Abstract:
The invention relates to a device ( 1 ) for dispensing or aspirating/dispensing liquid samples, comprising a pump ( 2 ) and a micro-ejection device ( 3 ), said micro-ejection device ( 3 ) having a pulse generator ( 4 ) with a chamber ( 5 ). Said pulse generator ( 4 ) is used to produce pressure waves in the liquid in order to cause the samples of a liquid to be dispensed. The micro-ejection device ( 3 ) also comprises an end piece ( 6 ) and a liquid line ( 7 ) which connects the pulse generator ( 4 ) to the end piece ( 6 ). The pulse generator comprises a micro-actuator ( 10 ) which is configured to function in the same direction as that in which the pressure wave leaves the chamber ( 5 ). The inventive devices are characterised in that the chamber ( 5 ), in the area of the end facing away from the end piece ( 6 ), or the corresponding connecting element ( 14 ) has a narrowed section ( 16 ) which restricts any expansion of the pressure waves in the direction of the pump ( 2 ). The invention also relates to systems with multiple devices of this type.

Description:
RELATED PATENT APPLICATIONS  
         [0001]    This application claims priority of the Swiss patent application No. 2252/00 filed on Nov. 17, 2000, the Swiss patent application No. 2314/00 filed on Nov. 29, 2000, the Swiss patent application No. 2413/00 filed on Dec. 12, 2000, and the international application PCT/CH01/00639 filed on Oct. 29, 2001.  
         FIELD OF THE INVENTION  
         [0002]    According to the preamble of the independent claim 1, the invention relates to a device for dispensing or aspirating/dispensing liquid samples, comprising a pump and a micro-ejection apparatus, with said micro-ejection apparatus having an impulse generator with a chamber and with said impulse generator being used to pressure waves in the liquid in order to cause the samples of a liquid to be dispensed, and with said micro-ejection apparatus further comprising an end piece and a liquid line which connects the impulse generator to the end piece and with the impulse generator comprising a micro-actuator which is configured to function in the same direction as that in which the pressure wave leaves the chamber. The invention further also relates to respective systems comprising such systems.  
         PRIOR ART  
         [0003]    It is known that drops with a volume of more than 10 μl can be dispensed very easily from the air because drops will leave the tip of the pipette automatically when the pipette is handled correctly. The size of the drop is then determined by the physical properties of the sample liquid such as surface tension or viscosity. The size of the drop thus limits the resolution of the quantity of liquid to be dispensed.  
           [0004]    The aspiration and dispensing, i.e. the pipetting, of liquid samples with a volume of less than 10 μl usually demands instruments and techniques which guarantee the dispensing of such small samples. The dispensing of a liquid with the tip of a pipette, i.e. with the end piece of an apparatus for dispensing or aspirating/dispensing liquid samples, can occur from the air (“from air”) or by touching a surface. This surface can be the solid surface of a vessel (“on tip touch”) to which the liquid sample is to be given. It can also be the surface of a liquid situated in this vessel (“on liquid surface”). A mixing process occurring subsequently to the dispensing is recommended especially in the case of very small sample volumes in the range of nanoliters or even picoliters, so that an even distribution of the sample volume within a diluent is ensured.  
           [0005]    Disposable tips substantially reduce the likelihood of an undesirable transmission of sample particles (contamination). Known are simple disposable tips (so-called “air-displacement tips”) whose geometry and material is optimized for the precise dispensing of very small volumes. The use of so-called “positive-displacement tips” which comprise a pump plunger on their inside is also known.  
           [0006]    For the purpose of automating the pipetting process of volumes below 10 μl it is necessary to distinguish two processes from each other: The defined uptake (aspiration) and the subsequent delivery (dispensing) of liquid samples. Between such processes the tip of the pipette is usually moved by the experimenter or an automatic machine, so that the place of aspiration of a liquid sample differs from its dispensing point. For the precision of dispensing only the liquid system is relevant which consists of the pump (diluter), liquid line and end piece (tip of pipette). Among the many possible pumps for high-precision aspiration and dispensing of liquids, commercially obtainable devices with the names “CAVRO XL 3000 Modular Digital Pump” or “CAVRO XP 3000 plus Modular Digital Pump” have proven their worth. They are supplied by Cavro Scientific Instruments Inc., Sunnyvale, Calif., USA. Such pumps comprise a cylinder with a plunger which is movable therein and a stepper motor for driving the plunger. The stepper motor operates with a voltage of 24 V and is triggered by an external computer or microprocessor. Further details are shown in the operating manual “Operators Manual P/N 724043C” of Cavro Scientific Instruments Inc.  
