Device and method for automatically analysing the constituents of an analyte Number:7,435,322 from the United States Patent and Trademark Office (PTO) owispatent

Title: Device and method for automatically analysing the constituents of an analyte

Abstract: The invention relates to a device and a method for automatically analysing the constituents of at least one analyte, especially by means of gel electrophoresis and/or isoelectric focussing, comprising at least one separating matrix, especially a gel. Each separating matrix is applied on at least one side in a supporting direction S of a supporting element, can be loaded with an analyte, and can be electrically contacted on opposite ends in a separating direction T in order to split the analyte into its constituents. The invention is characterised in that the supporting element is embodied in such a way that it enables a material-transferring process to take place from one side--in an access direction Z which is different from the support direction S and the separating direction T--essentially over the entire area of the separating matrix in which the constituents of the analyte are located after being separated, and in that the supporting element comprises a continuous sealing surface which essentially prevents said material-transferring process from taking place outside said area.

Patent Number: 7,435,322 Issued on 10/14/2008 to Schlichting

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Inventors:	Schlichting; Hartmut (Gilching, DE)
Appl. No.:	10/467,552
Filed:	February 1, 2002
PCT Filed:	February 01, 2002
PCT No.:	PCT/EP02/01098
371(c)(1),(2),(4) Date:	January 21, 2004
PCT Pub. No.:	WO02/061408
PCT Pub. Date:	August 08, 2002

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Foreign Application Priority Data

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Feb 01, 2001 [DE]			101 04 821
May 06, 2001 [DE]			101 22 323
May 29, 2001 [DE]			101 26 282

Current U.S. Class:	204/464 ; 204/459; 204/462; 204/604; 204/610; 204/614
Current International Class:	G01N 27/447 (20060101)
Field of Search:	204/614,464,608,457

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References Cited [Referenced By]

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U.S. Patent Documents

4204767	May 1980	Kato et al.
4337131	June 1982	Vesterberg
4374723	February 1983	Vesterberg
4810348	March 1989	Sarrine et al.
5019232	May 1991	Wilson et al.
5234559	August 1993	Collier et al.
5275710	January 1994	Gombocz et al.
5279721	January 1994	Schmid
5449446	September 1995	Verma et al.
5562813	October 1996	Mullaart et al.
5800691	September 1998	Kozulic
5954931	September 1999	Maracas et al.
5993627	November 1999	Anderson et al.
6171463	January 2001	Selby et al.
6201628	March 2001	Basiji et al.

Foreign Patent Documents

	2944127		May., 1980		DE
	0300924		Jan., 1989		EP
	0631132		Dec., 1994		EP
	02268272		Nov., 1990		JP

Primary Examiner: Noguerola; Alex
Attorney, Agent or Firm: IP Strategies

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Claims

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The invention claimed is:

1. Device for automatic analysis of the components of at least one analyte, by means of at least one of gel electrophoresis and isoelectric focussing, for use with at least one separation matrix, wherein the device comprises at least one support element and defines at least one transfer chamber; wherein the support element is adapted to carry the separation matrix in a support direction, on at least one side, such that the separation matrix is loadable with an analyte and is contactable electrically at opposite ends in a separation direction for separation of the analyte into its components; wherein the device is adapted to allow a mass transfer process with the transfer chamber, from one side in an access direction, over substantially the entire area of the separation matrix, in which the components of the analyte are contained after separation, wherein the support element has a continuous sealing face to the transfer chamber, wherein the sealing face substantially prevents the mass transfer process outside the area of the separation matrix, wherein the access direction is different than the support direction and the separation direction, and wherein the support element is adapted to be fixed to the separation matrix during separation and transfer.

2. Device according to claim 1, wherein the support element is adapted to carry a separate separation matrix for each said at least one analyte.

3. Device according claim 1, further comprising means for electrical contact of the separation matrix on both sides of the access direction, for transfer of the separated components of the analyte onto a membrane.

4. Device according to claim 1, further comprising means for measurement of reflection or transmission of the separation matrix in at least one of the access direction and the support direction.

5. Device according to claim 1, wherein the support element is further adapted to delimit the at least one separation matrix on both sides in the support direction.

6. Device according to claim 1, wherein the separation direction, the support direction, and the access direction are substantially perpendicular with respect to each other.

7. Device according claim 1, wherein the support element is further adapted to carry at least one additional separation matrix in contact with the at least one separation matrix, into which the components of the analyte can be transferred.

8. Device according to claim 1, further comprising a membrane that can be brought into contact with the at least one separation matrix in the access direction, onto which the components of the analyte can be transferred.

9. Device according to claim 1, further comprising one of a penetration and a channel disposed in the at least one support element transverse with respect to the separation direction, allowing for the mass transfer with the at least one separation matrix, and adapted for primary separation of the analyte.

10. Device for automatic analysis of the components of at least one analyte, by means of at least one of gel electrophoresis and isoelectric focussing, for use with at least one separation matrix, the device comprising a support element, wherein the support element is adapted to carry the separation matrix in a support direction, on at least one side, such that the separation matrix is loadable with an analyte, and is contactable electrically at opposite ends in a separation direction for separation of the analyte into its components, wherein the support element is adapted to allow a mass transfer process, from both sides in an access directions, over substantially the entire area of the separation matrix, in which the components of the analyte are contained after separation, wherein the support element on both sides in the access direction has a continuous sealing face, substantially preventing the mass transfer process outside the area of the separation matrix, and wherein the access direction is different than the support direction and the separation direction.

