Abstract:
The invention relates to a device for carrying out an almost simultaneous synthesis of a plurality of samples. The device is especially suitable for use in automated laboratory processes in the area of combinatorial chemistry. The aim of the invention is to provide a device of this type which enables the synthesis of a plurality of samples bonded to microbeads, said microbeads being provided in the cavities of a support plate. To this end, a plane support plate ( 1 ) is provided with a plurality of cavities ( 11 ) arranged regularly in an iterative grid. The cavities accommodate microbeads ( 12 ). A removable covering ( 2 ) is provided, said covering being provided with webs ( 21 ) which each cover at least one of a row of associated cavities ( 11 ) in such a way that a capillary gap ( 3 ) is formed between the microbeads ( 12 ) and the webs ( 21 ) and larger recesses ( 22 ) are left respectively between the adjacent webs ( 21 ). A dosed liquid dispenser ( 4 ) is allocated to each capillary gap ( 3 ).

Description:
BACKGROUND OF THE INVENTION 
     The invention relates to a device for a substantially simultaneous synthesis of a plurality of samples that are particularly for use in the automated laboratory work in the field of the combinatorial chemistry. 
     Sample particles, (“beads” or “Perlen”), have been used in separations and synthesis in the laboratory technical field for tenth of years. These particles mostly are glass or polymeric globules that have diameters of 0.01 mm up to 1 mm, typically about 0.1 mm, which are filled, dry or pre-swelled, as a loose material into a receptacle where they are then flushed by a liquid, whereby an adsorption process or a reaction process takes place between the solid phase surface of the particles and the liquid surrounding the particles. Methods of the column chromatography (for example, gel filtration), of the column extraction, of the immundiagnosis, of the bio-molecule purification (for example, DNA cleaning), as well as of the homogeneous and heterogeneous synthesis (of oligonucleotides, peptides or combinatorial substance libraries) utilize these techniques. In addition to the automation and miniaturizing of laboratory techniques, the parallelizing of the same is of great interest in obtaining a higher sample throughput and, hence, to speed up otherwise time-consuming procedures. To this end, samples are very often arranged in a raster so that the identity (origin, quality) of the sample can be associated to an area coordinate. Such coordinates are very easily to be detected in particular in automated systems of sample handling. 
     Therefore, so-called micro-titer plates have been developed for liquid samples, which support cavities in right-angular arrangements of 8·12 (96 samples) 16-24 (384) or 32·48 (1536). Thereby, the dimensions of the cavities of these sample holders depend on such volumes that can be reliably dosed by the commercially available devices (pipettes), and follow a miniaturizing continuously progressing with the dosing technology, what is simplified by the capability of an ali-quote (distribution of a mother-sample into different daughter-samples) of liquids at will. 
     Within the frame of work for miniaturizing laboratory procedures there is searched for possibilities to distribute sample particles, in analogy to the arrangement of liquid samples, in a two-dimensional raster. Since the miniaturizing of dosing liquids has already advanced very far, so the single particle becomes the smallest unit. Furthermore, there is the demand to handle high quantities, as it is common use when working with particles. 1-g polymer resin contains about 1 million particles. 
     There are different solutions known for filling micro-titer plates or reaction vessels. 
     So WO 98/24543 A1 describes a device for transferring liquids in which, inter alia, a micro-titer plate is provided, the chambers of which have at least one opening in their bottom area that is dimensioned in a way that, in the course of a filling operation, the passage of the liquid through this opening due to capillary forces is avoided. In WO 98/06490 A1 a device for an organic solid phase synthesis is described, in which the reaction vessel is arranged above a collective vessel for receiving liquids in such a way that the transfer of the liquid can be attained by generating a low pressure, whereas the liquid is held in the reaction vessel by a slight overpressure. From WO 97/19749 A1 there is a device for addressable combinatorial substance libraries known, in which one substance each is represented in a capillary tube, whereby the filling with liquids is obtained by capillary forces. In WO 97/37755 A1 a plate is described that contains a plurality of reaction cells being arranged in rows and columns, which are supplied with liquids by pumps. Furthermore, the specifications WO 97/43629 A1 and WO 98/16315 A1 describe distribution systems for liquids, which are comprised of a plurality of plates, whereby the liquid flow is operated and controlled by a capillary barrier and by electro-kinetic pumps, respectively. 
