Abstract:
Devices and methods for integrated packaging, shipping, storage and precise dispensing of extremely small volumes of liquids such as aqueous solutions and compounds dissolved in organic solvents are disclosed. Devices of the invention include a sealed reservoir with an integrated metering tap. The tap includes a metering tube, which is translatable between a fill position inside the reservoir and an expel position outside the reservoir. The metering tube includes: (1) a tube end closure in a lower portion of the tube, (2) a port above the tube end closure, and (3) a piston in an upper portion of the tube. The piston is movable between a down position that seals the side port and an up position above the port. Movement of the piston from the up position to the down position can displace from 10 nanoliters to 20 microliters, e.g., from 20 nanoliters to 2 microliters, or 50 nanoliters to 500 nanoliters. Integrated arrays of reservoir/tap units are suitable for use in automated, multiwell formats such as those commonly used for high-throughput screening.

Description:
This application claims priority from U.S. provisional application Ser. No. 60/185,810, filed Feb. 29, 2000. 
    
    
     TECHNICAL FIELD 
     This invention relates to microfluidics and laboratory automation. 
     BACKGROUND 
     The development of automated combinatorial chemistry systems and ultra-high throughput screening systems have dramatically increased the number of compounds per unit time being synthesized and screened in drug discovery programs. Such technology involves rapid handling of large numbers of very small samples. For example, thousands of new compounds per week may be produced, with each compound being concentrated in a total volume of only 50 microliters. Microliter amounts of sample often must suffice for hundreds of screening assays. Conventionally, aliquots of the concentrated, liquid sample are dispensed using “sip and spit” liquid handling technology, diluted in an appropriate medium, and re-dispensed into an assay mixture, again using sip and spit technology. This “reformatting” process adds complexity to the overall process, thereby increasing time and cost per assay. In addition, reformatting generates waste of valuable sample material. 
     SUMMARY 
     The invention features a method of packaging a multiplicity of liquids for shipment, storage and metered dispensing. The method includes: (a) providing an integrated array of isolated reservoir units alignable with an array of liquid-receiving units (LRUs); (b) dispensing the liquids into the array of reservoir units; and (c) incorporating a dispensing tap into each reservoir unit to form a reservoir/tap unit sealed against spillage or leakage of the liquids. Preferably, the reservoir units are also sealed against air and light. The array of LRUs can be a multiwell container such as a 96-well microtiter plate, a 384-well microtiter plate, or a 1536-well microtiter plate. In preferred embodiments, each tap includes a translatable metering tube, which contains a tube end closure, a port, and a translatable piston. In some embodiments, the liquid is a solution of one or more chemical compounds. In some embodiments, liquid-contacting surfaces of the reservoir and tap are resistant to damage by acids, bases, salts and organic solvents. 
     The invention also features a method for independently dispensing a metered amount of a plurality of liquids into an array of LRUs. The method includes: (a) providing an array of isolated, sealed, tapped reservoir units, the array of reservoir units including a reservoir for each LRU, each reservoir unit containing an integrated metering tap; (b) aligning the array of reservoir/tap units with the array of LRUs so that each tap is aligned with one LRU; and (c) actuating one or more taps in the array of reservoir units so that each actuated tap dispenses a metered amount of liquid into the LRU aligned with that tap. The metered amount dispensed into any particular unit in the array can be from zero nanoliters to 20 microliters, preferably from 20 nanoliters to 2 microliters, e.g., 50 nanoliters to 500 nanoliters. 
     Preferably no tap contacts an LRU surface, and the liquid dispensed from each tap breaks contact with the tap before contacting the LRU aligned with that tap or the contents of an LRU. Preferably, the reservoirs are sealed against air and light. The array of reservoir units can be aligned directly above the array of LRUs. In some embodiments, each tap can be actuated independently. Preferably, each tap contains minimal (or substantially zero) dead volume. Examples of suitable LRUs are multi-well containers such as a 96-well microtiter plate, a 384-well microliter plate and a 1536-well microtiter plate. 
