Abstract:
An air displacement pipetter comprises a guidance sleeve positioned within a hole in a cylinder block and a piston within a portion of the guidance sleeve a piston penetrating the cylinder block, a spring energized seal disposed within the cylinder block, a seal capture plate contacting the spring energized seal, a carrier plate, a pipette tip attached to the carrier plate, and a manifold plate between said carrier plate and said cylinder block.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to provisional application 60/778,759 filed on Mar. 3, 2006. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    This invention relates to air displacement pipetters. 
         [0004]    2. Description of the Related Art 
         [0005]    Air-displacement array pipetters are used extensively in the life-science and biotechnology arenas. These are designed to pipette in the Microliter range into and out of 96, 384, and 1536 assay plates that conform to the SBS microplate standard. 
         [0006]    As shown in  FIG. 1 , a typical array pipetter in the prior art consists of two parts. The first is a cylinder block  20  into which 96 or 384 pistons  22  are inserted into corresponding cylinders. The second is a tip array carrier  26  with pipetting tips  28  welded thereon. The tip array carrier  26  is mated to the cylinder block  20  with o-rings  30  to seal the tips  28  to their corresponding cylinders. This mating can be done statically by bolting the tip array carrier  26  to the cylinder block  20  or dynamically by using a robotic or other motorized mechanism to pick up the carrier  26  and fasten it to the cylinder block  20 . In use, the pistons  22  are coupled at their top ends to a robotic mechanism (not shown) that raises and lowers the pistons (typically all at once) to perform pipette operations to and from wells of multi-well plates. Typically, a pipetter includes an array of 96 or 384 
         [0007]    There are many disadvantages to this basic design. For example, a large number of o-rings  30  are needed to seal the tip array carrier  26  to the cylinder block  20 . When the array carrier  26  is removed from the cylinder block  20 , many of these o-rings  30  may stick to the mating surface on the cylinder block  20  and are thus removed from their seat grooves in the array carrier  26 . If automated tip array changing is implemented, any such loss of o-rings is unacceptable, as it will cause at least a partial loss of sealing. Even in the case of static mating, the situation is far from ideal because installing  96  or especially  384  small o-rings  30  is a burdensome task for the user. 
         [0008]    Furthermore, welding the tips  28  to an array carrier  26  results in a very rigid architecture. If one tip is damaged, the entire array carrier has to be replaced. Moreover, the array of 96 or 384 tips has to be used in its entirety. Selective pipetting to or from a subset of wells in a plate is not possible. For example, to perform serial dilution one may want to pipette from one row of wells to another row. This is impossible with a welded array carrier  26  that carries a complete rectangular array of tips  28 . 
       SUMMARY OF THE INVENTION 
       [0009]    The invention includes several different embodiments of air displacement pipetters and methods of making air displacement pipetters. 
         [0010]    In one embodiment, such an air displacement pipetter comprises a guidance sleeve positioned within a hole in a cylinder block and a piston positioned within the guidance sleeve. 
         [0011]    In another embodiment, an air displacement pipetter comprises a cylinder, a piston penetrating the cylinder, a spring energized seal disposed within the cylinder; and a seal capture plate contacting the spring energized seal. 
         [0012]    In a further embodiment, an air displacement pipetter comprises a carrier plate; 
         [0013]    a pipette tip attached to the carrier plate, and a manifold plate between the carrier plate and the pipette tip. 
         [0014]    A method of making an air displacement pipetter comprises inserting a pipette tip into a carrier plate, attaching a manifold plate to the carrier plate, and coupling multiple cylinders and/or pistons to the pipette tip. 
         [0015]    Another method of making an air displacement pipetter comprises inserting one or more sleeves into one or more corresponding cylinders of a cylinder block, inserting one or more seals on top of the sleeves, and inserting one or more pistons into the seals and sleeves. A first plate is placed over an upper portion of the pistons, the first plate containing holes through which the upper portions of the pistons extend. The one or more pistons are secured to the first plate. A second plate is placed over an upper portion of the pistons, the second plate containing holes through which the upper portions of the pistons extend. The one or more pistons are then secured to the second plate. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]    These and other aspects of the invention will be readily apparent from the description below and the appended drawings, in which like reference numerals refer to similar parts throughout, which are meant to illustrate and not to limit the invention, and in which: 
           [0017]      FIG. 1  is a cut-away view of a typical array pipetter. 
           [0018]      FIG. 2A  is a cut-away view of a pipetter of the present invention. 