11. Method for automatic analysis of the components of at least one analyte, by means of at least one of gel electrophoresis and isoelectric focussing, using at least one separation matrix, wherein each said separation matrix is carried, in a support direction, on at least one side by a support element, is loaded with an analyte, and is contacted electrically at opposite ends in a separation direction for separation of the analyte into its components, the method comprising: carrying out a mass transfer process from one side in an access direction, over substantially the entire area of the separation matrix containing the components of the analyte after separation, wherein the access direction is different than the support direction and the separation direction, wherein the support element has a continuous sealing face with a transfer chamber, substantially preventing the mass transfer process outside the area of the separation matrix, and wherein the separation matrix is fixed to the support element during separation and transfer.

12. Method according to claim 11, further comprising providing electrical contacts on both sides of the separation matrix in the access direction, for transfer of the separated components of the analyte onto a membrane.

13. Method according to claim 11, further comprising carrying out a mass transfer process from both sides of the access direction with the separation matrix.

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Description

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The invention relates to a device and a method for automatic analysis of the components of at least one analyte, in particular by means of gel electrophoresis and/or isoelectric focusing with at least one separation matrix, in particular a gel.

Gel electrophoresis is an already established semi-quantitative method for the detection of single DNA fragments or proteins from a mixture of the same in an aqueous solution. Such a mixture is termed as analyte in the following, and as components of the analyte merely proteins are named, even if always DNA fragments and/or proteins or similar substances should be understood thereby.

During gel electrophoresis, the analyte is separated according to molecular weight of the involved proteins and is either detected with antibodies or simply is stained. Thereby, besides the molecular weight, also the charge and the shape of the substances play a role. For separation, the analyte is applied locally onto a separation matrix which is moistened with an aqueous buffer solution. Common matrices are agarosic gels and poly acryl amide gels of different concentration. The analyte is applied at one end of the separation matrix. By applying a voltage of e.g. 50-2000 V, it is subsequently moved within the separation matrix in the direction of the voltage. The aqueous buffer thereby serves for maintaining the pH-value and the salt concentration. According to their molecular weight, shape, and charge, the proteins thereby are retained differently strong by the matrix and distribute themselves after some time (e.g. 1-10 h) within the separation matrix along the current path.

The proteins thus separated are subsequently identified by means of a biological chemical reaction with antibodies. This occurs in a specifically prepared membrane which takes up the separated substance. In most cases nitro cellulose or nylon are used. The transfer from the separation matrix results by applying voltage, and, in fact, extensive between the separation matrix and the membrane pressed there upon. This is called blotting. For acceleration, this can be supported by a pressure difference. After a while, strongly depending on the blotting conditions, the charged proteins have shifted into the membrane quantitatively. By adding a specific antibody, a certain protein then is bound in the protein mixture selectively. The antibody is detected in most cases by means of radio active, fluorescence or chromatic labeling and thus makes the protein identifiable ("visible").

If the position of the proteins within the separation matrix after the separation has already been determined in preliminary tests, it is sufficient to stain the proteins unspecifically. This occurs by dipping the whole separation matrix into a staining solution, which preferentially stains proteins. The separation matrix subsequently is again destained. Bands remain visible, indicating the current position of the proteins on the separation matrix. The position of the bands is a rough measure for the molecular weight of the proteins, the intensity of the bands is a rough measure for the amount of proteins present in the substance to be measured. As detector within the optical field, on the one hand serves the appearance, on the other hand a calibrated densitometer, registering the intensity of the stain. The staining step subsequent to the separation can be omitted, if the analyte has been treated with a suitable stain prior to separation. For this, mostly fluorescing stains are used, which subsequently are detected automatically by means of UV-scanners.

For the first step--the electrophoretic separation--several arrangements are established:

During vertical slab electrophoresis, typically 10-50 different analytes are applied in a very broad gel (50-150 mm long, 100-200 mm wide, 0.3-2 mm thick) side by side. They run parallel, but in separated paths. The gel commonly first lies between 2 glass- or plastic plates, as long as a voltage is applied. 2 reservoirs with buffer solution communicate respectively with the upper and lower end of the gel. After separation of the proteins, the gel with the glass plates is taken out of the device, the glass plates are removed, and the staining steps and the further transfer steps, respectively, are carried out for blotting. The gel is extremely instable mechanically: it can tear by its own weight, sucks onto plane basis and buckles immediately. Without a buffer solution it dries, and thereby modifies its shape. Because of this, the automatic handling of the gel without the supporting glass plates is impossible. At present 6 different appliances are common to support the above-mentioned processes: an apparatus for pouring of the gels, a separation chamber, a power supply, staining vats, and a densitometer. In case an immune reaction is carried out, additionally a blotting apparatus is required as well as further staining and buffer exchange vats.

By means of staining of the proteins prior to electrophoresis, one can achieve that the evaluation procedure can result without removing the glass plates. Thereby, a partial automation is possible with restrictions in the staining technique. The blotting process thereby cannot be automated.

During sub-marine electrophoresis, the gel lies on a horizontal plate which is lying in the vat. The vat is filled up with buffer up to slightly above the upper edge of the gel. The two electrodes are respectively arranged at the end of the gel in the buffer. For staining, evaluating and blotting, the gel is taken out of the vat. Also here, automation is not easily possible.

During the horizontal slab electrophoresis, the gel lies on a horizontal glass plate and is exposed topside. The buffer is provided to a limited extent from correspondingly thickened gel blocks at both ends of the gel. Here, in fact already partial automation is possible. The upside exposed gel enables staining after the separation. Also, a UV-scanner can have direct access to the gel. Blotting without removing the gel, however, is also not possible with these appliances, because for blotting, the gel has to be electrically contacted from both phases and additionally has to be in contact with a membrane from the exposed face. An electrode on the lower side of the gel would, however, render the 1.sup.st electrophoresis step impossible, because the applied voltage would be short-circuited. Blotting methods, solely based on diffusion, last too long.

