Abstract:
Nanoliter pipette assembly. The assembly includes a housing containing a working fluid in a working fluid chamber therein and includes a moveable piston within the housing, the piston moveable by a linear actuation mechanism for contact with the working fluid. A tip portion is provided that includes a diaphragm deformable to engage an inner portion of the tip. It is preferred that the diaphragm have a projecting three-dimensional structure for direct contact with a liquid.

Description:
[0001]    This application claims priority to provisional application Ser. No. 62/334,709 filed on May 11, 2016, the contents of which are incorporated herein by reference in their entirety. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    This invention relates to a pipetting device for drawing and dispensing small liquid volumes from approximately 1-1000 nanoliters. 
         [0003]    Handheld pipettes are a ubiquitous tool; they are found across industry and academia in essentially all wet laboratories, and are essential to accurate laboratory work in the fields of chemistry, biology, and medicine. They are used for manipulating small volumes of fluid, and still make up the largest percentage of the US liquid handling market. Hand held pipettes offer the convenience, flexibility, ease of use, and low cost that more complex liquid handling solutions cannot offer. 
         [0004]    Handheld pipettes operate under very simple physical principles. They consist of several main components: an internal piston, a spring loaded plunger, a disposable tip, an adjustable stop, and a fixed stop. The pipette is held in one hand by the user, and a volume is selected by moving the adjustable stop. The user then presses the plunger down to the adjustable stop. This causes the piston to displace a volume, V p . The pipette is then lowered into the fluid and the plunger is slowly released. As the spring forces the piston to move back to its initial position, the pressure in the tip lowers and a volume, V p , of fluid is drawn into the tip. The fluid can then be dispensed into another container by depressing the plunger to the second stop. 
         [0005]    As previously mentioned, their ease of use and low cost has led to their widespread adoption; however, there are several important limitations of current handheld pipettes. Pipetting volumes smaller than 1000 nanoliters is challenging and typically imprecise (accuracy ˜25%). Volumes smaller than 100 nanoliters is currently inaccessible. Under the current pipette operating principles, in order to achieve such small volumes, extremely small piston diameters must be used, which are in most cases not manufacturable. Because the piston diameter is fixed, the range of volumes a given pipette can dispense is limited by the piston range. As a result, labs will often have to purchase a variety of pipettes to dispense volumes in every range they may need. Pipette volume resolution is determined by the positional resolution of the hard stop and the diameter of the piston. When purchasing a pipette there is often a tradeoff between resolution and range. 
         [0006]    Therefore, current pipetting technology is not suitable for manipulating smaller volumes. Reducing the volumes that a pipette can aspirate and dispense will allow labs to conserve resources, lower their costs, and perform more experiments. 
       SUMMARY OF THE INVENTION 
       [0007]    The nanoliter pipette assembly according to the invention includes a housing containing a working fluid in a working fluid chamber therein. A piston is provided that is moveable within the housing by a linear actuation mechanism for contact with the working fluid. A tip portion is provided having a diaphragm therein, the diaphragm deformable to engage an inner portion of the tip. The diaphragm and tip portion each include an orifice for aspirating and dispensing a selected fluid wherein the linear actuating mechanism, diaphragm properties and housing properties are selected so that the piston displaces a first volumetric amount of the working fluid on one side of the diaphragm and in which the diaphragm displaces a second volumetric amount of the selected fluid on the opposite side of the diaphragm via direct contact with the selected fluid. The second volumetric amount is less than the first volumetric amount providing a deamplification ratio. In a preferred embodiment, the diaphragm orifice includes a projection that mates with an orifice in the tip. The diaphragm, projection and internal surface of the tip portion are wetted by the selected fluid. A suitable working fluid is a compressible gas such as air. 
         [0008]    In another preferred embodiment, the adjustable diaphragm parameters include diaphragm radius, diaphragm thickness, diaphragm shear modulus and diaphragm pre-stretch. In yet another preferred embodiment, the linear actuation mechanism includes a series of cams to provide repeatability and adjustability of the selected fluid volumes. 
