Patent Publication Number: US-6902680-B2

Title: Device for selectively filtering under reduced pressure and for vacuum drying sample liquids or drops of sample liquids as well as use of said device

Description:
This application is a divisional of co-pending application Ser. No. 09/424,537, filed on Nov. 24, 1999, now U.S. Pat. No. 6,315,902 and for which priority is claimed under 35 U.S.C. § 120. application Ser. No. 09/424,537 is the national phase of PCT International Application No. PCT/EP98/03130 filed on May 27, 1998 under 35 U.S.C. § 371. The entire contents of each of the above identified applications are hereby incorporated by reference. This application also claims priority of Application No. 197 22 02 1.5 filed in Germany on May 27, 1997 under 35 U.S.C. § 119. 
    
    
     The invention relates to a device for selectively filtering liquid samples in compartments comprising inlet and outlet openings of a carrier body under reduced pressure and vacuum drying drops present in the area of the outlet openings of the compartments. Furthermore, the invention relates to the uses of the device in the purifying of biopolymers, especially nucleic acids. 
     In molecular biology, in the analysis of samples, carrier bodies called microtitration plates are used comprising a plurality of compartments arranged two-dimensionally in lines and rows. These compartments are provided with inlet openings towards the upper side of the carrier body and comprise outlet openings on the lower side of the carrier body. In the compartments, there are filter bodies in the shape of frits or membranes provided to bond the substances of the liquid samples to be analyzed. 
     The sample liquids to be analyzed are introduced into the individual compartments of a carrier body (microtitration plates), if required after being processed. By applying a low pressure to the outlet openings of the compartments, it is provided that the sample liquids flow through the filter bodies of the compartments. In this process, the components to be analyzed and other components of the sample liquid get caught in the filter bodies. In order to obtain the components of interest of the liquid sample, the components which are not required have to be washed out of the filter bodies. For this purpose, wash buffers of various concentrations are pipetted into the compartments. By means of these wash buffers, a part of the components not of interest of the sample liquids is released form the filter bodies. By means of an elution buffer, the component of interest of the sample liquids is then washed out of the filter bodies. The pipetting and the flowing process of the wash buffers, the elution buffer and the sample liquids occur in a pipetting machine, which is computer controlled. These pipetting machines are provided with a vacuum chamber comprising an opening whereon the carrier bodies are substantially gastightly fitted to draw out the sample liquids or the wash buffers through the filter body. A device of the type described above is described in DE-A-41 07 262. In the filtration under reduced pressure, it always occurs that remaining liquids stay on the outlet openings of the compartments in the form of drops. Before the step of elution takes place, it has to be provided that these liquid drops have been removed. For the quantities to be analyzed occasionally are rather small so that a mixture with remaining liquid can lead to faulty results. What is more, components of the sample liquid which are not to be analyzed are present in the liquid drops, thus also adulterating the result of the analysis. In prior art, the carrier bodies, respectively microtitration plates, are dropped several times on a support having an absorbent material so that the liquid drops are virtually shaken off. However, this process cannot be integrated in the case of an automatic process, as in the case of a pipetting machine. 
     The invention is based on the objective to provide a device by means of which remaining liquid drops on the outlet opening of the compartments of the carrier bodies can be removed automatically and without manual interaction after a filtration process under reduced pressure has taken place. 
     To achieve this objective, the invention proposes a device for selectively filtering liquid samples in compartments comprising inlet and outlet openings of a carrier body under reduced pressure and drying drops of the sample liquid present in the area of the outlet openings of the compartments, the device comprising:
         a chamber comprising an interior space limited by a top wall, a bottom wall and lateral walls connecting them,   the top wall comprising an opening with an opening edge whereon a closing lid or the carrier body can selectively be put in a substantially gastight manner by means of outlet openings directed towards the interior space of the chamber,   one of the lateral walls being formed as an access lateral wall which can be opened and closed substantially gastightly for introducing the carrier body into the interior space of the chamber, and   the bottom wall comprising a drain for liquid exiting from the outlet openings of the compartments of the carrier body, and   a vacuum pump with a suction conduit terminating in the interior space of the chamber for producing a first low pressure for drawing liquid samples through the outlet openings of the compartments with the carrier body being placed on the opening edge of the top wall and the access lateral wall of the chamber being closed and for producing a second low pressure higher than the first low pressure for drying drops of liquid samples still present on the outlet openings of the compartments with the carrier body being located in the interior space of the chamber and the lid being placed on the opening edge of the top wall as well as the access lateral wall of the chamber being closed.       

