are selected as a function of application (especially the size and number of the separation capillaries to be detected) for reading in the analysis operation. Given a pixel size of 24 * 24 um for example, the slightest misadjustment (eg shifting of the separation capillaries) can cause the image on the CCD matrix to shift. To avoid this effect, the invention uses a search algorithm that determines the pixel groups which are to be read. This means that the controller 44 automatically selects the required pixels of the spectrally and locally resolved image of the detection window row. The algorithm for reading is a suitable one from image data processing, eg the socalled watershed algorithm (see S. Wegner et al. in "Spektrum der Wissenschaft", 1997, p 113) or what is called the chain code algorithm, explained below with reference to Fig. 5.
Using 100 capillaries and n fluorescence emissions for example, 100 * n pixel groups (regions of interest) are imaged on the CCD chip. The pixels of the groups must be binned and read out correlated. The pixel groups are defined in a predetermined manner or automatically determined with the data processing algorithm in a first trial phase. With the chain code (Fig. 5) only a starting point of the matrix coordinates is recorded and the remaining pixels of a group are detected and read with predetermined direction codes.
Eight direction codes can be provided as shown, for example, relating to the eight pixels that surround an observed pixel. By entering a numeric sequence corresponding to the numbered directions, all the pixels belonging to a pixel group can be uniquely identified. This entry is insensitive to slight image shifts (dislocation in the left part of the image) and is advantageous in allowing reduction of the memory requirement for characterizing a group.
Fig. 6 shows as a schematic side view details of the collector device (or outlet vessel) 70 (see Fig. 1). The collector device 70 consists of a pressure vessel 73 with a pH buffered carrier medium 74. The pressure vessel 73 is connected by a pressure line 75 to a pump device 77 that produces compressed air for loading the separation capillaries 10 with their exit ends in the carrier medium.
The pump device is controlled by the unit 44, 45. Also projecting into the carrier medium is an electrode 71, connected to a high-voltage power supply (not shown).
Between the electrode 71 and the exit ends of the separation capillaries 10 is a molecule trap 76, indicated by dashed lines, that is intended for collecting the separated samples. Through the effect of an electric field, the separated samples exit from the ends of the capillaries and drift to the electrode 71. In doing this they come up against the molecule trap in the form of a porous dividing wall (eg a membrane or gel) with pores of a characteristic diameter of approx. 10 to 100 nm. Collection of the separated samples or molecules is implemented by one of the following principles.
Seeing as the speed of motion under the influence of an electric field is substantially greater than the speed of diffusion motion, the probability of molecules passing through the molecule trap from the electrode to the exit ends during a turn-off time of the high voltage is very slight. In this case it is not necessary to select the size of the pores within very precisely defined limits. In an alternative mechanism it is assumed that the separated samples consist of field-dependent stretched molecules (polyelectrolytes) that pass the pores relatively simply in stretched form under the influence of the electric field, but in globular form can only pass the pores with difficulty when the field is turned off.
In a preferred embodiment of the invention the electrodes are directly attached to the capillaries at the input, as shown schematically in Fig. 7. For this purpose the inlet ends of the separation capillaries have an outer metal coating 14 (of silver or platinum for example), simultaneously fulfilling the electrode function. This allows substantial reduction of the minimum injection volume. Electrical contact is produced by a contact device 15 (eg clip).
The procedure of a separation analysis using the separation apparatus detailed above (see Fig. 1) is described in what follows. Firstly, for loading the carrier media, an empty plate or a vessel is moved under the injection ends of the separation capillaries and pressure is applied to the pressure vessel 73 of the collector device 70 so that the carrier medium 74 enters the exit ends of the separation capillaries and runs through them to the injection ends. The pressure is reduced as soon as sufficient separation medium has flowed through the capillaries. In the following step the samples reservoir 21 is moved from below to the - injection ends of the separation capillaries so that the injection ends are immersed in the samples arranged on the storage reservoir. Next a high voltage is applied to the separation capillaries for a certain loading time to load the samples, ie to inject very small amounts of sample into the ends. The loading time and the high voltage are selected so that the first millimeters of the separation capillaries are filled with the samples to be separated. In the case of the above mentioned capillaries with inner diameters between 50 and 100 um for example, and using common solvents, this is produced in a loading time of 1 to 20 s and with high voltage of approx. 100 to 400 V/cm (preferably approx. 10 kV). After sample loading the samples reservoir 21 is replaced by the storage reservoir 24 with a buffer solution (solution of electrolytes), with which the injection ends of the separation capillaries are in contact during the following separation process. For the actual separation process the high voltage is applied again for a time depending on the application, this being between 10 and 30 min or even amounting to hours.
During the entire separation process, automatically controllable by the unit 44, the samples to be separated (molecules, DNA fragments, proteins, etc) move to the exit ends of the separation capillaries. As a result of the separation medium there is "selection", ie the small molecules reach the detection windows faster than the large molecules, so that complete analysis of the sample composition is possible because of the locally and spectrally resolved detection on each window as a function of time. Separation of the molecules is also possible by other mechanisms, eg by socalled end-labeled free solution electrophoresis.
Experimental results of the separation apparatus according to the invention are presented in Fig. 8 through 11. Fig. 8 shows a comparison between illumination of the detection range by a cylindrical lens and a line generator (here the noisy signal is produced by the use of a multimode optical fiber instead of a monomode one). The cylindrical lens generates a socalled Gaussian profile, whereas the line generator produces a plateau-shaped profile and thus more homogeneous illumination. Fig. 9 shows a curve to illustrate the practical elimination of crosstalk between different separation channels (separation capillaries). The three maxima correspond to the detector signals from pixel groups assigned to adjacent separation capillaries. Crosstalk between adjacent capillaries is less than 1~, so that unique and reproducible assignment of the detector signals to the separation channels is possible. In the example shown the capillaries were arranged next to one another without a dividing wall. Crosstalk is reduced even more by the optical isolation of the capillary holder.
Fig. 10 illustrates the choice of a low-viscosity carrier medium (separation matrix) according to the invention. The curves show the dependence of carrier medium viscosity on the particular carrier medium concentration. The concentration is selected so that the viscosity amounts to 100 centiStokes (mm2/s) (corresponding to approx. < 100 cP).
Fig. 11 shows a control experiment to demonstrate the reproducibility of the separation apparatus according to the invention. 96 identical DNA samples were separated simultaneously. By way of example, the detector signals of eight capillaries are shown accumulated over a detection interval of approx. 30 min, demonstrating excellent correlation of the separation results in the different capillaries.
The invention is explained above with reference to fluorescence measurements. But, in analogous manner, optical detection can also be implemented based on absorption, reflection or transmission measurements.