         [0009]    It is also preferred that the liquid to be drawn completely wets an exterior side of the diaphragm and an inner surface of the tip in such a manner that when the liquid is drawn there is no air, and thereby no liquid-air interface inside the pipette tip. It is also preferred that the projection on the diaphragm (nipple) protrudes through the orifice in the tip to provide direct contact with the liquid, so as to prevent the volume of drawn liquid from being influenced by capillary pressure that is a primary limitation to pipetting smaller volumes with current handheld pipettes. It is also preferred that the outer surface of the pipette tip be non-wetting so that liquid does not stick to it. It is further preferred that the orifice be small, on the order or tens of microns in lateral dimension to minimize volume loss due to evaporation for volatile liquids. 
         [0010]    It is also preferred that the diaphragm is elastic and that the working fluid is a compressible gas such as air. The invention disclosed herein allows the volume displacement of the diaphragm to be a scaled amount of the piston&#39;s displacement based on the diaphragm&#39;s stiffness and air&#39;s compressibility. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         [0011]      FIG. 1  is a cross-sectional view, along with detail, of an embodiment of the invention disclosed herein. 
           [0012]      FIGS. 2 a , 2 b , 2 c  and 2 d    are cross-sectional views of device tips showing a sequence of diaphragm positions. 
           [0013]      FIG. 3  is a cross-sectional view of a tip portion and diaphragm according to an embodiment of the invention. 
           [0014]      FIG. 4  is an exterior, schematic view of a nanoliter pipette tip according to an embodiment of the invention. 
           [0015]      FIG. 5  is a cross-sectional view of another nanoliter pipette tip suitable for practice of the invention. 
           [0016]      FIG. 6  is a cutaway, exploded, view of the device showing details of a tip suitable for use in the invention. 
           [0017]      FIG. 7  is a cross-sectional view of the tip portion of another embodiment of the invention. 
           [0018]      FIGS. 8, 9 and 10  are graphs of a calculated relationship of relevant volumes in embodiments of the invention. 
           [0019]      FIG. 11  illustrates a pressure relief system used to calibrate the pipette of the invention before use or adjustment. 
           [0020]      FIG. 12  is a cross-sectional view showing the specifics of a piston cam mechanism according to a preferred embodiment of the invention. 
           [0021]      FIGS. 13 a -13 h    are perspective views illustrating an eight-step breakdown of the cam mechanism disclosed herein. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0022]    As will become apparent from the following disclosure, a pipette tip, including a diaphragm, in combination with a novel piston linear actuation mechanism, may be configured as pan of a high-resolution pipette assembly, that can dispense volumes of fluid as small as one nanoliter. The components function via a volume deamplification concept in which a pipette piston displaces a volumetric amount of a working fluid on one side of the diaphragm placed in the tip and in which the diaphragm displaces a smaller volumetric amount of fluid at an opposite side of the diaphragm via direct contact with the fluid. This displacement reduction from one side of the diaphragm to the other may be characterized by a deamplification ratio that can span multiple orders of magnitude. One or more portions of a fluid chamber that encloses the working fluid may undergo elastic deformation to facilitate the deamplification. Additionally or alternatively, the working fluid may be compressible to contribute to the deamplification. The deamplification ratio and resolution may also be adjustable. 
         [0023]    Referring to  FIG. 1 , a schematic cross-sectional view of the nanoliter pipette is shown. The pipette assembly  10  consists of a tip  12 , a housing,  14 , a piston and accompanying mechanism  16 , and a diaphragm  18  constrained in the tip. The piston and diaphragm define an adjustable fluid chamber,  20 . The working fluid  22  is in contact with the piston  16  and the chamber side  24  of the diaphragm. The piston  16  is movable and displaces the working fluid  22  within the chamber  20 . The illustrated embodiment is not to scale. The tip  12  consists of several separate pieces that are used to form a fluid tight seal with the diaphragm  18  and the housing  14 , using sealing methods known to those skilled in the art. 