With all methods using an extensive/flat gel and having several analytes running simultaneously, besides handling problems, the following further problems occur:--heat development due to the supplied electrical energy leading to different running periods at the edges and in the middle of the gel plate. Cooling by means of air or the carrier plate should provide remedy here. In case large glass plates are used, the gel often has a different thickness in the middle or at the edge, influencing the running velocity. Attempts to remove this are mechanical supports. Narrow placed paths can influence themselves mutually. An attempt to encounter this is sufficient spacing or separation of the path by means of grooves in the glass plate lying underneath. For loading special pipettes and loading slots are used to avoid errors caused by spacing which are too small. With horizontal gels, a shifting of the chemical environment during electrophoresis occurs due to the limited reserve of buffer, influencing additionally the running velocity.

Rod electrophoresis is a variant according to which the gel is enclosed in a glass tube. Here, prior to electrophoresis, staining is carried out. By this, automation, in fact, becomes possible easily; however, only as long as blotting is abandoned. For blotting, with sufficient diameter of the tube, the gel can be taken out by means of a piston. Due to the mentioned poor mechanic characteristics of the gel, unloading is a very hazardous procedure which, even if it succeeds, delivers poor reproducible results. Total automation thus is possible neither.

For capillary electrophoresis, a very thin and long capillary (e.g. 0.1 mm.times.50 cm) is used. The dimensions allow for a very high resolution of the molecular weight, improving with decreasing cross section and increasing length. The capillary is filled with gel automatically which is removed after the procedure by means of water under high pressure. With suitable capillary geometry, the gel can be omitted. The stain is applied during loading of the analyte. The substances are detected with an UV-scanner during passing a certain position in the capillary. This variant, therefore can be carried out automatically very well with respect to the detection of the different molecular weights. By a high number of capillaries, at the same time, a high throughput can be achieved. Blotting, however, is impossible with capillary electrophoresis, because the access to the gel from the side is impeded.

With capillary electrophoresis, the very high voltage (up to 30,000 V) and the heat development and the stability of the capillary are problematic. The surface specific heat development increases with the diameter and even stronger with the length of the capillary. This is encountered by cooling with air and water.

To take account of the problematic that proteins of different composition but identical molecular weights have similar running velocities in the gel, modifications of the electrophoresis were developed, which, however, as explained in the following, are not automated due to their complicated handling:

The gradient gel. The acryl amid concentration in the poly acryl amide gels substantially influences the running velocity of the proteins. Therefore, normally the concentration is complied with as exactly as possible. The concentration, however, can be consciously varied along the running direction of a slab gel, to achieve that high and low molecular weights of the proteins will be displayed well separated on the same gel.

The Ferguson gel. Besides the molecular weights, the different shape of the proteins influences their running velocity. This can be taken advantage of by using gel matrices of different pore size. If the concentration of the gel is varied transverse to the running direction and the same analyte is applied in all paths, then a Ferguson-plot is generated. In the two-dimensional image, proteins are represented by curves of different gradients. Due to the fact that two different characteristics of the proteins are detected this method is considerably more informative as the standard electrophoresis.

The isoelectric focusing. Thereby, as further characteristic, the isoelectric point of the proteins is retrieved. A pH gradient along the gel channel is generated in the gel. The analyte is applied at an arbitrary point, preferentially in the middle of the gel channel, and voltage is applied. Because only at the position with the matching pH-value, the charge of the proteins disappears, the proteins drift until they have reached this point and remain there. After sufficient time, an arrangement of the proteins in the gel is reached corresponding to their isoelectric point, which thereby is determined.

2D electrophoresis. This is a combination of isoelectric focusing with standard electrophoresis. In a first gel, mostly a rod, the proteins are separated according to their isoelectric point. The thus separated proteins are transferred to a second gel--a slab gel--and there they are further separated electrophoretically. Thus, a two-dimensional fingerprint of the analyte is obtained. In one dimension, there is the isoelectric point and in the other direction, the molecular weight is plotted.

At present, methods are established copying a "laboratory on the chip". These methods comprise techniques for the transfer of substances within the chips, for reaction with added reagents and for detection, including also mass spectrometry in vacuum.

As premise, these methods have the automation in small dimensions, and they try to carry out all known biochemical methods on this micro scale. In a carrier, mostly a translucent glass plate of e.g. 40.times.10 mm and 1 mm thickness, at least one channel of e.g. 5-50 .mu.m is recessed. This channel is closed at the top enduringly with a cover plate lying on the lower chip, whereby a closed channel having very small dimensions is built up. The electrophoresis takes place in this channel which firstly is filled with a carrier matrix, mostly a special gel with low viscosity. The contact results from top side through fine openings in the cover plate, through which the analyte is applied also. The evaluation results likewise to the capillary electrophoresis, in that the separated proteins pass by a detector. Thereby, mostly an optical detector is concerned, accessing from top side through the planes of the translucent glass plates. The adequate stain substances are added prior to the beginning of separation. Through side channels, running into the main channel, it is possible to pass through several analytes in the same channel subsequently and to analyze them in the separation matrix. Principally, also immune reactions can be carried out in that adequate reagents are passed through from one of the side channels. Hereby, the transport is carried out by adequately applied voltages. However, thereby the point of time has to be known, at which the reagents can be supplied meaningfully, because only one reaction with the part of the main channel prior to the channel outlet takes place at any time.