     The miniaturizing of the support plates mentioned goes along with the miniaturizing of the corresponding filling technologies and meets its critical geometric or time limits when conventional automated pipetting devices are used, since each single sample particle has to be supplied with liquids. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a device for a substantially simultaneous synthesis of a plurality of samples, which are bound to micro-beads provided in the cavities of a support plate. 
     The object is realized by the features of the first patent claim. Advantageous embodiments are covered by the dependent claims. 
    
    
     
       DETAILED DESCRIPTION OF THE INVENTION 
       In the following, the invention will be explained in more detail by virtue of schematical embodiments. There is shown in: 
         FIG. 1  a perspective view of a principle setup of an inventional device as well as a representation of an enlarges detail; 
         FIG. 2   a  a lateral view of a device according to  FIG. 1 ; 
         FIG. 2   b  a plan view of a device according to  FIG. 1 ; 
         FIG. 3  a plan view of a device according to  FIG. 1  with the position of an inventional cover in two synthesis steps; and 
         FIG. 4  a plan view of an embodiment of a cover with a plurality of operational sections. 
     
    
    
     Without limiting the invention thereto, it will be started in  FIG. 1  from a support plate  1 , in which micro-beads  12  are provided in each of the single cavities  11  in such a way that the micro-beads project from out of these cavities. When micro-beads of a diameter of 100 μm are used, these micro-beads in the sorted-in state will project from out of the surface of the support plate  1  by of from 20 to 50 μm. In the present example, each nine of such micro-beads belong to one sample receiving area, whereby all the sample receiving areas are aligned to one another in rows and columns (refer to FIG.  3 ). A detachable cover  2  is provided above the bead-filled support plate  1 , the cover  2  supports barriers  21  as most substantial elements. These barriers are so designed in their width and length that they capture all sample-receiving ranges of one row or column when attached to the support plate  1 . In this manner, due to the definedly preselectable projecting of the micro-beads  12 , a capillary gap  3  of preselectable height and of a defined width is produced, the latter by preselection of the width of the barriers  21 . In a further arrangement of the micro-beads, for example, a plurality of set back micro-beads in a common cavity, such a capillary gap can be also formed in that the support plate  1  is provided with spacers of a defined height in the range, where the barriers  21  are supported, and/or the barriers  21  themselves are provided with spacers of a defined height. 
     In order to determine the sides of the capillary gaps  3 , which are formed in this manner, the barriers  21  are spaced from one another by larger recesses  22 , which are dimensioned in such a way that capillary forces will not be active any longer in these recesses. 
     The height of the capillary gap  3 , at which the capillary forces will still be active in transporting liquids, depends on the surface tension of the material for the support plate  1  and for the barriers  21 , and on the liquids that are to be handled. When materials such as glass and metal are used, between which a gap is formed, and water is used as a liquid, the capillary forces will be active up to gap heights of 500 μm. When using water, a specific hydrophilizing of the glass and/or of the metal surface permits the flowing of a liquid due to capillary forces also at greater distances. Depending on the synthesis fluid that is to be handled, the barriers  21  in the range of their supporting area on the support plate can be provided with a hydrophilic or hydrophobic surface, whereby at least the side walls of the recesses  22 , which limit the barriers, should be provided with a respective oppositely acting surface covering. 
     The capillary gap, formed in the example by the projecting-out micro-beads  12 , opens up the possibility to have liquids intentionally flow along the gap. Thereby a structurized cover plate is used for the cover  2  in the example, whereby this cover plate is provided with parallel recesses  22 . By these recesses  22  a separation of two capillary gaps running in parallel to one another is obtained. 