         [0024]    In operation, still referring to  FIG. 1 , the piston  16  moves to displace a volumetric amount of working fluid  22  within the fluid chamber  20 . The volume displaced, V p , by the piston  16  is equal to the product of the surface area of surface  26  and the distance  28  the piston has moved. The piston  16  is shown in this displaced position after being moved from its initial position shown in dashed lines. The volume displacement, V p , causes a corresponding volume displacement, V d , by the diaphragm  18 . 
         [0025]    Due to the small volumes the tip  12  will be handling, a novel piston and accompanying mechanism  16  has been designed. Details of how the piston and piston mechanism  16  deflect the diaphragm  18  to aspirate and dispense fluid can be seen in  FIGS. 2 a -2 d   , cross-sectional schematics of the pipette tip. In step  1  in  FIG. 2 a   ,  30 , the operator sets the stop to determine volume to dispense, V d . In step  2  in  FIG. 2 b   ,  32 , the operator depresses the piston  16  to the bottom of its stroke, deflecting the diaphragm  18  to its maximum position. At this point, a three dimensional feature  34  contacts the fluid. Because the pipette is dispensing fluids as small as 1 nL, evaporation becomes a concern. If proper design considerations are not made, a large percentage of aspirated fluid can evaporate in the time it takes to aspirate the fluid and dispense it in the appropriate container. An orifice  36  on the pipette tip  12  was designed to be extremely small, limiting evaporation. However, as this orifice  36  becomes smaller and smaller, the more difficult it becomes to aspirate and dispense fluid accurately due to an increase in capillary pressure. Therefore, the fluid facing surface of the diaphragm  18  is designed with a three dimensional feature  34 . During step  2  ( FIG. 2 b   ),  32  of the pipetting process, this feature comes in contact with the working fluid. The diaphragm  18 , the three dimensional feature  34  and the interior cavity of the tip  12  are configured to be wetting such that retraction of the diaphragm to a controlled position allows fluid to fill the cavity defined by the deflection of the diaphragm  18  and the retraction of the piston mechanism  16 . This is the motivation behind the design of the piston mechanism  16 . The diaphragm  18  must be deflected to its maximum position  32  first in order to come in contact with the fluid. Then in step  3 ,  FIG. 2 c   ,  38 , the piston  16  is retracted to the position shown in order to aspirate fluid volume, V d . In Step  4 ,  FIG. 2 d   ,  40 , the diaphragm  18  is once again deflected to its maximum position to dispense all the fluid. 
         [0026]    The working fluid  20  may be a compressible fluid such as air or some other gas. The compressible working fluid  22  compresses when the piston  16  moves against the working fluid  22  to displace it, resulting in an increased fluid chamber pressure. Here, the working fluid acts to temporarily store a portion of the work energy transferred thereto by the piston. In one embodiment, the diaphragm  18  undergoes elastic deformation and the working fluid is compressed when the piston  16  moves against the working fluid  22  to displace it. Thus, diaphragm elasticity and working fluid compressibility may be used in various combinations to arrive at the desired deamplification ratio. 
         [0027]    A set of three nanoliter pipette tips  12  has been designed to exhibit the configuration stated above. Each tip  12  possesses different dimensions and initial conditions.  FIG. 3  illustrates which dimensions can be varied. The diaphragm radius,  42 , the diaphragm thickness,  44 , the diaphragm shear modulus.  46 , the diaphragm pre-stretch,  48 , and the size of the fluid chamber  20 . Changing the dimensions allows the pipette assembly  10  to behave differently based on which tip  12  is selected by the operator. Different tips can cause the pipette  10  to have different volume ranges and resolutions. 