For carrying out a comprehensive immune reaction, as they have been carried out in the already explained methods, it would be necessary, to bring immune reagents into contact with every part volume leaving the separation channel, and to evaluate the reaction. Such an immune reaction can comprise e.g. 5 or 10 biochemical steps, taking time respectively. Due to constructional reasons, these reactions come up sequentially, the screening of all separated components there is time consuming accordingly, in spite of the reaction velocity increased by the small size. As long as no knowledge concerning the molecular weight and therefore the running time of a protein searched for exists, therefore, only by means of many experiments, the "reactive position" searched for can be determined or in that conventional electrophoresis with blotting and immune detection is carried out first.

Therewith, the present chip constructions are no full substitute for conventional blot-techniques in spite of their high degree of automation, allowing for the transfer, and thus the immunologic detection of all separated substances simultaneously in one production step.

Accordingly, the object of the invention is to provide a device and a method for automatic analysis of the components of at least one analyte which, avoiding the drawbacks mentioned above, allow for an automatic gel electrophoresis including transfer of the separated analytes to a membrane or a further gel.

This object is solved by a device and a method, respectively, according to the independent claims. Further embodiments are defined in the dependent claims.

According to the invention, a device for automatic analysis of the components of at least one analyte, in particular by means of gel electrophoresis and/or isoelectric focusing, with at least one separation matrix, in particular a gel, is provided whereby each separation matrix 602 is supported on at least one side in a support direction S by a support element 601 such as a sample plate, is loadable with an analyte 626, and is electrically contactable at opposing ends 627, 628 in a separation direction T for separation of the analyte into its components, characterized in that the support element 315, 215, 102, 1, 601 is formed such that it allows for a mass transfer process from one side and, in fact, from an access direction Z, differing from the support direction S and the separation direction T, substantially over the entire area of the separation matrix 314, 214, 101, 2, 602, in which the components of the analytes after separation are contained, and the support element 315, 215, 102, 1, 601 has a continuous sealing face, preventing substantially this mass transfer process outside this area.

Thereby, it is achieved advantageously that the unstable separation matrix together with the separated components of the analyte can remain subsequent to the separation process within the robust support element, to be subjected to a plurality of process steps. By means of the one-sided access in access direction, it is possible to carry out mass transfer processes with each of the separation matrices separately, e.g. to stain, to de-stain, to change the buffer, to transfer the separated components of the analyte by diffusion onto a membrane. The band pattern can be read out in reflection. The pouring and loading of the separation matrices is also possible. Thereby, continuous sealing of the accesses enables automatic operating sequences.

In a preferred embodiment of the invention, the support element 315, 215, 102, 1, 601 can be formed such that it enables a mass transfer process from both sides in the access direction Z, respectively, subsequently over the entire area of the separation matrix 314, 214, 101, 2, 602, in which the components of the analyte are contained after separation, and the support element can have on both sides in the access direction (Z) respectively a continuous sealing face, preventing substantially the respective mass transfer process outside this area. By this additional access from the 2.sup.nd side, the efficiency of the mass transfer processes is essentially improved, the blotting process on a membrane can be substantially accelerated by current and pressure differences, the transfer onto a further separation matrix is enabled, and the band pattern can be read out in transmission. By this, all process steps occurring during the gel electrophoresis and the isoelectric focusing are possible at the separation matrix, and thus, can be automated while the separation matrix remains within the protecting and sealing support structure.

The invention also comprises the corresponding method for automation of the analysis of the components of at least one analyte as well as a device for transportation of substances between processing stations.

All embodiments of the invention have in common that each of the analytes to be investigated is analyzed in a single element. By combining many of such elements, it is possible to investigate as many sample substances simultaneously as desired. Subsequently, if desired, the familiar image of the parallel analysis as in a slab gel can be generated in an electronic manner, with which, also in chemical and biological respect, full consistency exists.

A further idea of the invention is that the separation matrix is very narrow in Z-direction and is free laterally over the complete length for staining, de-staining, blotting and for transfer of the separated analyte in a further separation matrix. Due to the small width of the access in Z-direction it is ensured that the support element, being adjacent from the S-direction, in fact stabilizes the mechanically instable separation matrix.

A further idea of the invention is that the optical evaluation of the stained components of the analytes can result either in S-direction and/or in Z-direction. Thus, either the optical distance within the gel can be made large, to increase the sensitivity, and the support elements can be made cheaper, because the optical elements are omitted or prior stained proteins can be observed during the separation.

A further idea is that the directions T, S and Z are respectively perpendicular with respect to each other whereby it is possible to separate the analyte within the separation matrix in T-direction, and in Z-direction to enable access for biochemical, chemical, or physical reactions or for transfer of the separated analyte on the complete length, and in S-direction, to guarantee by means of suitable support elements, the stability of the separation matrix and, if the support elements are translucent, simultaneously optical access. Thus, the removal of the mechanically instable gel from the support elements is unnecessary, and the possibility exists to completely automate all process steps including the blotting procedure over the entire gel length.