Claims (23)

1. Electrophoresis apparatus with a large number of separation capillaries (10), each having a detection range (10a), and a detector device (40) with an imaging device (41) and a detector camera (42), whereby the separation capillaries are arranged in such a way on a common holding device (50) that the detection ranges (10a) form a straight row (13) that is imaged on the detector camera by the imaging device.

2. Electrophoresis apparatus according to claim 1 in which an illuminating device (60) is provided for uniform, simultaneous illumination of the row of detection ranges.

3. Electrophoresis apparatus according to claim 2 in which the illuminating device comprises a light source (61) and an optical system (62) that form a line-type illuminating field.

4. Electrophoresis apparatus according to claim 3 in which the optical system (62) is a Powell lens.

5. Electrophoresis apparatus according to one of the preceding claims in which the detector camera (42) comprises at least one row of detector elements onto which the row (13) of detection ranges is imaged.

6. Electrophoresis apparatus according to claim 5 in which the detector device contains a dispersion element (43) and the detector camera (42) comprises a two-dimensional detector matrix onto which the row of detection ranges is locally resolved in a first direction and spectrally resolved in a second direction.

7. Electrophoresis apparatus according to one of the preceding claims in which the holding device (50) is a capillary holder (51) that has grooves (52) to hold separation capillaries with mutual optical isolation.

8. Electrophoresis apparatus according to claim 7 in which the grooves are formed by webs (53) on a surface of the capillary holder whose spacings are essentially the same as the outer diameter of the separation capillaries.

9. Electrophoresis apparatus according to one of the preceding claims in which the collector device (70) contains a supply of the separation medium for loading the separation capillaries under the influence of pressure.

10. Electrophoresis apparatus according to one of the preceding claims in which the collector device contains a molecule trap (76).

11. Electrophoresis apparatus according to one of the preceding claims in which the injection ends of the separation capillaries (10) are mutually aligned like samples on a samples reservoir (21).

12. Electrophoresis apparatus according to claim 10 in which the samples reservoir is a microtiter plate.

13. Electrophoresis apparatus according to claim 12 in which the microtiter plate (21) has 96, 384 or 1536 recesses for holding samples.

14. Electrophoresis apparatus according to one of the preceding claims in which the viscosity of the separation medium is less than or equals 100 cP.

15. Electrophoresis apparatus according to one of the preceding claims in which samples are automatically fed.

16. Electrophoresis apparatus according to one of the preceding claims in which the detector device (40) is adapted to detect at least 96 detection ranges (10a) simultaneously locally and spectrally resolved.

17. Method for capillary electrophoretic sample separation in which a large number of separation capillaries are simultaneously loaded from the exit ends with a separation medium and then from the injection ends with samples to be separated and a separation process is subsequently performed so that the passage of the samples to be separated by a large number of detection ranges is simultaneously detected with time, local and spectral resolution.

18. Method according to claim 17 in which detection is performed with a two-dimensional detector camera with a matrix arrangement of detector elements, whereby the detector elements are read in groups corresponding to predetermined regions of spectral interest.

19. Method according to claim 18 in which the detector elements to be read are determined using a chain code algorithm.

20. Method according to one of the claims 17 through 19 in which the individual steps work under fully automatic control.

21. Method according to one of the claims 17 through 20 in which the samples to be separated are arranged in samples reservoirs and transported with these to the injection ends of the separation capillaries.

22. Method according to one of the claims 17 through 21 in which the separation process and detection are performed without movement of mechanical parts or fluids.

23. Method according to one of the claims 17 through 22 in which the capillary electrophoretic sample separation with the arrangement of samples in samples reservoirs, the loading of the separation capillaries with the samples, the electrophoretic separation and replacement of the separation medium for all samples in the samples reservoirs and for all separation capillaries simultaneously is performed fully automatically.