     The device according to the invention is provided with a low pressure chamber comprising a top wall, a bottom wall and lateral walls connecting them. One of the lateral walls is formed as an access lateral wall and can be opened and closed so that it is possible to introduce a compartment carrier body (subsequently called microtitration plate) via this lateral wall into the interior space of the chamber. Furthermore, the chamber comprises an opening in the top wall thereof being closable by means of a lid. As an alternative to the lid, a microtitration plate can also be placed onto the opening so that the chamber can then be used for filtering the sample liquid of the compartments under reduced pressure. The chamber comprises a drain in the bottom wall so that the flowing liquids can be drained. 
     The low pressure in the interior space of the chamber is produced by a vacuum pump connected to the interior space of the chamber by means of a suction conduit. By means of this vacuum pump, a first and a second low pressure can be produced alternatively in the interior space of the chamber. The first low pressure is required to draw in the sample liquid through the outlet opening of the compartments of the microtitration plate. Thus, the first low pressure is used for filtering under reduced pressure. The second low pressure higher than the first low pressure serves to dry sample liquid drops which might be adhering to the outlet openings of the microtitration plate at a vacuum. In the case of vacuum drying, the opening in the top wall of the chamber is closed by means of a lid, while the microtitration plate itself is located in the interior of the chamber. By lowering the pressure in the chamber, the evaporation point of the liquid drops is reduced, whereby they can be removed by evaporation. 
     By means of the device according to the invention, it is possible to use one and the same low pressure chamber both for filtering and for removing sample liquid drops. The manipulations to be executed on the chamber for this purpose, which are described above, can be put into practice without a problem by means of the gripping device of a pipetting machine so that manual interference is no longer necessary. 
     In an advantageous development of the invention, it is provided that the chamber comprises a support mounting for receiving the microtitration plate for putting it in the interior space of the chamber. Preferably, this support mounting is located on the interior side of the access lateral wall of the chamber directed towards the interior space, which wall is formed to be movable from the other walls of the chamber or towards them for this purpose. In this context, it is advantageous if the access lateral wall is provided with at least one cam element connected to the access lateral wall. The cam element can be moved by means of a driving device, whereby the access lateral wall is also moved. In this context, the cam element and the access lateral wall work in the manner of an automatic drawer which can be opened so far that the microtitration plate can be inserted from the top into the reception mounting by means of a gripping arm and can be taken out of the support mounting from the top, respectively. 
     In an advantageous development of the invention, it is further provided that the chamber comprises at least one heating element for heating the interior space. By means of this heating element, an appropriate temperature level can be set in the interior space of the chamber. The interior space of the chamber can also be heated during filtering under reduced pressure. In any case, heating the interior space is advantageous in the case of vacuum drying, as the conditions in the chamber can then be set more quickly such that liquid drops which might still be present are evaporated. 
     In order to simplify placing the lid or the microtitration plate on the opening edge of the top wall, it is suitable if the opening edge is provided with introduction surfaces extending angularly to the outside for centering the lid or the microtitration plate when it is put on. This has the advantage that the opening of the top wall can be closed automatically either by means of the lid or the microtitration plate, even if the positioning of the gripping arm is less accurate. 
     In order to be able to seal the interior space of the chamber against the environment without having moved the microtitration plate into the interior space of the chamber before and having closed the opening in the top wall by means of the lid, it is advantageous to insert a sealing body or a sealing mat consisting of a substantially gastight material and being placed on top of the inlet opening of the compartments of the microtitration plate, if it is located in the opening of the top wall. After a filtration under reduced pressure has taken place, the microtitration plate can remain in the opening of the top wall to for vacuum drying liquid drops which might be adhering to the outlet openings. 
     The device according to the invention can especially be employed for isolating, separating and/or purifying of biopolymers, as is described in EP-A-0268946; this document is incorporated by reference in the disclosure of the application. 
    