In the following the invention describes a new variant of the gel electrophoresis combining the advantages of the known processes, without having new drawbacks. By means of the device according to the invention, it is enabled to combine the entire procedure--the pouring of the gel, the applying of the sample substance, the electrical separation of the proteins, the staining and de-staining, the optical evaluation as well as the blotting of all separated substances--within one apparatus. Except for the loading of the analyte--principally this can also result automatically--no manual interferences are necessary until the result is available, in common manner electronically. In case the apparatus is used for a blotting procedure, also the insertion and removal of the membrane is ensued, which can also be automated. By small dimensions of the gel paths as well as active temperature stabilization during some or all process steps, very fast and precise results are possible. The method can be carried out in parallel working in any way whereby a very high throughput becomes possible. Nothing has to be changed in the chemical procedure of the established slab electrophoresis--the gels, the stains, the labeling, the buffers. The blotting procedure also is carried out under the conventional chemical conditions on the same membrane materials. Therefore, new measured values are compatible with conventionally determined measure values, and the existing data stock remains compatible with the use of the new apparatus. The invention therefore replaces several of the apparatuses used up to now, shortens the time until a result is available, and, therefore, increases substantially the throughput. Due to omitting human interventions, the total procedure can be easily validated, is practically not influenced by individual handling of different operators, and is reliable and suitable for constant use.

For the investigation of DNA-fragments, the staining step also results automatically. This is of particular importance because thereby usually as stain ethidium bromide is used. This substance is strongly mutagen; therefore contact with the skin has to be avoided strictly. Therefore, automation is especially important.

The direction in which the proteins are separated, depends on the embodiment and results e.g. from top to bottom (T-direction), the proteins are subsequently stained from left to right (Z-direction), again de-staining and eventually are evaluated optically from front to back (S-direction). In case blotting is carried out, this results from left to right (Z-direction). Each other initial position is of course possible; essential is the definition of the directions T, Z, S.

According to the invention, therefore the optical evaluation and also later the blotting in the same arrangement can result. After the blotting, one embodiment additionally allows for the immune detection within the blotting membrane. Thus, in the separation matrix all 3 dimensions are used, and it is possible to leave the support elements connected to the separation matrix during the whole procedure. The blotting procedure therefore is fully automated because the critical step of the stripping of the separation matrix from the support elements is omitted.

The electrophoretic currents, voltages and times which are used for optical investigation of the analyte, can be adapted unchanged for the immunologic investigation of the analyte, whereby an unambiguous assignment of the results is possible.

As detector any sensor element qualifies, responding to the characteristics which characterize the position of the different proteins:

For example, CCD-array of a CCD-camera, in case that the light source illuminates the entire length of the gel channel simultaneously, a single photo diode with appropriate collector, in case the light source scans the gel channel, a film or semiconductor array sensitive to radio activity, in case the gel is labelled radio active instead of the staining procedure, a detector for fluorescent radiation, in case the staining is carried out with a fluorescent active stain, a pH-sensitive sensor, mechanically sensing the channel.

As light source in a broad sense, all sources can be considered, the emission of which is influenced with or without a preceding pre-treatment by the proteins in reflection or transmission. These are amongst others a filament lamp with or without focusing, an array of light emitting diodes with or without focusing, a laser expanded or not, a UV-light source with or without focusing, an infrared light source with or without focusing, a radio active source.

To enable a good coupling of sensor and detector to the limiting glass plates, it can be meaningful, to flood this volume with an appropriate liquid. Thus, a liquid having a similar index of refraction as the glass plate or the gel can prevent reflections and can compensate for damages of the surfaces.

In one embodiment, the matrix channel is substantially longer in T-direction than it is wide in S-direction and deep in Z-direction, and has a rectangular cross section. It is open and accessible in Z-direction and is covered in S-direction by means of thin glass plates. In Z-direction, the thickness of the separation matrix is sufficiently small, to enable the effective diffusion of stains. In S-direction, the thickness of the separation matrix is sufficiently small, to enable the heating and cooling of the separation matrix and its temperature control through the thin glass plates. The temperature control of the separation matrix enables the acceleration of the diffusion of the stain/de-staining liquids as well as control of thermally instable substances. The gel channel is fixed to a supporting structure together with the glass plates. Such an arrangement is termed as "stick" in the following.

A stick can be manufactured at very low costs, e.g. as injection molded plastic part. The stick can be thrown away after the measurement; the gel never has to be removed therefrom. Therefore, the gel can be as instable mechanically nearly arbitrarily.

As alternative for glass plates all types of foils or plates can be considered which are translucent for the radiation used, in particular the same materials which are listed in the following embodiment for the analyzing plates. With sufficient stability of the gel, also one of the covers can be omitted.

According to the stability of the gel used, it can be useful to slightly reduce the access to the gel in Z-direction directly at the gel, to avoid that the gel is displaceable laterally. Also coatings for an increase of the adhesion of the gel channel to the glass plates are possible.

In a further embodiment, instead of a complete opening of the gel channel in Z-direction, a partially but continuous opening of the gel channel, however, still over the entire length (T-direction) is used. This can be done in that one capillary having a substantially circular cross section is provided with a high number of small openings on opposing sides. These openings can have macroscopic as well as microscopic dimensions. Through these, the staining and de-staining substances can reach the gel just as well as in the case of a complete opening. Also blotting is possible. According to this embodiment, the non-perforated sides of the capillary correspond to the S-direction. Thereby, it can be useful, to shape the capillary oval, to avoid problems with the refraction index in the optical path.

The main apparatus for handling of these sticks is provided with its own evaluation sensor mechanism and corresponding staining and de-staining means for each stick. Thereby, the stick can remain at the same place in the apparatus during the whole procedure rendering the main apparatus very simple. By combining an arbitrary number of sticks and the also relatively cheap sensor mechanism, any number of substances can be measured simultaneously, equivalent to the typical 10-20 paths of the slab electrophoresis. The individual sensor mechanism, however, is not compulsory, also a common evaluation means, to which the single stick can be transported, can be meaningful or a sensor, sampling the sticks sequentially.