           [0007]    An apparatus of this kind and a respective method are known from U.S. Pat. No. 5,763,278. It concerns automatic pipetting of small volumes, with the apparatus comprising a pipetting needle, a diluter with a liquid output with a syringe and a valve. The syringe comprises a plunger and a plunger drive. A tubing connects the needle and the liquid output of the diluter, with the diluter and the tubing containing a substantially incompressible liquid. An impulse generator is arranged in the apparatus and connected with the incompressible liquid in the tubing, so that mechanical impulses with a force of at least 0.01 Ns can be emitted directly into the liquid of the tubing. Such an impulse is used to drive the liquid from the needle. The drop size is defined by a purposeful advance of the diluter plunger and the drop is ejected from the needle with an impulse. As a result of the definition of the volume with the diluter, the size of the drop and its reproducibility depends on the resolution of the diluter and is limited by the same.  
         OBJECTS AND SUMMARY OF THE INVENTION  
         [0008]    It is the object of the present invention to provide a device for dispensing or aspirating/dispensing liquid samples up to the picoliter range in which the dispensed drop size and its reproducibility does not depend on the resolution of the diluter.  
           [0009]    This object is achieved by the features of the independent claim 1. Additional features arise from the dependent claims.  
           [0010]    This invention relates to a device ( 1 ) for dispensing or aspirating/dispensing liquid samples, comprising a pump ( 2 ) and a micro-ejection apparatus ( 3 ), with said micro-ejection apparatus ( 3 ) having an impulse generator ( 4 ) with a chamber ( 5 ). Said impulse generator ( 4 ) being used to produce pressure waves in the liquid in order to cause the samples of a liquid to be dispensed. The micro-ejection apparatus ( 3 ) further comprises an end piece ( 6 ) and a tubing ( 7 ) for the liquid which connects the impulse generator ( 4 ) to the end piece ( 6 ). The impulse generator comprising a micro-actuator ( 10 ) which is configured to function in the same direction as that in which the pressure wave leaves the chamber ( 5 ). The inventive devices are characterized in that the device ( 1 ), in the area of the end of the chamber ( 5 ) averted from the end piece ( 6 ), comprises a narrowed section ( 16 ) which is open during the production of the pressure waves and which restricts any propagation of the pressure waves in the direction towards the pump ( 2 ). 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0011]    The present invention will now be explained in more detail by reference to schematic drawings which illustrate preferable embodiments and shall not limit the scope of the present invention, wherein:  
         [0012]    [0012]FIG. 1 shows diagrammatic view of a device for dispensing or aspirating/dispensing liquid samples;  
         [0013]    [0013]FIG. 2 shows a sectional view through an impulse generator according to a first embodiment;  
         [0014]    [0014]FIG. 3 shows a sectional view through an impulse generator according to a second embodiment;  
         [0015]    [0015]FIG. 4 shows a sectional view through an impulse generator according to a third embodiment;  
         [0016]    [0016]FIG. 5 shows a sectional view through an array of impulse generators according to a fourth embodiment. 
     
    
     DESCRIPTION OF THE INVENTION  
       [0017]    [0017]FIG. 1 shows a diagrammatic view of a device for dispensing or aspirating/dispensing liquid samples according to a first embodiment. Said device  1  comprises a pump  2  and a micro-ejection apparatus  3 . The micro-ejection apparatus comprises an impulse generator  4  with a chamber  5  and is filled completely with a liquid which thus forms a continuous liquid column. The impulse generator  4  is arranged in such a way that, for the purpose of producing the dispensing of liquid samples, pressure waves can be produced in the liquid. The micro-ejection apparatus  3  further comprises an end piece  6  and a liquid tubing  7 . The liquid tubing  7  has a length of approx. 1 m in an embodiment and connects the impulse generator  4  with the end piece  6 . The inside diameter of said tubing  7  is 0.8 mm in this embodiment and the wall thickness measures 0.6 mm.  
         [0018]    Preferably, the pump  2  is a reciprocating pump or a diluter of type “CAVRO XP 3000 plus Modular Digital Pump”. The cylinder  8  of the diluter has a volume in the range of 50 to 500 μl and the resolution of the diluter lies in the region of 3,000 steps or 6,000, 12,000 or 24,000 partial steps per entire stroke. The diluter is used in order to fill the tip of the pipette or the end piece  6 . This comprises both the aspiration of a sample volume as well as the compensation of a dispensed volume during dispensing. The diluter or the pump  2  and the impulse generator  4  are mutually connected by way of a liquid tubing  9 . The inside diameter of the tubings  7 ,  9  which are preferably made of tetrafluoroethylene-hexafluoropropylene-copolymer (FEP) is preferably 0.1 to 4 mm, with a diameter of 0.1 to 1 mm being especially preferable. For all tubings  7 ,  9  (“tubing”) a wall thickness of 0.3 to 1.2 mm is preferable.  