         [0028]      FIGS. 4-7  illustrate our initial embodiment of the nanoliter pipette tip  12 . This tip can dispense volumes ranging from 1-10 nl.  FIG. 4  is a front view of the tip  12 . The tip  12  will screw onto the housing  14  and form a fluid tight seal.  FIG. 5  is a cross section of the 10 nl tip  12 . The tip  12  is composed of several key components, all critical to the assembly and functionality of the tip. The tip  12  is composed of two main pieces, tip bottom  50  and tip top  52 . An exploded view of tip bottom  50  and its mating components can be seen in  FIG. 6 . The diaphragm  18  is secured to the membrane clamp  54  via adhesive  56 . The adhesive makes assembly easier and holds the diaphragm pre-stretch  48 . A detailed view of tip bottom  50 , membrane clamp  54 , the diaphragm  18 , the adhesive  56 , and the three dimensional diaphragm feature  34  can be seen in  FIG. 7 . In one embodiment, the raised feature  34  on the diaphragm is a glass microsphere that will be secured to the diaphragm via an adhesive, or can be formed as a monolithic feature of the diaphragm such as by a molding technique.  FIG. 5  also features many other components in an example embodiment. A machined nut  58  secures the assembly and provides pre-load by compressing a spring  60 . The preload allows for fine tuning of the compressive forces on the membrane  18 . A PET washer  62  acts as a thrust bearing to prevent any torsional stress from getting to the membrane  18  via the nut  58 . A gasket  64  acts to seal tip top  52  and tip bottom  50 . 
         [0029]      FIG. 12  delineates the specifics of the piston cam mechanism  92 - 98  and the chamber  78 , which corresponds with the housing  14  in  FIG. 1 . The chamber  78  holds a sealed working volume, V o , that comes in direct contact with the diaphragm  18  in the tip  12 . V o  can be adjusted to the correct volume via a side screw  76 . The side screw  76  is preloaded via the side spring  74  to ensure that the screw does not move during operation. Additionally, a side o-ring  66  provides a fluid seal to ensure that there is no leakage in the system. The chamber  78  is connected to the tip  12  and the exterior body  86  via threads, M6 and ⅞″-14 respectively. Similarly to the side screw  76 , the tip  12  is sealed via a gasket  64  and the exterior body is sealed via the top o-ring  80 . Above the chamber is the dynamic portion of the mechanism as parts  82 ,  84 ,  88 ,  90 ,  92 ,  94  and  104  are all in motion, both vertical and rotational. The piston  82  fits into the chamber  78  and when its motion is directly coupled to that of V p . It is also press fit into the interior cap  88 . 
         [0030]    Around the piston  82 , is the piston spring  84 . The piston spring  84 , compresses during operation and provides an upward bias to the cams,  92  &amp;  94 , via the interior cap  88  and lead screw  104 . The lead screw  104  is fitted into the top of interior cap  88  and is mated with the threaded bushing  90 . The threaded bushing  90  is press fit into the variable cam  92 . The motion and dynamics of four cams mechanisms,  92 - 98  are described below. The exterior cams  96  &amp;  98  are held in place via a shoulder in the exterior body  86  and a top spacer  100 . The top spacer is bolted into the exterior body  86  via four 4-40 screws of length 0.3125″  102 . The thumb push  110  is coupled to the thumb connector  106  via a bearing  108  that is press fit onto both pieces. 
         [0031]    In the exterior body  86  rests the cam mechanisms  92 - 98 , which along with the actual piston  82  correspond to  16  in  FIG. 1 . These series of cams provide the repeatability and adjustability required to handle the small volumes of fluid. The cam mechanisms are made up of the exterior cam top  98 , exterior cam bottom  96 , interior cam  94  and the variable cam  92 . The exterior cams  96 ,  98  fit together with mirrored offsets and rest on a shoulder in the exterior body  86 . These two cams do not move during the pipetting process. During operation the interior cam  94  and variable cam  92  move up and down and rotate about the vertical axis. Before operation, the two cams can move independent of each other through the use of the lead screw  104  and the threaded bushing  90 . Rotation of the lead screw, which is done manually by turning the thumb connector  106 , moves the variable cam  92  up and down relative to the interior cam  94 . 