     The capillary gaps will be filled in that a liquid is pipetted at the leading face of the cover, into a respective start of the gap, as is schematically indicated by way of a gap and by means of a dosable fluid dispenser  4  in FIG.  1 . 
     The filling of each capillary gap with a different liquid requires a very careful pipetting of the liquids in order to prevent the mixing of two liquids. In order to ensure a simultaneous and equally dosed filling of all capillary gaps  3 , it is more advantageous to provide the liquid supply via bores  13  in the support plate  1  or via bores  25  in the cover  2 , which are respectively pre-positioned to a row and column of the cavities arranged in a line. A connection is provided via hose-like connections  5  or fitting pieces connected to these bores, to a liquid supply means, for example, a jet-pump, not shown in more detail in the following. This liquid supply means is associated to a respective row or column, whereby it is advantageous to apply a common and equally defined pressure to all liquid supplies. Such connections are adapted to feed now well-dosed liquids into the capillary gaps. It is, however, necessary to very precisely adapt the pumping rate of the jet-pump to the speed of flow of the liquid, produced by the capillary forces. This has to be detected by experiment for the individual case, in order to avoid a crossover of the liquid from a capillary gap to an adjacent one. 
     As already hinted at, it is also possible to realize the filling of the capillary gaps via the cover plate  2 . Then the bores for the connections of the hoses have to be produced in the cover plate, centrally to the barriers  21 . Such an embodiment also permits to use an adapter for a micro-titer plate instead of the hose connection, and to realize the filling of the capillary gaps by exploiting hydrostatic differences in pressure. One advantage in using jet-pumps connected to hose connections is the compactness of the system so that evaporations are avoided. Such a closed system is also advantageous in those cases where chemicals are used which must not get into contact with air. 
     The described modifications permit the filling of any desired number of lines with liquids. Presently, in adaptation to the micro-titer plates available, the described device permits the simultaneous filling of 96 lines. There are, however, no limits as to the number of lines, and with an increasing use of micro-technical machining processes there can be realized far more than 100 lines. 
       FIG. 2   a  shows an embodiment of the device according to  FIG. 1  in a lateral front view, thereby the support plate  1  is provided with bores  13 , only five of which are represented in  FIG. 2   a . Hose-like connections  5  are connected to these bores  13 , said connections lead to not shown liquid supply means  4 . The support plate  1 , in turn, is mounted on a displacement table V, which enables a lateral displacement in parallel to the normal of the sheet. Furthermore, the support plate  1  and the cover  2  are connected to each other via a guiding means  6 . Such a design permits to incorporate a further structurized cover  2 , as it is shown in plan view in  FIG. 2   b  in more detail. In addition to the transparent cover  2 , described hereinbefore, including the barriers  21  provided to the same, the cover  2  further comprises a porous portion  23 , which is followed by a plane section  24 . The extensions of this plane section  24  are dimensioned in such a way that the plane section is capable of covering the entire support plate  1  under formation of a capillary gap capturing all the sample areas, provided that the covering range has been moved over the support plate  1 . A complete design of only such a coverage alone is shown in  FIG. 4 , in which the bores  25 , as an alternative, are there allocated to the first part of the cover  2 . 
     Under use of the device described, the actual synthesis of the desired samples is performed as follows: The support plate  1 , having a size of 250·250 mm2, which is filled with micro-beads  12  that, at a suitable porosity, can take, for example, a sample liquid volume of 0.25 nl, is brought into contact with the cover  2  containing the recesses  22 . The support plate  1  and the cover  2  are aligned to each other by means of an adjustment device, not shown in detail, so that one projecting barrier  21  each rests upon a row of reaction chambers filled with beads. The barriers  21 : connect, for example, one row with 96 sample fields that comprise 864 micro-beads  12 . Moreover, the support plate in the example has two-times 96 through-bores  13  that lie in the extension of the rows and columns of the arrays of beads. The above-described hoses  5  are secured to these bores on the rear side of the support plate. The 192 hoses lead to the liquid supply means, for example syringes, which is, respectively, are filled with chemicals. A pressure is simultaneously applied to the syringes by a syringe drive, and the liquids are transported to the support plate  1  via the hoses  5 . The capillary gaps  3  are filled with one chemical each. This is achieved in that the end of each barrier is placed accurately above one bore, from out of which the liquid is ejected for entering into the capillary gap. The pumping rate, which is controlled by the syringe drive, has to be correlated to the speed of flow of the liquids, driven by the capillary forces. A filling operation will take about 3 min., when a capillary gap of a height of about 30 μm, a width of about 1000 μm and a length of 250 mm is used. 
