Abstract:
A micromechanical pipetting device comprising an integrally built pipetting module which has an inlet/outlet which may be connected to a removable pipetting tip or integrally built with a pipetting tip. The pipetting module comprises a micromechanical structure which is integrally built on a silicon wafer. In order to improve the accuracy of the pipetted volume the device is characterized in that it comprises 
     a) a first chamber located within said pipetting module, the volume comprised within said first chamber being alterable by displacement of a membrane which is a portion of a wall of said chamber, said first chamber having an opening, said opening being permanently open and allowing fluid flow into and from the interior of said first chamber, 
     b) a channel located within said pipetting module, said channel establishing a direct, valveless and permanent fluidical connection between said opening of the first chamber and the inlet/outlet of the pipetting module, 
     c) actuator means for displacing said membrane, and thereby aspiring or expelling a volume of air or of a liquid into or from said first chamber, which in turn causes aspiring or expelling a volume of a liquid sample into respectively from said pipetting tip, and 
     d) first sensor means for generating a first output signal related to the displacement of the membrane.

Description:
BACKGROUND OF THE INVENTION 
     The invention relates to a micromechanical pipetting device for pipetting liquid volumes in a range between a minimum value smaller than a microliter and a maximum value of about 10 microliters. The device comprises a pipetting module which has an inlet/outlet which may either be connected to a removable pipetting tip or may have a pipetting tip integrally built into the module. The integrally built pipetting module comprises a micromechanical structure which is integrally built on a silicon wafer. 
     Some micromechanical structures are known for the purpose of dispensing very small volumes of liquid. A micromechanical pump comprising valves can be used for this purpose, but high accuracy of the dispensed volumes cannot be attained, mainly due to the reflow caused by the operation of the valves and dead volumes and leaking problems associated with the use of valves. Moreover such micropumps normally pump a number of liquid portions until the desired volume to be dispensed is approximately attained. Thus the accuracy of the total volume dispensed depends from the accuracy of the volume portion transported by each pumping step. 
     A similar approach can also be implemented by dispensing microdrops, as in ink-jet printers, until the desired volume to be dispensed is approximately attained. Also in this case the accuracy of the total volume dispensed depends from the accuracy of the volume of each microdrop. The accuracy of pipetted volume obtained by this approach is limited, in particular because it depends on the properties of the liquid being pipetted. 
     Another known approach for dispensing very small volumes of liquid is the use of a micromechanical pump controlled by a feedback loop comprising an anemometric flow sensor and an integrator of the output signal of this sensor. The function of the feedback loop is to measure the volume pumped by the micromechanical pump and to control it accordingly. Thus in theory the feedback loop would control the micromechanical pump in such a way that the latter pumps a steady flow of liquid over an interval of time until the desired volume to be dispensed is attained and then the operation of the pump is stopped. This approach has several important disadvantages. There is always a delay between the measurement of the pumped volume and a corresponding control of the micromechanical pump. Thus a correction of the operation of the pump via feedback loop only happens after the pumped volume is already larger than the desired value. Such a device is therefore not accurate enough for pipetting very small volumes with high accuracy. The operation of anemometric flow sensors requires heating of the liquid pumped. Thus, such a device cannot be used for pumping thermally sensitive liquids of the kind to be pipetted e.g. in clinical chemistry analyzers. 
     SUMMARY OF THE INVENTION 
     The aim of the invention is therefore to provide a micromechanical pipetting device for pipetting with high accuracy very small volumes of liquids and with which the above-mentioned disadvantages of known prior art devices can be avoided. 
     According to the invention, this aim is attained with a device of the type described herein, which device is characterized in that it comprises. 
     a) a first chamber located within a pipetting module, wherein the volume contained within said first chamber may be modified by displacement of a membrane which is a portion of a wall of said chamber, said first chamber having only one opening, said opening being permanently open and allowing fluid flow into and from the interior of said first chamber, 
     b) a channel located within said pipetting module, said channel establishing a direct, valveless and permanent fluidical connection between said opening of the first chamber and the inlet/outlet of the pipetting module, 
     c) actuator means for displacing said membrane, and thereby aspirating or expelling a volume of air or of a liquid into or from said first chamber, which in turn causes aspiration or expulsion of a volume of a liquid sample through said pipetting tip, and 
     d) a first sensor means for generating a first output signal related to the displacement of the membrane. 
     The main advantage of the device according to the invention as compared with the prior art devices is that it makes it possible to pipette very small volumes of liquid with high accuracy, reproducibility, reliability and fast performance. 