         [0032]      FIGS. 13 a   - 13 - h  show an 8-step breakdown of the cam mechanism in operation. The interior and variable cams  92 ,  94  start in position I) continue as follows: 1) the thumb push  110  is depressed causing the interior and variable cams  92 ,  94  to move down until the top face of the exterior cam  96  comes in to contact with the face of the interior cam  94  2) the interior and variable cams  92 ,  94  continue to move down and rotate 22.5 degrees due to the angular face mate to arrive at position C, 3) the thumb push  110  is released and the bottom face of the exterior cam  96  comes in to contact with the face of the variable cam  92 , 4) the interior and variable cams  92 ,  94  continue to move up and rotate another 22.5 degrees to arrive at position E, 5) the thumb push  110  is again depressed and the interior and variable cams  92 ,  94  move down until the top face of the exterior cam  96  comes in to contact with the face of the interior cam  94 , 6) the interior and variable cams  92 ,  94  continue to move down and rotate 22.5 degrees due to the angular face mate to arrive at position G, 7) the thumb push  110  is released and the bottom face of the exterior cam  96  comes in to contact with the face of the interior cam  92 , 8) the interior and variable cams  92 ,  94  continue to move up and rotate another 22.5 degrees to arrive back at position I). Note: all rotation is counterclockwise. 
         [0033]      FIG. 11  shows the pressure relief system that is used to calibrate the pipette before every use or adjustment. It is necessary for the pipette to have this capability so that the desired deamplification ratio can be achieved. The relief slider  68  can be easily pulled down to expose a relief cavity that connects directly to the inside of the sealed working fluid  20  in the chamber  78 . This relief cavity is sealed by an o-ring that is not pictured in the figures. The relief slider  68  is held in place by a thin shim  70  that is mounted to the chamber  78  via two 4-40 screws with a length of 0.25″  72 . 
         [0034]    The volume deamplification principles described above and the design of a pipette tip  12  and piston mechanism  16  in accordance with the present teachings is guided by a mathematical model detailed in our earlier patent application US20130283884 A1. Using this model, pipette tip values can be selected to achieve desired pipetting performance. 
       Example 
       [0035]    Three different pipette tips have been designed and manufactured. All three tips are compatible with the same chamber  78  and piston/cam mechanism  82 ,  92 - 98 . The first tip has the ability to dispense fluids in the range of 1-10 nl. A graph of the calculated relationship V d  vs V p  can be seen in  FIG. 8 . It has a volume deamplification ratio V d /V p ===49600. The second tip was designed to dispense volumes within the range of 10-100 nl. Its graph can be seen in  FIG. 9 . It has a volume deamplification ratio V d /V p =4960. Finally.  FIG. 10  presents the final pipette tip which can dispense volumes of 100-1000 nl. It possesses a volume deamplification ratio of V d /V p =496. As can be seen in the  FIGS. 8-10 , the function V d (V p ) is most accurately modeled as a third order polynomial, but with careful selection of pipette tip parameters, the diaphragm radius  42 , the diaphragm thickness  44 , the diaphragm shear modulus  46 , the diaphragm pre-stretch  48 , and the initial volume of the fluid chamber  20 , V d (V p ) acts approximately linear over the entire stroke of the pipette. Linearity of this function is crucial to making the pipette intuitive to use, simplifying mechanical design, and thus lower costs. 
         [0036]    It can be appreciated that, based on the principles above and using suitable fabrication methods known to those skilled in the art, the design may be scaled to manipulate volumes smaller or larger than ˜1-1000 nl. The pipette device could also be used to manipulate materials other than liquids, or liquids containing soft solids, for example biological cells. Other considerations may include electrical contact to the diaphragm and/or tip, such that electrical signals can be applied when the tip is in contact with solids and/or liquids. The design may also be employed in other configurations, such that multiple tips are arrayed in close proximity, driven by one or more piston mechanisms, which may be manual or motorized. In one example, an array of diaphragms, each within its own tip, is in contact with a single piston via a common volume of working fluid. The characteristics of the diaphragms within the array may be chosen to be the same, or to vary in a prescribed manner. 
         [0037]    Additional information about the present invention may be found in “Universal Handheld Micropipette” Review of Scientific Instruments 87, 115112(2016) and in United States published patent application US2013/0283884. The contents of both of these references are incorporated herein by reference in their entirety. 
         [0038]    It is recognized that modifications and variations of the present invention will be apparent to those of ordinary skill in the art and it is intended that all such modifications and variations be included within the scope of the appended claims.