     In some embodiments of the dispensing method, each tap includes a translatable metering tube, which can contain a tube end closure, a port and a translatable piston. Actuating the tap can include translating the tube so that the port is inside the reservoir; drawing liquid from the reservoir through the port and into the tube; translating the tube so that the port is outside the reservoir; and expelling liquid from the tube through the port and into a fluid output channel. The liquid can be drawn into the tube and expelled from the tube by translating the piston. Some embodiments include propelling the expelled liquid away from the port. Propelling the expelled liquid can be achieved by applying a propelling fluid to the expelled liquid. The propelling fluid can be a propelling liquid, e.g., an aqueous liquid or an organic solvent; or a propelling gas, e.g., air, nitrogen or argon. Some embodiments of the method include providing a curtain of forced gas surrounding the fluid output tip, with the forced gas moving in the same direction as the liquid exiting from the fluid output tip. 
     The invention also features devices for storing, shipping and dispensing metered, nanoliter or microliter amounts of liquid into a liquid receiving unit. 
     An offset nozzle-type device includes: an array of isolated, sealed, reservoir/tap units, each unit containing an integrated metering tap, each tap including: (a) a metering tube translatable between a fill position inside the reservoir and an expel position outside the reservoir. The metering tube includes (1) a tube end closure, e.g., a plug, in a lower portion of the tube, (2) a port above the tube end closure, and (3) a piston in an upper portion of the tube. The piston is movable between a down position that seals the port and an up position above the port; and (b) a fluid output channel having an upper portion in fluid communication with the port when the tube is in the expel position and a lower portion terminating in a fluid output tip. A compressed gas path in fluid communication with the fluid output channel at a point upstream of the port when the tube is in the expel position can be used to apply a gas stream to propel the expelled liquid through the fluid output channel. Some embodiments include a compressed gas path terminating in an annular opening surrounding the fluid output tip. 
     An in-line nozzle embodiment of the device includes an array of isolated, sealed reservoir/tap units, each unit containing an integrated metering tap, each tap including: (a) a metering tube translatable between a fill position inside the reservoir and an expel position outside the reservoir. The metering tube contains (1) a tube end closure in a lower portion of the tube, (2) a port above the tube end closure, and (3) a piston in an upper portion of the tube. The piston is movable between a down position that seals the port and an up position above the port; and (b) a nozzle containing a fluid output channel through which the tube extends when in the down position, the fluid output channel having an upper end in fluid communication with a compressed gas path, and a lower end terminating in a nozzle tip. 
     A nozzleless-type device includes an array of isolated, sealed reservoir/tap units, each unit containing an integrated metering tap. Each metering tap including a metering tube translatable between a fill position inside the reservoir and an expel position outside the reservoir. The metering tube contains (1) a tube end closure in a lower portion of the tube, (2) a port above the tube end closure, and (3) a piston in an upper portion of the tube. The piston is movable between a down position that seals the port and an up position above the port. Each unit contains a compressed gas path, which includes one or more compressed gas outlets located above the port so that it can deliver a downward gas stream across the port, when the metering tube is in the expel position. 
     In each of the above devices, movement of the piston from the up position to the down position can displace, for example, 10 nanoliters to 20 microliters, preferably from 20 nanoliters to 2 microliters, e.g., 50 nanoliters to 500 nanoliters. The array of reservoir units can be arranged so that each tap aligns with one well of a multi-well container such as a 96-well microtiter plate, a 384-well microtiter plate or a 1536-well microtiter plate. However, with suitable equipment, any particular tap can be positioned to dispense into any chosen well. 
     As used herein, “liquid-receiving unit” (LRU) means: (a) a defined or addressable area on a flat liquid-receiving surface, e.g., a glass slide; (b) a depression or well in a liquid-receiving container, e.g., a microtiter plate, or (c) a receptacle, e.g., a test tube, vial or bottle. 
     As used herein, “reservoir/tap unit” means a single tapped reservoir. 
     The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. 