     In particular the inclusion of a first sensor means for generating a first output signal related to the displacement of the membrane makes possible a highly accurate and real-time monitoring of the operation of the device which is suitable for the fast forward and reverse flow in a pipetting device according to the invention. 
     Moreover, the device according to the invention advantageously differs from prior art devices in that it makes it possible to pipette the entire volume to be pipetted by a single stroke of the actuator means. 
     A preferred embodiment of the device according to the invention further comprises a control means for controlling the operation of the actuator means in response to the first output signal generated by the first sensor means. The micropipetting module according to the invention and the means for controlling the operation of the actuator means are preferrably configured and dimensioned so that the total volume to be aspirated and dispensed with the pipetting tip is aspirated into the pipetting tip by means of a single stroke of the displacement movement of the membrane. 
     Another preferred embodiment of the device according to the invention is characterized in that a portion of the membrane is part of the first sensor means and the first output signal generated by this sensor means is related to or representative of the displacement of the membrane. 
     A further preferred embodiment of the device according to the invention is characterized in that a portion of the channel forms a second chamber and is part of a second sensor means for generating a second output signal representative of the pressure in the channel, and the means for controlling the operation of the actuator means is responsive to both the first and the second output signals. 
     A further preferred embodiment of the device according to the invention is characterized in that a portion of the channel forms a second chamber and is part of a second sensor means for generating a second output signal representative of the fluid flow through the channel, and the means for controlling the operation of the actuator means is responsive to both the first and the second output signals. 
     The above mentioned preferred embodiments which include the association of multifunctional sensors located close to the pipetting tip make possible a direct and highly accurate monitoring of very small pipetted volumes and early and active recognition and avoidance of malfunctions of the micropipetting module. 
     A further preferred embodiment of the device according to the invention is characterized in that a portion of the channel forms a third chamber which is located between the pipetting tip and the first or the second sensor means, said third chamber serving to prevent pipetted fluid from contacting the portion of the channel which comprises said first sensor means or said second sensor means. 
     A further preferred embodiment of the device according to the invention is characterized in that said actuator means comprises an electrostatic actuator or a piezoelectric actuator or an electromechanical actuator. 
     A further preferred embodiment of the device according to the invention is characterized in that said first sensor means is a capacitive or an electro-optical sensor. 
     A further preferred embodiment of the device according to the invention is characterized in that said second sensor means comprises a pressure or a flow measurement sensor. The use of an integrated pressure sensor according to the instant invention ensures that the pipetting module operates in the normal range (e.g. of viscosity) for which the system is designed. 
     A further preferred embodiment of the device according to the invention is characterized in that a plurality of said pipetting modules is integrally built on a silicon wafer. 
     A further preferred embodiment of the device according to the invention is characterized in that the pipetting tip is a silicon pipetting tip integrally built with the pipetting module. 
     Exemplified embodiments of the invention are described below with reference to the accompanying drawings. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 a  is a schematic view of an array of micromechanical modules formed on a silicon wafer. 
     FIG. 1 b  is a schematic view of a micromechanical pipetting module according to the invention. 
     FIG. 2 is a schematic representation of a longitudinal section along the line II—II in FIG. 1 b.    
     FIG. 3 is a partial representation of a cross-section along the line I—I in FIG. 1 b.    
     FIG. 4 a  is a schematic view of an array of micromechanical modules formed on a silicon wafer. 
     FIG. 4 b  is a schematic view of a second embodiment of a micromechanical pipetting module according to the invention. 
     FIG. 5 is a partial representation of a longitudinal section along the line II—II in FIG. 4 b.    
     FIG. 6 is a partial representation of a cross-section along the line I—I in FIG. 4 b.    
     FIG. 7 is a partial representation of a cross-section along the line III—III in FIG. 4 b.    
     FIG. 8 is a longitudinal section of a third embodiment of a micromechanical pipetting module according to the invention. 
     FIG. 9 is a longitudinal section of a fourth embodiment of a micromechanical pipetting module according to the invention. 
     FIG. 10 is a longitudinal section of a fifth embodiment of a micromechanical pipetting module according to the invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     FIG. 1 a  a shows schematically a silicon wafer on which an array of micromechanical modules  14  has been formed. Each of such modules can be used as a component of a first embodiment of a micromechanical pipetting module according to the invention. 
     FIG. 1 b  shows schematically a first embodiment  11  of a micromechanical pipetting module according to the invention. Liquid volumes in a range between a minimum value smaller than a microliter and a maximum value of about 10 microliters can be pipetted with such a module. Module  11  is an integrally built pipetting module comprising a micromechanical structure which is integrally built on a silicon wafer  14 . 