In a further embodiment, a number of fixedly mounted gel channels which can be automatically filled up with gel, is standing in the main apparatus, in that the channels at first being laterally open, are sealed on both sides and subsequently are released. By pressing of a membrane, besides the separation and stain steps, blotting also is possible.

A further idea of the invention is to build the support elements in S-direction such that it is possible to carry out many different working steps by means of different parts of the main apparatus at a separation matrix in that only the support elements are displaced laterally, preferentially in S-direction. The gel channel, thus, no longer is stationary between different process steps but rather is transported to a new position for carrying out each new procedure by a very simple mechanism:

For this purpose, in one embodiment a penetration having the dimensions of the gel is cut through a glass plate. This glass plate is two-dimensionally enclosed between two further glass plates from its top and from the bottom, providing for the sealing. The two glass plates are stationary, while the middle glass plate can be moved transverse with respect to the direction of the penetration, i.e. of the gel channel. The middle glass plate with the gel channel is termed as sample plate in the following; the two external plates are termed as analysis plates.

By a displacement mechanism the sample plate can be brought into several discrete positions relative to the stationary analysis plates, which are termed stations. In each of the stations, a special procedure can be carried out with the sample plates. Typical examples for procedures are: pouring of the gel--applying of the sample substance and electrophoresis-staining, de-staining of the gel and detection of the bands--blotting-immune detection--2D electrophoresis--transfer to a further separation matrix--cleaning of the gel channel. For this purpose, corresponding devices are provided at the analysis plates at each station.

During the individual process steps, the sample plate can be moved pressure-free during the displacement between the analysis plates. In the working position, the analysis plates are pushed against the sample plate whereby the gel channel is completely sealed at the top and at the bottom, if no corresponding station in the analysis plates is accessing. The processing of the gel mainly is carried out in the stations in Z-direction transverse to the displacement direction. Preferentially, the upper analysis plate is mounted flappable, to allow for a very simple maintenance of the sample plate.

In one embodiment, pouring is carried out in station (V-A) through 2 openings in the lower analysis plate. The sample substance is applied into the station (V-B) through a small funnel which is attached onto the upper analysis plate. Together with a second funnel, in this position also the buffer is brought into contact with both gel ends and the electro analysis is carried out. In station (V-C) the staining is carried out by small chambers which communicate via slots in the two analysis plates from Z-direction with the gel. In this station also the band pattern is read out from S-direction. For this, one respective mirror is arranged in the sample plate on both sides next to the gel channel, allowing for an optical path in the direction perpendicular to the sample plate. The optical path e.g. starts at a light source below the analysis plate, runs through the gel, and is deflected in a detector, for example underneath the analysis plate. In station (V-D) it is possible to blot the proteins in Z-direction from the gel by means of current onto the membrane. For this, below the lower analysis plate, a chamber with buffer and electrode, and above the upper analysis plate, a further chamber with a removable membrane, which is pushed onto the gel is present. The latter also is mixed with buffer and contacted electrically. In station (V-E) the readily blotted membrane which previously was deposited on the sample plate, is brought into contact with immune reagents and in station (V-F), this membrane is analyzed. In a further station (V-X) which is not illustrated, the gel channel is brought into contact with a second gel, being perpendicular to the upper analysis plate. The 2.sup.nd two-dimensional gel allows for electrophoresis in a direction perpendicular to the original direction. For this, beneath the analysis plate, a chamber is arranged, containing the buffer and an electrode. The two-dimensional gel also is equipped with a buffer chamber and an electrode at its opposing edge. In position (V-R), the rinsing of the gel from the channel and its cleaning is provided for. In the sample plate, for this two further short channels are provided perpendicular thereto, independently from the gel channel. The gel which is polymerized after pouring into the inlets and outlets can thus be rinsed via adequate openings in the analysis plate.

A further plate for the supply capillaries to the funnels, a mechanism for pressing of the upper analysis plate, for pressing of the sample well plunger, for pressing of the blotting plunger, for pressing of the 2.sup.nd gel during the 2D electrophoresis as well as for the electrode supplies can be attached above the upper analysis plate.

In a further embodiment, besides the access as it results in station (V-C) and (V-E) simultaneously on the entire length of the gel channel and/or the membrane, an access results on the selected part sections, to save reagents (not shown). Therefore, the slots are necessary in the analysis plates which comprise only a part of the full channel length. In the extreme case, this can be a punctual opening.

In a further embodiment, the reading out of the band pattern from Z-direction results in that for this, a further station is provided. The optical mirrors are omitted. Therefore, in the case of transmission measurements through the separated and stained proteins in the gel, the optical absorption is increased against the detection from S-direction, because the optical path in the gel is increased. With diffuse radiation from the band pattern, the steradian effective for the detector is increased, because the distance to the lens can be decreased. Thereby, smaller amounts of proteins can be detected. These advantages also persist for an application of non-optical detector methods. Further, the distance between the gel channels can be reduced, and their number can be increased. The optical path can be widened adequately by means of lenses, to guarantee a better utilization of a CCD-array. For the manufacturing of this sample plate, no high quality optical components and phases are necessary. The sample plate can be manufactured at extreme low costs, for example as a slotted plastic injection molded part.

In a further embodiment, the reading out of the band pattern from a direction lying between the S-direction and the Z-direction is carried out e.g. at 30 degrees, seen from the S-direction. The optical mirrors are omitted. The refraction index of the sample plate is selected adequately, to hit the channel under a preferably advantageous angle.

In a further embodiment, the transport of the membrane results by means of an additional membrane plate instead of the sample plate. The sample plate then comprises a number of channels; preferentially one for each separation matrix and the membrane plate comprises a number of grooves, preferentially one respectively, for the deposit and transport of the corresponding membranes after blotting.