    
     
       An exemplary embodiment of the invention will now be described with reference to the figures. 
         FIG. 1  shows a lateral view of a pipetting machine with a gripping arm and a low pressure chamber, 
         FIG. 2  shows a longitudinal section through the low pressure chamber in the closed state and with the carrier body being placed on top, 
         FIG. 3  shows a section along line III—III of  FIG. 2 , 
         FIG. 4  shows a longitudinal section through the low pressure chamber in the opened state to show the introduction of the carrier body into the low pressure chamber, and 
         FIG. 5  shows a longitudinal section through the low pressure chamber in the closed state with the opening in the top wall being closed and the carrier body being located in the interior of the low pressure chamber for vacuum drying. 
     
    
    
       FIG. 1  shows, strongly schematized, a pipetting robot  10  comprising a movable bracket  12 . The bracket  12  is movable along the rail  14  and comprises one or multiple pipetting needles  16  as well as a gripping device  18  with two gripping arms  20 . The pipetting needles  16  and the gripping device  18  are movable along the bracket  12 , thus being able to move in the X and Y directions relative to the table  22  of the machine  10 . 
     Various utensils and objects required for the pipetting process are located on the table  22 , which are not elaborated on for reasons of simplicity. Among other things, a low pressure chamber  24  is located on the table  22  which can be employed both for filtering under reduced pressure and for vacuum drying remaining liquid which might still be adhering to the carrier bodies  26  after a filtering process. 
     The construction of the low pressure chamber  24  and the carrier body  26  will subsequently be described with reference to  FIGS. 1  to  5 . As can be seen in  FIG. 2 , the carrier body  26  comprises a plurality of tubular compartments  28  each comprising an inlet opening  30  and an outlet opening  32 . Filter bodies  34  in the form of porous matrix materials, frits or membranes are located in the compartments  28 . The carrier body  26  comprises a rim  36  extending circumferentially laterally over the arrangement of the compartments  28 . 
     The chamber  24  comprises a bottom wall  38  and a top wall  40  parallel thereto which are interconnected via four lateral walls  42 ,  44 , three lateral walls of which (designated by  42  in the figures) are formed to be fixed and the fourth lateral wall  44  is formed movably to enable access to the interior space  46  of the chamber  24  (subsequently called access lateral wall). An opening  48  is located in the top wall  40 , the opening edge  50  thereof comprising a sealing surface  52 . Above the sealing surface  52 , the opening edge  50  comprises an expanding centering zone  54 . 
     The access lateral wall  44  is provided with a support mounting  56  comprising a circumferential frame  58  defining an insertion opening  60  and being fixedly connected from the inside to the access lateral wall  44 . The frame  58  is arranged parallelly to the top wall  40  and the bottom wall  38 . As can be seen especially in the case of  FIGS. 3 and 4 , two cam elements  62  project from the access lateral wall  44  which are guided axially displaceably in guiding rails  64  located in a base plate  66  supporting the bottom wall  38 . A driving motor  68  is flanged on the plate  66  comprising a driving element  70  in the form of a cog wheel combing with a rack  72  connected to one of the two cam elements  62 . In this manner, the access lateral wall  44  can be moved in the direction of the arrows  73  by means of the fixed lateral walls  42  or can be moved towards them. On the interior side of the access lateral wall  44 , a circumferential seal  74  is located abutting on the fixed lateral walls  42 , the top and the bottom walls  40 ,  38  and seals the interior space  46  of the chamber  24 . 
     As can further be seen in the figures, the bottom wall  38  comprises an inclination  76  towards a drain pipe  78  to direct the liquid to a reception container  80 . A vacuum pump  82  is connected to the drain pipe  78  via a suction conduit  81 , through which pump air is drawn out of the interior space  46  to produce a low pressure. As can be seen in  FIG. 5 , the opening  48  in the top wall  40  of the chamber  24  can be closed by means of a lid  84  comprising a projection  86  for gripping the lid  84  by means of the gripping device  18  of the pipetting machine  10 . Apart from the pressure, the temperature  46  of the chamber  24  can also be set, with at least one of the fixed lateral walls  42  being provided with one or multiple heating elements  87 . 