     The support plate  1  is mounted upon the displacement table V, which permits movements in parallel to the barriers  21 , whereas the cover in the example should be safely arrested during the entire synthesis. Following the first synthesis step, the support plate I will be displaced underneath the cover  2  and along the barriers  21 , in the course of which the micro-beads are conducted past by the porous portion  23  of the cover  2 . Thereby the size of the pores in the porous portion has to be significantly smaller than the diameter of the micro-beads. The porous region absorbs the synthesis chemicals and these chemicals are drained from there by means of a device operating with low pressure and which is not represented in more detail; the micro-beads  12  are dried in this way. The support plate  1  is completely moved past below the porous area, until the entire support plate has arrived at the back end portion of the cover that is given a plane surface. In this section  24  the support plate  1  is subject to a rotation of 90°, which is necessary for performing the second synthesis step. Also in this case the support plate  1  is moved by means of a rotary table, while the cover remains safely clamped. After the support plate  1  has been rotated, the micro-synthesis beads are subject to a flushing. The flushing-out solution will also be transported by capillary forces to the beads. Again a drying of the beads is carried out by wiping over the porous portion  23  of the cover as described hereinabove. Then the cover  2  is again in a position for synthesis. The second synthesis step now proceeds in a same manner, the support plate  1 , however, lies under the cover plate rotated by 90° and a further coupling step follows. There are further coupling steps possible, as many as desired. Thus, after the two-stage synthesis described, and starting from 96 rows and 96 columns, 9216 different combinations of twice 96 substances result. Further synthesis steps, after a respective rotation of the plates relative to each other, are feasible in any desired number. Two of the synthesis positions described are exemplified in  FIG. 3  by example of sixteen rows (1 to 16) and sixteen columns (A to Q). 
     The use of borofloat glass for the cover  2  has proven as being particularly advantageous. In order to be able to visually supervise the liquid stream, which forms in the capillary channels mentioned, a transparent material should always be selected for the cover  2 . Additionally, glass is distinguished by a high degree of flatness, which is is an important criterion for realizing a capillary gap of uniform thickness extending over a length of 250 mm. By use of diamnond cutting tools, 97 recesses  22  of a distance of 2.25 mm have been worked into a first range of the cover, under the condition that there are 96 rows and columns, respectively. The depth and width of a recess is so dimensioned that the recess  22  itself does not act like a capillary any more. To this end a width of 1000 μm and a depth of 1500 μm are selected in the embodiment described, thus barriers  21  of a width of 1.25 mm remain between two adjacent recesses. It lies within the scope of the invention to use other materials for the cover. 
     The design of the cover  2  described hereinabove is the most advantageous one, as concerns handling and stability of the device. It, however, also lies within the scope of the invention to realize the provided barriers by use of an arrangement of parallel stripes. To this end single stripes of glass are used correspondingly dimensioned in length and width. The height of the stripes can be selected as desired, and only depends on the stability requested from the device. The single stripes will be arranged parallel to each other at the distance of the sample receiving ranges, and they will be fixed relative to each other by sticking their ends onto a supporting stripe or to a support plate. 
     List of Reference Numerals 
     
         
           1 —support plate 
           11 —cavities 
           12 —micro-beads 
           13 , 25 —bores 
           2 —cover 
           21 —barriers 
           22 —recesses (between the barriers  21 ) 
           23 —porous portion 
           24 —plane section 
           3 —capillary gap 
           4 —liquid supply means 
           5 —connections (hoses) 
           6 —guiding means