     The micromechanical pipetting module  11  shown by FIG. 1 b  comprises three layers arranged one above the other and connected to one another unreleasably by means of anodic bonding: a first glass layer  31 , a second glass layer  32  and a silicon wafer layer  14  arranged between glass layers  31  and  32 . Silicon wafer layer  14  is unreleasably connected to glass layers  31  and  32  by means of anodic bonding. Silicon wafer layer  14  in FIG. 1 b  has a surface of approximately 25×10 mm for the smaller volumes of the target range (minimum value smaller than a microliter and a maximum value of about 10 microliters). 
     Silicon wafer layer  14  comprises a chamber  15  and a channel  18  formed by micromachining on wafer  14 . The bottom wall of chamber  15  is a membrane  16  which is part of silicon wafer  14 . Chamber  15  has only one opening  17  which is connected to one end of channel  18 . The opposite end of channel  18  forms an inlet/outlet  12  of pipetting module  11 . A pipetting tip  13  is connected to inlet/outlet  12  by means of a sealing film  29 . 
     In an alternative embodiment layers  31  and  32  are also fabricated in silicon. This offers the advantage of reducing undesirable temperature effects. In this alternative embodiment the bonding process is called “silicon direct bonding”. The disadvantage of this kind of bonding as compared with anodic bonding with glass wafers is the higher temperature needed for performing the bonding process. A compromise to overcome this difficulty is to sputter a thin layer of pyrex glass onto a silicon wafer and then to perform anodic bonding. Within the scope of the invention the material of layers  31  and  32  can thus be either glass or silicon, whereby for silicon two different bonding processes are possible. 
     FIG. 2 shows a longitudinal section on the line II—II in FIG. 1 b.  As shown by FIG. 2 a micromechanical pipetting module  11  shown by FIG. 1 b  comprises chamber  15  having membrane  16  as bottom wall, channel  18 , actuator means  19  for displacing membrane  16  and sensor means  21  for generating an output signal related to the displacement of the membrane  16 . A portion of the membrane  16  is part of the sensor means  21  and the output signal generated by this sensor means is representative of the displacement of the membrane  16 . Components of sensor  21  are located in a chamber  51  delimited by membrane  16 , silicon wafer  14  and glass plate  32 . 
     Sensor  21  is preferably a displacement sensor. Sensor  21  in FIG. 2 may comprise an electrical capacitor as measuring element. Sensor  21  in FIG. 2 may alternatively be an electro-optical sensor. 
     The volume contained within chamber  15  may be increased or decreased by displacement of a membrane  16 . Chamber  15  has only one opening  17  which is permanently open and which allows fluid flow into and out of the interior of chamber  15 . 
     Channel  18  establishes a direct, valveless and permanent fluidical connection between opening  17  of chamber  15  and the inlet/outlet  12  of the pipetting module  11 . 
     Actuator means  19  may be an electrostatic actuator as schematically represented in FIG. 2, or a piezoelectric actuator. The electrical connections of actuator  19  are not shown in the Figures. Membrane  16  can also be displaced by a pressure increase or decrease of a gas in the chamber formed by membrane  16 , silicon wafer  14  and glass plate  32 . This pressure change can be achieved on the chip e.g. by a thermopneumatical actuation, that is by heating and cooling a gas or by evaporation and condensing of a liquid. 
     FIG. 3 shows a partial representation of a cross-section on the line I—I in FIG. 1 b.  FIG. 3 shows in particular the cross-sectional shape of chamber  15  and an example of the width and depth of wafer layer  14  of pipetting module  11 . The broken line in FIG. 3 shows the position taken by the membrane  16  when it is displaced e.g. by means of an actuator located below membrane  16 , but not represented in FIG.  3 . 
     In order to perform a pipetting operation with the pipetting module  11 , actuator means  19  are activated to displace membrane  16  for aspirating or expelling a volume of air or of a liquid into or from chamber  15 . Such a displacement of membrane  16  causes a corresponding aspiration or expulsion of a volume of a liquid sample from said pipetting tip  13 . 
     When a pipetting module according to the invention is used to perform pipetting operations the interior of the pipetting module is filled either with air or with a liquid, e.g. water, separated from the pipetted liquid by an air segment. Sample or reagent is aspirated or expelled from the pipetting tip when actuator  19  displaces membrane  16 . While pipetting, the pipetted liquid, for instance a biological liquid sample or a reagent for performing a clinical chemistry test, does not enter channel  18  but remains within the pipetting tip. 