In a further embodiment, the membranes assigned to the channels are not separated but rather form a large continuous membrane. In this case blotting results simultaneously from all channels on separated paths of this membrane. The membrane plate for transport of the membrane comprises a groove for receiving the whole membrane.

In a further embodiment, the continuous membrane is situated on a movable plunger underneath the blotting station (VI-D). In this station, in which at first blotting was carried out from the sample plate, after displacing the sample plate, all steps of liquid supply to the membrane can be carried out (also multiply), in particular buffer exchange, loading with immune reagents, rinsing, washing, cleaning, carrying out reaction. The detection of the resulting reaction is carried out by means of a detector plate which is slid laterally over the membrane, preferentially from the opposing side as the sample plate.

In a further embodiment, in this station (VI-D) additionally the reading out of the membrane is enabled by means of a laterally inclined arrangement of the detector components or liquid supplies.

In a further embodiment, at least one penetration is accommodated transverse to the direction of the separation channels in the sample plate. In this transverse channel, the analyte can firstly be separated by means of e.g. isoelectric focusing and the separated proteins can be transferred in a further station by means of connecting this transverse penetration with the separation channels. The type of separation is similar to the one of 2D electrophoresis.

In a further embodiment, the channels of the sample plates are formed at the edges such that the cross section of the penetration close to the openings is slightly reduced in Z-direction, to increase the adhesion of the separation matrix in the channel.

In a further embodiment, staining and de-staining is respectively carried out in separated chambers.

In further embodiments, further stations are conceivable for additional procedures: drying of the gel alternative detection methods, e.g. radioactive detectors, UV-sources and detectors, laser or scanner, the wave lengths of which are not compatible with the common glass types chemical biological reactions prior to evaluation and prior to blotting, respectively.

It should be emphasized that the specifications for the directions are only to be understood exemplary and for an easier understanding. The actual orientation of the analysis and sample plates as well as additional means and also the order of the stations are arbitrary.

The term glass plate only is meant as an example. As material for the sample plates, all materials are considered which have a sufficient rigidity and flatness and are resistant against the chemicals used. The optical translucence is not necessary, if the optical detection is omitted during the other process steps. To be considered are, amongst others, glass, ceramics, sapphire, silicon, poly acryl amide, polystyrene, polyester, polyimide, polyurethane, polycarbonate, polyurethane, polyamide, polyethylene imines, polyarylen sulfide, polysiloxane, polyacetat, polysulfide and other plastics.

The material for the analysis plates additionally has to have high abrasion resistance and good thermal conduction. The above listed materials do qualify here also, but also locally optimized materials as e.g. glued plates for better heat conduction can be considered. The analysis plates typically have a thickness of 0.5-3 mm, which are enforced on the side facing away from the sample plate by sandwich-like additional plates, at least in the areas in which no peripheral means are attached.

In the following, the invention is described by means of further embodiments which are explained in detail with the drawings.

FIG. 1 The first embodiment I is a device according to the invention in side view. A separation chamber is integrated into a support structure.

FIG. 2 The device according to FIG. 1 in top view.

FIG. 3 A further embodiment II and III, respectively, of a device according to the invention in top view.

FIG. 4 The device according to FIG. 3 in side view.

FIG. 5 The blotting plunger according to FIG. 3 in top view.

FIG. 6 The device according to FIG. 3 with the blotting plunger being inserted according to FIG. 5 in top view.

FIG. 7 A further embodiment IV of the device according to the invention in top view.

FIG. 8 The device according to FIG. 7 in side view.

FIG. 9 The device according to FIG. 7 in front view.

FIG. 10 A further embodiment V of a device according to the invention, constituted of a sample plate in top view.

FIG. 11 The sample plate according to FIG. 10 between the associated analysis plates with associated stations.

FIG. 12 The sample plate according to FIG. 10 in top view.

FIG. 13 The upper analysis plate according to FIG. 11 in top view.

FIG. 14 The sample plate according to FIG. 10 in spatial illustration.

FIG. 15 The analysis plates according to FIG. 11 in spatial illustration with membrane plunger.

FIGS. 16a-e Detailed drawings of the analysis plates according to FIG. 11.

FIG. 17 A further embodiment VI of a device according to the invention, constituted of a sample plate in spatial illustration.

FIG. 18 The sample plate with pouring plates according to FIG. 17 in side view.

FIG. 19 The upper analysis plate according to FIG. 17 in top view.

FIG. 20 The sample plate with the analysis plates in separated position according to FIG. 17 in side view.

FIG. 21 The sample plate with the analysis plates in staining position according to FIG. 17 in side view.

FIG. 22 The sample plate and a membrane plate with the analysis plates in blotting position according to FIG. 17 in side view.

FIG. 23 The membrane plate with the analysis plates in reaction position according to FIG. 17 in side view.

FIG. 24 The membrane plate with the analysis plates in washing position according to FIG. 17 in side view.

FIG. 25 The membrane plate with the analysis plates in detection position according to FIG. 17 in side view.

Single Element Embodiment I

A gel channel 314 (in FIG. 1 in top view and in FIG. 2 in side view), having a rectangular cross section of typically 1.times.0.5 mm and a length of typically 100 mm, is enclosed from two opposing sides of 2 plates 315 being just as wide. The plates, preferably from glass or similar material, have a typical thickness of 0.05-0.5 mm and are optically completely translucent.

This gel channel is integrated with the glass plates into a plastic carrier, to close the different inlets to the gel channel against each other and to stabilize the system mechanically. The carrier has in profile (FIG. 2) a shape similar to a butterfly with opened wings. The wings are 311, 312, 316, 317. Cavities 330 and 331 are defined by these wings, serving for receiving the staining and de-staining agents. The cavities have direct contact to the gel channel.