     Subsequently, the operation and the use of the chamber  24  by means of the pipetting machine  10  will be elaborated on. 
     For the liquids present in the compartments  28  of the carrier body  26  to pass through the filter bodies  34 , the carrier body  26  is inserted into the opening  48  of the top wall  40  of the chamber  24 , the projecting rim  36  being placed on the sealing surface  52 . The carrier body  26  is inserted into the opening  48  by means of the gripping device  18 , with the carrier body  26  being centered in the opening  48  by the inlet zone  54  of the opening edge  50 . During this procedure, the access lateral wall  44  is closed, with no carrier body  26  being located in the insert opening  60  of the support mounting frame  58 . Then a first low pressure is produced in the interior space  46  of the chamber  24  due to which the liquids flow through the filter bodies  34 . In this process, the low pressure amounts to about 400 to 800 mbar. During this phase, the heating elements  87  can be operated to pre-heat the interior space  46  of the chamber  24 . The liquid flowing out of the outlet openings  32  of the compartments  28  gets into the reception container  80  via the drain pipe  78 . The liquid can therefore be disposed of by other means, as the residues remaining in the filter bodies  34  are of interest for the analysis. 
     To make these residues available for analysis, they have to be released from the filters  34 . For this purpose, the carrier body  26  has to be placed onto a mounting  88  with a plurality of individual reception containers  90  in which the substances being released from the individual filter bodies  34  are received (see FIG.  1 ). As the quantities of the substances to be analyzed, respectively the components of the liquids, are rather small, and for the analysis to be performed correctly, it has to be provided that only the components to be analyzed get into the reception containers  90 . Liquid drops which might still be adhering to the outlet openings  32  from the preceding filtering step can adulterate the measuring results, which is why they have to be removed before the elution process. For this purpose, the carrier body  26  located in the opening  48  of the top wall  40  is elevated by means of the gripping device  18  to introduce the carrier body  26  from the top into the insertion opening  60  of the frame  58 . For this purpose, the motor  68  is actuated to move out the access lateral wall  44  until the frame  58  is completely moved out of the interior space  46  of the chamber  24 . This situation is shown in  FIG. 4 , by means of which it becomes clear how the carrier body  26  is now inserted in the insertion opening  60  from the top, the edge thereof, like the opening edge  50 , comprising an inlet zone  92 . In this process, the control of the gripping device  18  can be selected such that the carrier body  26  is dropped shortly after being elevated and moved out of the opening  48  so that liquid drops which might still be adhering to the outlet openings  32  are released by the vibrations resulting therefrom. After the insertion of the carrier body  26  into the frame  58 , the access lateral wall  44  is moved back towards the fixed lateral walls  42 . Additionally, the gripping device  18  grips the lid  84  to close the opening in the top wall  40  with it. 
     Then the interior space  46  now substantially gastightly sealed is exposed to a low pressure which is substantially greater than the low pressure to draw the liquid through the filter body  34 . The low pressure now establishing amounts to about 50 to 100 mbar. Additionally, a temperature range between 40 and 60° C. is set by correspondingly controlling the heating elements  87  in the interior space  46 . That way, the evaporation point of the liquid which might still be adhering to the outlet openings  32  is shifted, the liquid thus evaporating. After this vacuum drying process, the chamber  24  is re-aerated, and the access lateral wall  44  is moved so that the carrier body  26  liberated from liquid drops on the outlet openings  32  can be moved onto the mounting  88  for applying the elution buffer. Then the carrier body  26  is moved onto the opening  48  of the top wall  40  of the chamber  24 . A carrier body with compartments is inserted into the frame  58 , in which compartments the eluate of the compartments of the carrier body  26  is received, when it is drawn out therefrom by means of low pressure.