     FIG. 4 a  shows schematically a silicon wafer on which an array of micromechanical modules  44  has been formed. Each of such modules can be used as a component of a second embodiment  41  of a micromechanical pipetting module according to the invention. 
     FIG. 4 b  shows schematically a second embodiment  41  of a micromechanical pipetting module according to the invention. Liquid volumes in a range between a minimum value smaller than a microliter and a maximum value of about 10 microliters can be pipetted with such a module. Module  41  is an integrally built pipetting module comprising a micromechanical structure which is integrally built on a silicon wafer  14 . 
     As can be appreciated from FIG. 4 b  micromechanical pipetting module  41  is very similar to micromechanical pipetting module  11  shown by FIG. 1, but differs therefrom in that in module  41  a portion of the channel  18  forms a chamber  23 . 
     FIG. 5 shows a longitudinal section of micromechanical pipetting module  41  shown by FIG. 4 b.  Micromechanical pipetting module  41  shown by FIG. 5 comprises chamber  15  having membrane  16  as bottom wall, channel  18 , actuator means  19  for displacing membrane  16 , a chamber  23  formed by a portion of channel  18 , and sensor means  22  for generating an output signal related to the displacement of the membrane  16 . A portion of channel  18  is part of sensor means  22  and the output signal generated by this sensor means is related to the displacement of membrane  16 . Components of actuator means  19  are located in chamber  51  delimited by membrane  16 , silicon wafer  14  and glass plate  32 . Components of sensor  22  are located in a chamber  52  delimited by a membrane  36  which is part of silicon wafer  14 , silicon wafer  14  and glass plate  32 . 
     Sensor  22  is for instance a pressure sensor or a flow measurement sensor. Sensor  22  in FIG. 5 is pressure sensor comprising an electrical capacitor as measuring element. Sensor  22  can also be a piezoresistive sensor or any other type of pressure sensor. 
     Sensor  22  can also be used for liquid level detection before aspiration of liquid to be pipetted. 
     FIG. 6 shows a partial representation of a cross-section along the line I—I in FIG. 1 b.  FIG. 6 shows in particular the cross-sectional shape of chamber  15  and an example of the width and depth of wafer layer  14  of pipetting module  41 . The broken line in FIG. 6 shows the position taken by the membrane  16  when it is displaced e.g. by means of an actuator located below membrane  16 , but not represented in FIG.  6 . 
     FIG. 7 shows a partial representation of a cross-section on the line III—III in FIG. 4 b.  FIG. 7 shows in particular the cross-sectional shape of chamber  23  of module  41 . 
     The operation of micromechanical pipetting module  41  is very similar to the operation of module  11  described above with reference to FIGS. 1 a,    1   b,    2  and  3  with exception of the fact that in module  41  sensor  22  for producing an output signal related to the displacement of membrane  16  is located under chamber  23 , that is at a distance from membrane  16 , whereas in module  11  sensor  21  for producing such an output signal is located under chamber  15  and directly under membrane  16 . 
     FIG. 8 shows a longitudinal section of a third embodiment of a micromechanical pipetting module according to the invention obtained by modifying the embodiment shown by FIG.  5 . The embodiment shown by FIG. 8 differs from the embodiment shown by FIG. 5 in that the embodiment shown by FIG. 8 comprises an additional sensor  21  located adjacent to actuator  19  in chamber  51 . Like in the embodiment shown by FIG. 2., Sensor  21  in FIG. 8 is preferably a displacement sensor. Sensor  21  in FIGS. 2,  8 ,  9  and  10  is a displacement sensor comprising an electrical capacitor as measuring element. Sensor  21  in FIGS. 2,  8 ,  9  and  10  can be replaced by an electro-optical sensor. 
     FIG. 9 shows a longitudinal section of a fourth embodiment of a micromechanical pipetting module according to the invention obtained by modifying the embodiment shown by FIG.  8 . In addition to the features of the embodiment shown by FIG. 8, in the embodiment shown by FIG. 9 a portion of the channel  18  forms a third chamber  24  which is located between outlet  12  of the pipetting module and chamber  23 . Chamber  24  serves to prevent pipetted fluid from contacting the portion of the channel  18  which comprises sensor  22 . 
     In a modified version of the embodiment shown by FIG. 2 a chamber similar to chamber  24  in FIG. 9 is located between outlet  12  of the pipetting module and chamber  15 . Such a modified version is not represented in the drawings. 