The cavities 330 and 331 in operation are closed by 2 plates 321 and 322 fluid tight, which, however, are not part of the carrier but rather of the rest of the apparatus. They are pressed via rubber rings against the wings of the carrier. The carrier is closed at its end by 2 plates 310, 313 and is sealed. Also the plates 321, 322 of the main apparatus seal against these plates 310, 313 of the carrier. By this, said cavities 330, 331, leading to the gel 314, are completely closed. Via connectors 327, 328, being embedded into the wall 321, 322 respectively from top side and from bottom, it is possible to pass liquid into the cavities and to drain them again. The bottom 313 is inclined outwards for this purpose to facilitate a complete draining.

From top side, the gel channel is contacted by means of the reservoir 332. In its inside during operation, the electrolysis buffer for the source side is contained. The container 332 is part of the carrier, and has a base running conically to the centre. The opening in the base communicates with the gel channel and serves for loading of the gel, also for adding of the measuring substance. Subsequently, the beaker is filled with buffer liquid. Thereby, it can be beneficial, to obviate by means of a possibly perforated plate (not shown), which prevents the direct access to the applied measurement substance, the rinsing of the latter during filling of the buffer. Also, a further narrowing of the cross section of the inlet can be useful. Alternatively, the loading procedure can result wet i.e., at first the buffer is filled and in a 2.sup.nd step e.g. by means of a pipette, the measuring substance is brought into a dent.

From below, the gel channel is contacted by means of the chamber 333. Thereby, a closed vessel 323 is concerned, belonging to the main apparatus, and being connected with the base plate 313 of the carrier via a rubber ring. It also contains electrolysis buffer. During attaching of the carrier, the reservoir at first is not filled. During the subsequent filling, at first, due to gravity, an air bubble is built up in the little tube 329, which would prevent wetting of the gel. By means of the capillary 324, this air bubble is sucked off at the highest position or can escape, respectively. Possibly it can be beneficial to embed a lateral dent into the base plate 313, which lies higher than the gel channel, but is not filled, and from which air can be sucked in an optimal manner. In the two reservoirs 332 and 333, respectively, the electrode is accommodated, serving for power supply during the electrolysis.

In FIG. 1, two further inlets 334, 335 to the gel channel 314 are visible. The gel 314, here, is covered with two glass plates 315.

From both sides 334, 335, the optical evaluation is carried out. This can result in transmission, then on one side 334 a light source is present, and on the other side 335 a detector, or in reflection, then detector and light source are on the same side, e.g. 335. As detector, an arbitrary sensor element, responding to the characteristics, can be considered which indicate the position of the different proteins.

Single Element Embodiment II

In FIG. 3 and in FIG. 4, an alternative embodiment of the main apparatus is illustrated. The plates 321, 322 of the main apparatus here are replaced by the vats 225, 226. These vats also close the cavities 230 and 231 of the carrier. The advantage lies within the very simple and save method for providing the sealing. The vats are respectively closed by a rubber membrane which can be retracted into the vat by negative pressure, and can be pressed against the carrier by overpressure uniformly. A pump, if necessary, also two pumps for overpressure and negative pressure, are sufficient, to close the cavities simultaneously at all carriers in the main apparatus.

The respectively double arranged capillaries 227 and 228 communicate with the cavities 230 and 231, and allow for the supply of liquids at the top into the cavities, and their discharge at the bottom.

The vats 225, 226 can be cooled and heated (not shown) from the side opposing the carrier. For better heat coupling to the liquid in the cavities 230, 231, it is possible to respectively provide the liquid inlets 227 and 228 with a large surface, and to let them protrude (not shown) into the cavities.

Single Element Embodiment III

In case blotting shall be carried out with a carrier subsequent to electrophoresis, thus a plunger 240 (see FIG. 5) is necessary. The plunger has the same vertical extension as the carrier, and fits precisely into the chamber 230 of the carrier (FIG. 6). At the tip, it carries as active element a blot membrane 241, which approximately is as wide as the gel channel 214. The gel channel also is covered on one side over the entire length and width by the membrane. At the beginning of the electrolysis procedure, the plunger is loosely placed in the chamber 230, without the membrane contacting the gel channel. As the vertical electrolysis procedure is completed, the membrane 241 of the plunger is pressed against the gel channel 214 by inflating of the rubber membrane of the vat 225. Buffer liquid can be supplied via several channels in the plunger from the capillary 227 into the vertical channel 242 of the plunger. This liquid Vets the membrane 241 from backwards. Simultaneously, buffer liquid is introduced into the chamber 231.

By this, the gel channel can be wetted on both sides by buffer on the long side 214 at fitting membrane 241. In both vets, electrodes are accommodated (not shown), dipping into the buffer and by means of which the current for blotting can be applied.

The main apparatus according to embodiment III, the main apparatus I, II are substantially equal.

In the main apparatus, respectively, two pumps are accommodated, to generate overpressure and negative pressure in the vats 225, 226. Thereby, the cavities 230 and 231 are sealed, or the plunger is pressed in blotting mode.

A pump with reservoir is present, to fill the tank 233 simultaneously at all carriers. A valve enables the closure of the capillary 224.

A pump with reservoir enables the filling of the container 232.

For each staining and de-staining and developer solution, which can be filled into one of the two cavities 230 and 231, a pump with reservoir is present (all carriers commonly).

A central effluent container is provided.

A central computer controls all procedures, it is connected to a calculator via a network interface