         [0019]    The impulse generator  4  comprises a chamber  5  and a micro-actuator  10  and is arranged in the device  1  for dispensing or aspiring/dispensing liquid samples between diluter or pump  2  and disposable tip or end piece  6 . The micro-actuator  10  can comprise a piezo-element (e.g. as a stacked actuator or as a bimorph element), a magnetic element or a thermal actuating system. The common aspect of all these micro-actuators is that they produce a pressure wave in the liquid, such that a mechanical impulse is transmitted onto said liquid. Whereas piezo-actuators act through a sudden expansion and magnetic actuators accelerate a solenoid, thermal actuators suddenly heat a gas. Said gas is situated in a chamber enclosed with a membrane and suddenly expands when heated, so that the membrane moves and produces a pressure wave. The chamber  5  comprises an input channel  11  and an output channel  12 . The micro-actuator  10  is preferably triggered by an electric square wave signal.  
         [0020]    Every short pulse of this rectangular signal produces a sudden change in shape and/or position of the micro-actuator  10  which is transmitted as an impulse onto the liquid in chamber  5 . Said impulse which is produced by the impulse generator  4  whose strength Δp was calculated in an embodiment with 0.016 Ns initiates a pressure wave in the liquid which preferably spreads in the direction towards the output channel  12  of chamber  5  and leaves the chamber  5  there.  
         [0021]    A chamber  5  which is preferably provided with rotationally symmetrical configuration and whose axis of symmetry  13  is arranged co-axially to the output channel  12  allows a substantially even propagation of said pressure wave in the chamber  5 . Preferably, the micro-actuator  10  is arranged in the chamber in such a way that it acts in the same direction in which the pressure wave leaves the chamber  5 . Since the entire micro-ejection apparatus  3  is always completely filled with a continuous liquid column, the volume of a dispensed liquid sample is defined alone by the parameters of a single impulse produced by the impulse generator  4 .  
         [0022]    In order to enable a substantially continuous propagation of the pressure wave through tubing  7  to the end piece  6 , the chamber  5 , the liquid tubing  7 , the end piece  6  and all other connection elements  14  which may also be additionally provided comprise substantially continuous transitions and a similarly constant inside diameter. Said inside diameter is preferably always larger than the opening diameter  18  of the end piece  6 . Under these preconditions the preferred wall thickness at the end of tip adapter  15  which carries the end piece  6  is less than 0.5 mm in the region of the transition to the tubing  7  and to the end piece  6 . Preferably, all other transitions between impulse generator  4  and end piece  6  are adjusted accordingly.  
         [0023]    In order to obstruct the propagation of the wave in the direction towards the pump  2 , the chamber  5  is preferably provided in the region of its end averted from the end piece  6 , i.e. in the zone of input channel  11 , with a narrowed section  16 . If the chamber  5  and the tubing  9  are connected to an additional connection element  14 , said connection element  14  can also be provided with such a narrowed section  16 .  
         [0024]    The end piece  6  is arranged as a disposable tip of type “air-displacement tip”, consists of a polymer material which is injection-molded for example and can be replaced after use as required. The end piece  6  preferable sits on a tip adapter  15  which forms a piece of the tubing  7  and is held and guided by an experimenter or a robot arm  17 . The opening diameter  18  of the end piece  6  or the disposable tip is preferably 20 to 150 μm for the dispensing of samples in the nanoliter range. A diameter of less than 50 μm is especially preferable for dispensing samples in the picoliter range. Said opening diameter  18 , as also the other geometry of the end piece  6 , can be adjusted as needed to the properties of the liquid to be pipetted or the intended volume of the samples to be separated. In an embodiment the opening diameter  18  is approx. 50 μm.  
         [0025]    [0025]FIG. 2 shows a sectional view through an impulse generator  4 , according to FIG. 1 and according to a first embodiment. The axis of symmetry  13  lies co-axially to the output channel  12 . At a right angle to the axis of symmetry  13  there is a micro-actuator with a bimorphous piezo-element  21  which closes off the chamber  5  on the rear side. The input channel  11  is provided on its transition to the inner wall  19  of chamber  5  with a narrowed section  16 .  