    
    
     DESCRIPTION OF DRAWINGS 
     FIG. 1 is a perspective view of a device for integrated storage and single-channel dispensing of small volumes of liquids. The depicted device contains a 96-unit array of integrated reservoir/tap units. The 96 units are arranged so that each of the 96 tips aligns with one well of a conventional 96-well microtiter plate. 
     FIG. 2 is a sectional view of a single reservoir/tap unit. The unit has a metering tube which is in the up position. FIG. 2 depicts an offset nozzle embodiment. 
     FIG. 3 is a sectional view of the reservoir/tap unit shown in FIG. 2, but with the metering tube in the down position. 
     FIG. 4 is a detail from FIG. 2 . The enlarged detail view shows the tube in the up position, and a piston in the tube. The piston is in the down position, where it rests against a tube plug. 
     FIG. 5 is the same as FIG. 4, except that the piston is raised into an up position. 
     FIG. 6 is an enlarged, front view of a metering tube. A tube port, through which liquid enters and leaves the tube is visible near the lower end of the tube. 
     FIG. 7 is a detail enlarged from FIG. 6, showing the tube port. 
     FIG. 8 is an enlarged sectional view (rotated 90° relative to FIG. 6) showing a lower portion of the tube, the tube plug, and a lower portion of the piston. 
     FIG. 9 is a sectional view (detail) of a reservoir/tap unit in which the metering tube is in the down position, and the piston is in an up position. 
     FIG. 10 is a sectional view (detail) of a reservoir/tap unit in which the metering tube is in the down position, and the piston is in a down position. 
     FIG. 11 is the same as FIG. 10, except that it shows a metered amount of liquid in an upper region of a fluid flow path, and arrows indicating flow of compressed gas through a compressed gas path. 
     FIG. 12 is the same as FIG. 11, except that it shows the metered amount of liquid in a middle region of the fluid flow path, and arrows indicating flow of compressed gas sweeping the liquid down the fluid flow path. 
     FIG. 13 is the same as FIG. 12, except that it shows the liquid in the lowermost portion of the fluid flow path, where the liquid is exiting from a flow path tip. 
     FIG. 14 is a sectional view of a single reservoir/tap unit. The unit has a metering tube in the up position. FIG. 14 depicts an in-line nozzle embodiment. 
     FIG. 15 is a sectional view of the reservoir/tap unit shown in FIG. 14, but with the metering tube in the down position. 
     FIG. 16 is a detail from FIG. 14 . The enlarged detail view shows the tube in the up position, and a piston in the tube. The piston is in the down position, where it rests against a tube plug. 
     FIG. 17 is the same as FIG. 16, except that the piston is raised into an up position. 
     FIG. 18 is a sectional view (detail) of a reservoir/tap unit in which the metering tube is in the down position, and the piston is in an up position. 
     FIG. 19 is a sectional view (detail) of a reservoir/tap unit in which the metering tube is in the down position, and the piston is in the down position. FIG. 19 shows a bolus of expelled liquid emerging from a port in the side of the tube. 
     FIG. 20 is a sectional view (detail) of a reservoir/tap unit (in-line nozzle) in which the metering tube has been withdrawn from the down position to the up position, after expulsion of a bolus of liquid. The liquid has been drawn into the fluid output channel in the nozzle. 
     FIG. 21 is a sectional view of a single nozzleless tapped reservoir unit with the metering tube in the up position. 
     FIG. 22 is a sectional view of a nozzleless reservoir/tap unit with the metering tube in the down position. A bolus of expelled liquid is shown at the port. 
     FIG. 23 is a sectional view of a nozzleless reservoir/tap unit with the metering tube in the down position. A bolus of expelled liquid is shown in flight after being propelled from the tip of the metering tube. 
     FIG. 24 is a detail from FIG.  21 . The enlarged detail view shows the metering tube in the up position and the piston in the down position. 
     FIG. 25 is the same as FIG. 24, except that the metering tube is in the up position and the piston is in the up position. 
     FIG. 26 is the same as FIG. 24, except that the metering tube is in the down position and the piston is in the up position. 
     FIG. 27 is the same as FIG. 24, except that the metering tube is in the down position and the piston is in the down position. 