           [0019]      FIG. 2B  is a detail cut-away view of the seal of  FIG. 2A   
           [0020]      FIG. 3  is a cut-away close-up view of a tip array carrier of the present invention. 
           [0021]      FIG. 4A  is a combination top view of a manifold plate and a cut-away relief view of the manifold plate along the line A-A. 
           [0022]      FIG. 4B  is a top view of an upper gasket mat for the manifold plate of  FIG. 4A . 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    Many of the above described drawbacks of the prior art are resolved by the embodiments of the invention illustrated in  FIGS. 2-4 .  FIG. 2  illustrates an embodiment of a cylinder block and actuating portion of a pipetting device.  FIGS. 3 and 4  illustrate embodiments of a tip array carrier portion of a pipetting device. The features of these two portions are advantageously utilized together in a single pipetter, but the features described herein have separate utility, and can be used in any of various combinations with each other and in pipetters that include otherwise conventional features. 
         [0024]    Referring now to  FIG. 2A , the upper portion of the pipetting device includes an actuating portion  34  that is attached to a set of pistons  36  in a cylinder block portion  38 . In operation, the actuating portion, described in greater detail below and as shown by arrows  32 , moves up to raise the pistons and aspirate liquid from the wells of a multi-well plate, and moves down to dispense aspirated liquid into wells of a multi-well plate. 
         [0025]    The cylinders  40  of the cylinder block  38  are provided with guidance sleeves  42  that line a portion of each cylinder  40 , and the pistons  36  are sealed at the top of the cylinders  40  with spring energized u-cup seals  46  instead of o-rings. In this embodiment, each seal  46  may made of Teflon and incorporates a preloaded spring that provides a constant grip on the piston  36 . A cross section of such a u-cup seal  46  is illustrated in  FIG. 2B . A polymer material  48  such as Teflon is formed with a channel  50 . A spring  52  resides in the channel. Seals with this construction are commercially available from, for example, Bal Seal Engineering of Foothill Ranch Calif. 
         [0026]    Sealing of the pistons  36  is accomplished through the use of these u-cup seals  200  because each cylinder&#39;s bore is large enough such that there is no contact between it and its cognate piston  36 . It is not economically feasible to use a tight fit between the piston and the metallic cylinder block—both of which are metallic, to achieve sealing action. Because the bore  40  has a larger diameter than the piston  36 , if the design only consisted of piston  36 , cylinder block  38 , and u-cup seal  46 , when the piston  36  is at its bottom-most position near the tip mating surface  54 , the end of the piston might well be significantly off centered with respect to the cylinder. For reasons described more fully below, it is advantageous for the tip of the piston at this position to be as centered as possible within its cylinder  40 . To help center the piston within the cylinder, the guidance sleeve  42  is provided. The inner diameter of this sleeve  42  is less than that of the cylinder and is very close to that of the piston  36 . This closer fit around the piston is not enough to provide sealing action but is sufficient to force the piston  36  to be reliably concentric with the cylinder. The sleeves  42  are made of a low-friction material such as nylon or acetal (Delrin) because if there is some residual concentric misalignment, slight rubbing between the two parts may occur. In addition to providing piston  36  alignment at the bottom-most position, the guidance sleeve  42  also helps with alignment at the seal position, protecting the u-cup seal  46  from damage that might occur if there were significant non-concentricity between seal  46  and piston  36 . The u-cup seals  46  are captured between the guidance sleeves  42  and a seal capture plate  56  that has an array of openings matching the array of bore holes  40 . 
         [0027]    This portion  38  of the apparatus is manufactured by machining bore holes through the cylinder block which are deeply countersunk to hold the guidance sleeves. The guidance sleeves are inserted into the bore holes to the bottom of their channels. The u-cup seals are installed into the bore holes onto the top of the guidance sleeves. The seal capture plate  56  may then be bolted onto the top of the cylinder block with its holes aligned with the bore holes in the cylinder block. 
         [0028]    The tops of the pistons  36  are captured between an upper movable plate  58  and a lower moveable plate  60  via an o-ring  62 , a washer  64 , and an e-clip  66 . When the two moving plates  58 ,  60  are attached (usually bolted), together, the o-ring  62  is compressed and the e-clip  66  is forced against moving plate  60 . The through holes in both moving plates  58 ,  60  are large enough to allow the pistons  36  to float laterally. At the top of the assembly is a flexible membrane  67  that covers the tops of cleaning channels  38  that are formed through the pistons. The flexible membrane  67  is used to allow a flow of cleaning fluid from the tops of the pistons through the channels  38  and out the bottom of the pistons to clean the pipette tips attached to the pistons. When the flexible membrane  67  is pulled away from the upper plate  58  during flow-through washing, cleaning fluids flow into the cavity and down through the pistons and out of the pipette tips. This aspect of the system illustrated in  FIG. 2  is described in detail in U.S. patent application Ser. No. 10/833,496, now U.S. Pat. No. ______, filed on Apr. 27, 2004, and hereby incorporated by reference in its entirety. 