         [0026]    [0026]FIG. 3 shows a sectional view through an impulse generator  4  according to a second embodiment. The axis of symmetry  13  lies co-axially to the output channel  12 . A micro-actuator  10  which operates on a magnetic basis is arranged at a right angle to the axis of symmetry  13 . A membrane  22  for transmitting impulses onto the liquid in the chamber  5  is arranged between the actuator arranged as a solenoid  20  and the chamber  5 . Said membrane  22  closes off the chamber  5  at the rear side. Instead of a solenoid working on a magnetic basis it would also be possible to use a stack of piezo-elements (not shown). The input channel  11  is provided at its transition point to the inner wall  19  of chamber  5  with a narrowed section  16 .  
         [0027]    [0027]FIG. 4 shows a sectional view through an impulse generator  4  according to a third embodiment. The axis of symmetry  13  lies co-axially to the output channel  12 . A piezo-element  21  is arranged as a micro-actuator  10  at a right angle to the axis of symmetry  13  and is glued onto a silicon plate  23 . The chamber  5  is thus closed off on the rear side with the silicon plate  23 . The silicon plate  23 , which is arranged between the micro-actuator  10  and the chamber  5 , transmits the impulses onto the liquid in chamber  5 . In contrast to the previously explained examples, the input channel  11  is arranged parallel to the output channel  12 . The input channel  11  is connected with the chamber  5  via a connection channel  24  which is etched into the silicon plate  23 . Said connection channel  24  has a very small cross section, so that in addition to the function of guiding the liquid it also fulfils the function of a constriction  16 . Moreover, the inner diameter of the input channel  11  is far smaller than that of the output channel  12 . The entire arrangement according to said third embodiment is held by an adapter  25 . Said adapter  25  also comprises the electric contacts  26  for the piezo-element  21  of the micro-actuator  10 . Preferably, a compartmenting structure  31  which reaches up to the upper side of the components  29  forming the chamber  5  is connected in a sealing fashion with the components  29 . Said compartmenting structure  31  preferably is a glass plate with compartments  32  incorporated therein for receiving the components  29 . The use of a glass plate comes with the advantage that a plane surface  33  is created for receiving the silicon plate  23 . The silicon plate  23  and the glass plate can thus advantageously be mutually connected in an anodic fashion without any use of adhesives.  
         [0028]    A system for dispensing or aspirating/dispensing liquid samples such as an automatic pipetting machine can comprise one (see FIG. 1) or several devices  1  (not shown). Automatic pipetting machines with eight channels are preferred, i.e. with eight end pieces  6 , with which standard microtiter plates™ (trademark of Beckman Coulter, Inc., 4300 N. Harbour Blvd., P.O. Box 3100 Fullerton, Calif., USA 92834) or microplates with 96 wells with liquid samples can be charged.  
         [0029]    Such systems can comprise the combination of 1 to n pumps 2 and 1 to n impulse generators  4  with the same number each. Similar systems can comprise the combination of a single pump  2  with several impulse generators  2 . Preferred are automatic pipetting machines with eight channels, i.e. with eight impulse generators  4  and eight end pieces  6 . The end pieces  6  are preferably arranged in a one-dimensional array in the form of a row, so that it is possible to work with 8, 32, 72 or 128 channels simultaneously and parallel. Similar systems can comprise a two-dimensional array of end pieces  6  in the form of a grid, so that microplates with 384, 864, 1536 or even more flat-bottom blocks can be charged simultaneously. Combinations of the aforementioned systems are also possible, so that a system will simultaneously comprise end pieces  6  which are arranged and/or movable in a linear or planar array. If a system comprises several impulse generators  4 , they can be arranged on a single component, e.g. in the form of an array of units. In a further preferred embodiment of a system in accordance with the invention for dispensing or aspirating/dispensing liquid samples, the end pieces  6  are attached directly to the impulse generators  4  which are arranged on a two-dimensional array and on a common component (see FIG. 5).  
         [0030]    Preferably, the individual impulse generators  4  and pumps  2  are each addressable and controllable in such systems via a control unit, e.g. via a personal computer which is equipped with the respective hardware and software.  
         [0031]    A three-way valve  27  and at least one storage reservoir  28  which is connected to the same is preferable especially for multi-channel systems with a large liquid throughput. System liquid or even a liquid to be dispensed can be situated in the storage reservoir. Several storage reservoirs are preferably used in systems with several pumps.  