     FIGS. 28A-28F are sectional views depicting a device and sequence of events in a preferred packaging method. 
    
    
     Throughout the various drawings, like reference numbers indicate like elements. 
     DETAILED DESCRIPTION 
     The invention provides methods and devices for integrated packaging, shipping, storage, and dispensing of extremely small volumes of liquids, e.g., aqueous solutions and compounds dissolved in organic solvents, in an automated, multi-well format of the type used in high throughput screening (HTS) or ultra-high throughput screening (UHTS). By virtue of a metering tap integrated with each reservoir in an array of reservoir/tap units, the invention advantageously avoids the use of conventional sip and spit technology. Consequently, multi-well plate assays can be performed without reformatting, i.e., transferring aliquots of concentrated samples from storage plates to working plates, diluting on working plates, transferring diluted samples from working plates to assay plates, etc. This maximizes speed and efficiency. Entire sets of samples, e.g., compounds for screening, can be stored and/or shipped conveniently in a single cassette, which can be plugged into an HTS or UHTS system, where nanoliter volumes of concentrated sample can be dispensed directly onto assay plates without reformatting. Because the reservoir/tap units in an array are isolated from each other, single-channel dispensing is achieved, and each reservoir/tap unit is individually addressable. Because each reservoir/tap unit in an array (cassette) can be sealed against air, moisture and light, labile compounds can be stored and handled under favorable conditions. 
     FIG. 1 is a perspective view of a device  10  according to the invention for storing and dispensing liquid into a conventional 96-well microtiter plate  12 . Protruding from lower surface  13  of device  10  are 96 flow tips  11  arranged so that when device  10  is aligned above 96-well microtiter plate  12 , each tip  11  is above a different one of the 96 wells  14  in plate  12 . On the upper surface  15  of device  10  are 96 mechanical interfaces  16  for tap actuation. Operation of each interface  16  actuates a tap whose flow path tip  11  is located beneath that interface  16 . 
     FIG. 2 is a sectional view of a single reservoir/tap unit  20 . The unit  20  contains a reservoir  21  formed by a cylinder wall  22 , sliding seal  23  and lower seal  24 . The unit  20  also contains a metering tube  25 , tube handle  26 , tube handle spring  27 , piston handle  28 , piston  29 , and piston handle stop  30 . The embodiment depicted in FIG. 2 is an example of an offset nozzle embodiment, because nozzle tip  11  is not directly in line with metering tube  25 . FIG. 2 shows the tube  25  and tube handle  26  in the up position. Tube handle  26  and piston handle  28  are included in each mechanical interface  16  shown in FIG.  1 . 
     Reservoir  21  contains minimal air space. Therefore, liquid in reservoir  21  is essentially coextensive with the volume of reservoir  21 . As liquid is metered from reservoir  21 , sliding seal  23  slides downward reducing the volume of reservoir  21  so that remnant liquid in reservoir  21  remains coextensive with the volume of reservoir  21 . FIG. 3 is a sectional view of a single unit  20  in which the tube  25  is in the down position. 
     FIG. 4 is a detail from FIG. 2, in which tube  25  is in the up position and piston  29  is in the down position. In the down position, piston  29  rests against tube plug (tube end closure)  31  so that piston  29  closes and seals tube port  32 , thereby blocking entry of liquid from reservoir  21  into tube  25 . FIG. 5 is the same as FIG. 4, except that piston  29  is raised into an up position. Raising piston  29  opens tube port  32  and draws a metered amount of liquid from reservoir  21  into tube  25 , with the metered amount depending on the height to which piston  29  is raised. 
     FIG. 6 is an enlarged, front view of metering tube  25 , showing tube port  32 . FIG. 7 is a detail from FIG. 6, showing tube port  32 . FIG. 8 is an enlarged sectional view (rotated 90° relative to FIG. 6) showing a lower portion of tube  25 , tube plug  31 , and a lower portion of piston  29 . 