     FIG. 10 shows a longitudinal section of a fourth embodiment of a micromechanical pipetting module according to the invention obtained by modifying the embodiment shown by FIG.  8 . In addition to the features of the embodiment shown by FIG. 8, the embodiment shown by FIG. 10 has an additional pressure sensor  27  for measuring the pressure on the actuator side, that is in channel  28  and in the chambers fluidically connected therewith, and an additional chamber  25  delimited by a membrane  26  which is part of silicon wafer  14 , silicon wafer  14  and glass plate  31 . Chamber  25  located above membrane  26  is a reference chamber for the operation of sensor  27 . Channel  28  connects a chamber  53  where pressure sensor  27  is located with the chamber  51  where actuator  19  is located. Pressure sensor  27  is used for monitoring the pressure applied in the case of pneumatic actuation principle, according to which a certain pressure is maintained in the actuator chamber, e.g. by thermopneumatic actuation. This pressure differs from the pressure measured by sensor  22  in that the signal of sensor  22  is modulated by the dynamics and the force of the actuator membrane and the fluidic behavior (i.e. dynamics and gravity) of the aspirated sample liquid and of the system fluid. Sensor  27  is used as part of a control system for maintaining a given (static) pressure by a direct feedback system and monitoring the behavior of the liquid by means including sensors  21 ,  22  and  27  analyzing the signals obtained with these sensors and if necessary feeding back a correction signal to the primary control system. The advantage of the supplementary pressure sensor  27  over displacement sensor  21  which measures the displacement of membrane  16  is that the output signal provided by sensor  27  is independent from the pressure in the channel system formed e.g. by chamber  15 , channel  18  and chamber  23  in the embodiment shown by FIG. 8, which pressure is measured by sensor  22 , and it is independent from temperature changes of membrane  16  and its environment, temperature changes which can be caused, for example, by a thermopneumatic actuation of membrane  16 . 
     In addition to one or more micromechanical pipetting modules of the type described above, a micromechanical pipetting device according to the invention comprises control means (not represented in the enclosed drawings) for controlling the operation of the actuator means  19  in response to one or more output signals generated by sensor means which produce an output signal representative of the displacement of membrane  16  for effecting pipetting operations. Such output signals are for instance the output signal of sensor  21  in FIGS. 2,  8  and  9 , the output signal of sensor  22  in FIGS. 5,  8  and  9 , and the output signal of sensor  27  in FIG.  10 . 
     In the preferred embodiment described above with reference to FIGS. 1 a,    1   b,    2  and  3  the control means control the operation of the actuator means  19  in response to the output signal generated by sensor means  21 . 
     In the preferred embodiment described above with reference to FIGS. 4 a,    4   b,  and  5  to  7  the control means control the operation of the actuator means  19  in response to the output signal generated by sensor means  22 . 
     In the preferred embodiments described above with reference to FIGS. 8 and 9 the control means control the operation of the actuator means  19  in response to the output signals generated by sensor means  21  and  22 . 
     In the preferred embodiment described above with reference to FIG. 10 control means control the operation of the actuator means  19  in response to the output signals generated by sensor means  21 ,  22  and  27 . 
     In a preferred embodiment of the invention the micropipetting module  11  and the means for controlling the operation of the actuator means  19  are so configured and dimensioned that the total volume to be aspirated and dispensed with the pipetting tip  13  is aspirated into the pipetting tip  13  by means of a single stroke of the displacement movement of the membrane  16 . 
     The control means for controlling the operation of the actuator means  19  may be at least partially integrated in the structure of a micropipetting module according to the invention or they may be partially or to a large extent located outside the micropipetting module. 
     According to a further aspect of the invention several sensors like  21 ,  22  and  27  optimized for different measuring ranges can be integrated into a compact micropipetting module according to the invention. 
     According to a further aspect of the invention combined use of the output signals provided by sensors  21 ,  22  and  27  improves the reliability in the interpretation of the signals provided by the sensors and enables active monitoring of the liquid dispensed, which is important in order to avoid malfunctions of the micropipetting module, which can occur for instance due to clotting of the pipetting tip. Pipetting of air bubbles, which has to be avoided in medical diagnosis tests, can be detected by the use of pressure sensors, before the test is performed. Processing of information obtained with the pressure sensors during aspiration of the sample allows recognition of highly viscous patient samples. These pathogen samples often represent a problem for the correct interpretation of medical test results. 
     According to a further aspect of the invention a plurality of micromechanical pipetting modules like the above described modules  11  or  41  are integrally built on a silicon wafer  14 . 
     According to a further aspect of the invention, pipetting tip  13  is a silicon pipetting tip integrally built with the pipetting module  11  or  41 .