         [0029]    To complete the assembly of both sections  34  and  38  of the apparatus, the pistons  36  are first inserted through the holes in the seal capture plate  56  and into the u-cup seals  46 , where they are allowed to find their optimal lateral positions within the u-cup seals  46 . Then the assembly of the two moving plates  58 ,  60  is undertaken. First, openings in the lower plate  60  are aligned with the pistons  36  and the lower plate  60  is placed over the pistons to rest on the seal capture plate  56 . The pistons are then retained on top of the lower plate  60  by plastic or metal deforming e-clips that are pressed into a slot on the piston above the lower plate  60 . The e-clips have an outer diameter larger than the holes in the lower plate so that when the lower plate  60  is raised, the pistons rise with it. Washers  64  and o-rings  62  are then placed on shoulders of the pistons. Then the upper plate  58  is placed over the pistons and onto the lower plate, compressing the o-rings between the bottom of a countersunk cavity in the upper plate and each washer  64 . The upper plate is then bolted or otherwise secured to the lower plate  60 . Because the through holes in these two moving plates have diameters larger than the portion of the pistons they are placed over, this second assembly will not perturb the optimal lateral positions that the pistons  36  have found for themselves when first inserted into the u-cup seals  46 . In addition to securing the floated pistons  36  in place, the compressed o-ring  62  also provides sealing between the piston body  36  and the upper moving plate  56 , thus maintaining continuity of the backwash cleaning system described above. This manufacturing process is advantageous as there are many sources of concentricity errors in the cylinder block  54 . The u-cup seals  46  cannot be guaranteed to be perfectly concentric. The center-to-center distance between cylinders cannot be perfectly uniform across 384 units, the perpendicularity among cylinders must have some imperfection, etc. 
         [0030]    This embodiment fulfills three main requirements. First, when 384 pistons arrayed in a rectangular matrix must penetrate into 384 cylinders, precise alignment is difficult. Any minor machining error in the piston array will result in misalignment for at least some of the pistons, which will cause excess wear and tear in the piston seals and/or leakage. The sleeves  42  and u-cup seals  46  help minimize these problems. Second, o-rings are conventionally used to provide sealing of the pistons but they are prone to failure. Replacing just one o-ring would require dismantling the entire pipetting head. The u-cup seals are more reliable than o-rings. Third, especially when flow-through washing capability is implemented, it is desirable to make their tips as concentric with the cylinder cross section as possible at the lower portion of their travel path. 
         [0031]    In preferred embodiments, the cylinder block/piston actuator described above is coupled to a novel tip array carrier. This is illustrated in  FIG. 3 . In this embodiment, the tip array carrier comprises four main components. These are a carrier plate  68  holding the pipette tips and into which the piston ends are inserted, an intermediate manifold plate  70 , two gasket mats  72 ,  74 , and the pipette tips  69  themselves. Instead of using individual o-rings around each pipette tip, the gasket mats  72 ,  74  perform the sealing function between the cylinder block and the tip array carrier. These may be made out of DMSO-resistant materials such as Chemraz and laser cut to achieve a desired hole pattern. In essence, one could think of these mats  72 ,  74  as o-rings that have been combined together into a single unit. In this embodiment, pipetting tips  69  are not soldered or welded to the carrier plate  68  but only inserted into counterbored through holes. Thus, individual tips  69  can be easily replaced or even omitted to allow for varying configurations of the same basic rectangular array. 
         [0032]    An important aspect of the embodiment illustrated in  FIG. 3  is the manifold plate  70 . The manifold plate contains channels that couple the pistons to the pipette tips. When the cylinder block and the pipette tip array are the same, the channels can be simply through holes connecting a piston to a corresponding cylinder. However, even though a tip array carrier with 384 tip mounting points can be fitted with 24, 96 or 384 tips, it is not feasible to design cylinder blocks with variable numbers of cylinders and pistons  102 . Hence the cylinder block and piston arrays are designed as separate fixed rectangular matrices of 24, 96, or 384 units. The manifold plate  70  is used to couple tip carriers with fewer than 384 tips to a cylinder block  54  with 384 pistons. 