     In illustrating operation of device  20 , FIG. 9 is sequential, following FIG.  5 . In FIG. 9, metering tube  25  has been translated downward into the down position, with piston  29  remaining in the up position, i.e., same position relative to tube  25 . In FIG. 9, downward translation of tube  25  through lower seal  24  has taken port  32  out of reservoir  21  and placed port  32  in fluid communication with fluid output channel  33 . The next sequential step is lowering of piston  29  into the down position, in which piston  29  rests against tube plug  31 . This lowering of pistion  29  expels liquid (not shown) from tube  25  and into fluid output path  33 . 
     FIG. 11 is the same as FIG. 10, except that it shows the expelled liquid  40  in an upper region of fluid output channel  33 , and arrows A indicating flow of compressed air through a compressed gas path  34 , and exit of the compressed air from an annular compressed gas outlet  35  surrounding nozzle tip  11 . The exiting air forms an annular curtain of air moving downward and surrounding a droplet of liquid that will exit from nozzle tip  11 . The annular curtain of air facilitates controlled movement of the droplet into the correct well, and effectively isolates all droplets and corresponding wells from each other. 
     FIG. 12 is the same as FIG. 10, except that it shows a metered amount of expelled liquid  40  (bolus), in a middle region of fluid output channel  33 , and arrows B indicating flow of compressed air in fluid output channel  33 . Air flowing from compressed gas inlet  59  through fluid output channel  33  sweeps liquid  40  down fluid output channel  33 . FIG. 13 shows liquid  40  in the lowermost portion of fluid output channel  33 , where it is exiting nozzle tip  11 . 
     FIG. 14 is a sectional view of a single reservoir/tap unit  20 . The unit  20  contains a reservoir  21  formed by a cylinder wall  22 , and lower seal  24 . The unit  20  also contains a metering tube  25 , tube handle  26 , piston handle  28 , piston  29 , and piston handle stop  30 . The embodiment depicted in FIG. 14 is an example of an in-line nozzle embodiment, because nozzle tip  11  is directly in line with metering tube  25 . FIG. 14 shows the tube  25  and tube handle  26  in the up position. Tube handle  26  and piston handle  28  are included in each mechanical interface  16  shown in FIG.  1 . FIG. 15 is a sectional view corresponding to FIG. 14, except that tube  25  is in the down position. 
     FIG. 16 is a detail from FIG. 14, in which tube  25  is in the up position and piston  29  is in the down position. In the down position, piston  29  rests against tube plug  31  so that piston  29  closes and seals tube port  32 , thereby blocking entry of liquid from reservoir  21  into tube  25 . FIG. 17 is the same as FIG. 16, except that piston  29  is raised into an up position. Raising piston  29  opens tube port  32  and draws a metered amount of liquid from reservoir  21  into tube  25 , with the metered amount depending on the height to which piston  29  is raised. 
     In FIG. 18, metering tube  25  has been translated downward into the down position, with piston  29  remaining in the up position, i.e., same position relative to tube  25 . In this in-line nozzle embodiment of the invention, tube  25  passes through fluid output channel  33  as it translates between the up position and the down position. In the down position, port  32  is beneath nozzle tip  11 . The next sequential step is lowering of piston  29  into the down position, in which piston  29  rests against tube plug  31 , as shown in FIG.  19 . This lowering of piston  29  expels liquid from tube  25  through port  32 . 
     A bolus of expelled liquid  40  is shown in FIG.  19 . Expelled liquid  40  clings to the side of tube  25  as a result of surface tension and adhesion. As tube  25  is retracted, nozzle tip  11  forces expelled liquid  40  to slide down the outside of tube  25 . During retraction, when bottom end  42  of tube  25  reaches lower end  44  of fluid output channel  33 , expelled liquid  40  migrates to bottom end  42  of tube  25  and clings there. As tube  25  is further retracted, expelled liquid  40  follows bottom end  42  of tube  25  upward through fluid output channel  33  (FIG.  20 ). When bottom end  42  of tube  25  reaches upper end  43  of fluid output channel  33 , expelled liquid  40  detaches from bottom end  42  of tube  25  and remains in upper portion of fluid output channel  33 . When tube  25  is fully retracted into up position, compressed air enters compressed gas path  34  and pushes expelled liquid  40  downward, so that it exits nozzle tip  11  and falls into a well in a microtiter plate (not shown). 