         [0032]    [0032]FIG. 5 shows a sectional view through an array of impulse generators according to a fourth embodiment. The individual chambers  5  of the adjacently arranged impulse generators  4  are arranged in a component  29  which consists of a polymer material which is integrally injection-molded or turned individually from solid material for example. Needles  30  are arranged co-axially with the axis of symmetry  13  of chambers  5  and form the output channel  12  of the same. Preferably, said needles  30  consist of stainless steel and taper to a point at their free end in such a way that they can either be used directly as end pieces  6  (i.e. as pipette tips) or they each can receive a disposable tip.  
         [0033]    In a partial or even close contact with said component  29 , a compartmenting structure  31  is sealingly connected with the component  29  on its upper side. Said compartmenting structure  31  preferably consists of a glass plate from which compartments  32  are arranged in such a way that they correspond in the register with the chambers  5 . The use of a glass plate leads to the advantage that a plane surface  33  is created. Advantageously the silicon plate  23  and the glass plate can be connected with each other in an anodic fashion without using any adhesives.  
         [0034]    To said surface  33  a thin silicon plate  23  is applied into which connecting channels  24  are etched. Micro-actuators  10  in the form of piezo-elements  21  are arranged on the side of the silicon plate  23  which is averted from the chamber, which piezo-elements correspond with respect to their distribution to the pattern of the underlying chamber arrangement. The impulses required for ejecting a sample are thus produced by the piezo-elements  21  and transmitted onto the liquid via the silicon plate  23 . All chambers  5  are connected with each other and with the input channel  11  leading to the diluter or pump  2  by way of the connecting channels  24  which have a cross section which is small in relationship to the output channels  12 , so that all said hollow chambers as well as the needles  30  (and if disposable tips are used they too) are always filled completely with a liquid column. This ensures on the one hand that the connecting channels representative of a constriction obstruct a propagation of the pressure waves in the direction towards pump  2  and that the volume of the liquid samples issued from the end pieces  6  is defined solely by the parameters of the impulses generated by the impulse generators  4 .  
         [0035]    The electronic system required for triggering the individual piezo-elements  21  as well as the respective electrical feed lines can be arranged on a common component  34  also in a manner corresponding to the distribution of the chambers  5  and the micro-actuators  10 . A housing (not shown) which preferably consists of a bottom shell  35  and an upper&#39;shell  36  preferably comprises the entire array with the impulse generators  4  and the associated electronic system on the component  34 . Such an array may comprise a number of eight impulse generators for example or even a surface area of  96  or  384  impulse generators for example.  
         [0036]    A system for dispensing or aspirating/dispensing liquid samples works as follows for example:  
         [0037]    1. The robotic arm  17  takes up a disposable tip from a storage location, with the conical, mutually adjusted shape of tip adapter  15  and disposable tip guaranteeing a favorable fit and sealing.  
         [0038]    2. The disposable tip is filled completely with a system liquid (e.g. with distilled or de-ionized water) by means of the diluter or by means of pump  2 .  
         [0039]    3. The robotic arm  17  moves to a receptacle in which the liquid to be pipetted is located and is lowered there until the end piece  6  or the pipette tip touches the liquid surface. A defined volume of the liquid is received by the end piece  6  (aspiration) with the pump  2 .  
         [0040]    4. The robotic arm  17  moves to a predetermined delivery point where the impulse generator  4  emits a precisely defined, short impulse. This initiates a pressure wave in the liquid which propagates in the chamber  5  of the impulse generator  4  in the direction towards the output channel  12 , through the tubing  7  and finally through the end piece  6 . The end piece  6  forms the narrowest point on the entire route which the pressure wave needs to cover so that the same is subjected to an acceleration in the end piece  6 . Once the speed of the pressure wave is so high that it overcomes the surface tension forces of the liquid, sample volumes of a specific and defined magnitude are ejected. The pipette diameter, i.e. the opening diameter  18  of the end piece  6 , has an influence that cannot be neglected on the resulting sample volume or drop size which lies in the range of 0.01 to 10 nl. The dispensed total volume is obtained from the volume of a single separated sample (drop) and the number of the impulses emitted by the impulse generator (dispensing).  
         [0041]    5. During the dispensing, the piston of the pump  2  follows up, so that substantially the dispensed volume of liquid is compensated.  
         [0042]    6. After dispensing a predetermined quantity of sample liquid, the robotic arm  17  moves the end piece  6  over a waste collection point where the disposable tip is ejected. A new tip is then picked up.  
         [0043]    The respective parts were provided with the same reference numerals in all figures.