         [0033]      FIG. 4  illustrates a manifold plate  70  that can be used to mate a 96-tip array to the 384-unit cylinder block.  FIG. 4A  illustrates how each tip  69  is addressed by four cylinders. The dark gray area is above the hatched area and represents a seal boss  82  used to reduce the force needed to provide proper sealing (described further below). The light gray area is below the hatched area and forms the channel that allows four cylinders to address the single pipetting tip. This embodiment illustrates the tip location  86  and its corresponding four piston locations  88 . Looking down on the manifold plate as in  FIG. 4A , during use, the pistons travel up and down together, with each group of four pistons  88  aspirating and dispensing fluid into and out of a single pipette tip couple to the pistons through the opening  86  in the manifold plate. 
         [0034]    This manifold plate  70  provides a lot of flexibility in configuring the tip array. Almost any arrangement of tips  69  can be made to work. There are assay plates whose footprints conform to the SBS standard but whose well arrays do not. In particular, the 24 wells of a Caco-2 assay plates are arranged in a rectangular array with a pitch of 19.3 mm, which is non-standard as the SBS basic pitch is 9 mm or multiples and fractions thereof. It is very easy to design a 24-tip array of this non-standard pitch and use an appropriate manifold plate  70  to mate it to the 384-unit cylinder block  54 . Using a manifold plate  70  is advantageous not only because it allows fewer than 384 tips to be addressed by 384 cylinders and pistons, but also because the manifold plate  70  allows several cylinders and pistons to be coupled to one tip  106 , thus increasing the volumes that can be pipetted. 
         [0035]    While the use of gasket mats  72 ,  74  overcomes the problem posed by individual o-rings, given the large surface area involved, a high compression pressure would be required to achieve proper sealing. If automated tip array changing is implemented, the tip array carrier is dynamically attached to the cylinder block, and this need for a high compression pressure can be a major obstacle as the required force may be beyond what a reasonable mechanism can achieve. 
         [0036]    This issue is addressed in two ways. First, the manifold plate is attached  70  (in this embodiment bolted) to the carrier plate  68  in a non-dynamic manner. Thus, the compression pressure required for the lower gasket mat  74  does not have to be provided by any mechanism needed for automated tip array changing. In order to precisely define the compression percentage of the lower gasket mat  74  and to keep the relatively thin manifold plate  70  flat, compression stops  80  are machined into the carrier plate. Compression stops  80  are raised islands on which the manifold plate  70  rests. The heights of the compression stops  80  are less than the uncompressed thickness of the lower gasket mat by a precise amount and thus precisely control the lower gasket  74  compression. The compression of the lower gasket mat  74  is determined not just to provide a tight seal but also to prevent any loose motion by the tips  69 . 
         [0037]    Second, as sealing is only needed in the areas around the cylinders, the manifold plate is machined with seal bosses  82  in those areas. Thus the effective compression area is greatly decreased, resulting in a significant reduction in the force needed to achieve sealing. 
         [0038]      FIG. 4B  illustrates the upper gasket mat  72  for the manifold plate  70  in  FIG. 4A . In this embodiment, the upper gasket mat  72  has a piston access hole  92  and a seal relief area  94 . The upper gasket mat  72  provides a seal during the above described tip washing procedure. During washing, fluids flow through the piston&#39;s hollow core and into the pipetting tip. But these fluids may wet the inside of the cylinder if they become trapped, and thus, might cause contamination and/or corrosion. One way to avoid this is to rest the end of the piston  36  on the upper gasket mat  72  so that a seal is formed there. This requires that the tip be well aligned with the corresponding hole in the upper gasket mat  304 , a requirement that can be satisfied if the piston  36  is concentric with the cylinder at its bottom-most position near the tip mating surface. 
         [0039]    With a pipetting system capable of automated tip array changing, multiple tip array and manifold configurations can be used within the same pipetting protocol, all driven by the same 384-piston head assembly. For example, to do compound titration in a 96-well plate, a tip array with a single column of 8 tips  106  is first used to perform serial dilution across the 12 columns. This tip array carrier is then exchanged for a full 96-tip array to replicate the master plate into several daughter plates. Such a protocol would be impossible without individually configurable tip arrays and flexible manifold design. 
         [0040]    It will be appreciated by those skilled in the art that various modifications and changes may be made without departing from the scope of the invention. Such modifications and changes are intended to fall within the scope of the invention, as defined by the appended claims.