     In in-line nozzle embodiments of the invention, nozzle  45  preferably is made of an elastomeric material, with fluid output channel  33  having an inside diameter slightly smaller than the outside diameter of tube  25 . Fluid output channel  33  expands slightly to accommodate tube  25 , as the tube passes through the fluid output channel. This promotes an airtight seal between tube  25  and fluid output channel  33 , when the tube is in the channel. Selection of a suitable elastomer is within ordinary skill in the art. 
     FIGS. 21-27 depict a nozzleless tapped reservoir. In FIG. 21 reservoir/tap unit  20  has metering tube  25  in the up position and piston  29  in the down position. FIG. 24 is a detail from FIG. 21 in which tube  25  is in the up position and piston  29  is in the down position. In the down position, piston  29  rests against tube plug (tube end closure)  31  so that piston  29  closes and seals tube port  32 , thereby blocking entry of liquid  40  from reservoir  21 . FIG. 25 is the same as FIG. 24, except that piston  29  is raised into an up position. Raising piston  29  opens tube port  32  and draws a metered amount of liquid from reservoir  21  into tube  25 , with the metered amount depending on the height to which piston  29  is raised. In FIG. 26, metering tube  25  has been translated downward into the down position, with piston  29  remaining in the up position. In this nozzleless embodiment of the invention, no fluid output channel or nozzle is necessary, and port  32  and fine point  46  are exposed (FIG.  22 ). The next sequential step is lowering of piston  29  into the down position, where it rests against tube plug  31 , (FIG.  27 ). This expels a precisely metered amount of liquid  40  through port  32 . Expelled liquid  40  is then swept downward by a downward flow of air from compressed gas outlet  35 . FIG. 23 shows expelled liquid  40  dropping from bottom end  42  of metering tube  25 , which is tapered to a fine point  46 . Fine point  46  facilitates release of expelled liquid  40  from bottom end  42  of metering tube  25  in a controlled manner. In some embodiments of the invention, a shroud  57  surrounds or partially shields lower end  48  of metering tube  25 , which extends downward when tube  25  is in the expel position. 
     FIGS. 28A-28F depict a device and sequence of events in a preferred packaging method according to the invention. Two reservoir/tap units in an array are depicted. In FIG. 28A fill pin  51 , which has an outside diameter equal to that of metering tube  25 , extends upward through lower seal  24 . This permits dispensing of liquid  40  into reservoir  21  from reservoir filling device  52  positioned above the reservoir. In FIG. 28B cap seal  53  is installed on top of reservoir  21 . In FIG. 28C metering tube  25  is aligned directly above fill pin  51 , so that tube bottom end  42  contacts upper end  54  of fill pin  51 . In FIG. 28D metering tube  25  is lowered so as to push fill pin  51  downward. By this process, metering tube  25  replaces fill pin  51  without allowing leakage of liquid  40  from reservoir  21 . In FIG. 28E metering tube  25  is seated against lower seal  24 . In FIG. 28F protective cover  55  is installed on bottom of device  20  for storage. In FIG. 28E, it can be seen that the lower portion of the device serves as a shroud  49  around lower end  48  of metering tube  25 . 
     Devices according to the invention can be designed for compatibility with various liquids, including aqueous buffers, organic solvents, e.g., dimethylsulfoxide, acids and bases. Compatibility is achieved by selection of suitable materials for fabrication of components that contact the liquid. Exemplary materials for fabrication of components are stainless steel, nylon, polyethylene, polypropylene, EPD rubber and polytetrafluoroethylene (PTFE; Teflon®). Selection of suitable materials and fabrication of components is within ordinary skill in the art. 
     It is to be understood that various modifications on the above-described embodiments can be made without departing from the spirit and scope of the invention. For example, to form a liquid reservoir, sliding seal  23  and lower seal  24  can be replaced with an expandable bladder. Accordingly, other embodiments of the invention are within the scope of the following claims.