Abstract:
Substrate beads for combinatorial synthesis are selected individually from a mixture by suspending the mixture in an electrically conductive liquid, in a bead selection vessel, causing the liquid to flow at a controlled rate through an aperture in the side wall of a pipette extending through the vessel, and detecting the passage of a bead through the aperture by monitoring an electrical resistance across the aperture. In an alternative embodiment, beads are passed through a tube into a collection passage in which a continuous laminar flow takes place. Detection takes place at the tip of the tube, and, in response to the detection of a bead, the flow through the collection passage is diverted to cause the bead to be deposited. In both cases, the selected bead is deposited into a well of a plate having rows and columns of wells in a rectangular array, while a vacuum is drawn through a filter in the bottom of the well. The bead selection head is moved from well to well in each column, and the well plate is indexed to position the columns successively underneath the path of the bead selection vessel. A plate handling mechanism retrieves plates from a supply stack, moves them laterally underneath the bead selection vessel, and elevates them into another stack.

Description:
This application is a 371 of PCT/GB97/02883 filed Oct. 17, 1997 which is a continuation of Ser. No. 08/734,228 filed Oct. 21, 1996 Abandon. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to chemical synthesis, and more particularly to an improved apparatus and method for distributing microscopic beads of the kind used as substrates in combinatorial chemistry. 
     BACKGROUND OF THE INVENTION 
     In combinatorial synthesis, it is often desirable to be able to distribute beads into a two-dimensional array, so that each variant in a combinatorial library can be identified by its position in the array. The array can consist of a set of plates, each having rows and columns of wells, with one bead, or some other predetermined number of beads, in each well. The beads are typically made of polystyrene, and serve as substrates for different compounds produced in the process of split and combine synthesis. Ultimately, the synthesized compounds are stripped from the beads and tested for activity. The identity of an active compound is determined by spectrographic analysis, in the light of the information available concerning the reaction histories of the beads being distributed. 
     The beads are spherical and of extremely small size, e.g. 300 mm in diameter. Consequently, they are difficult to handle, and it has been very difficult to separate a single bead from a mixture of beads. 
     SUMMARY OF THE INVENTION 
     The principle object of this invention is to provide an apparatus and method for selecting individual beads, or preselected numbers of beads from a mixture of beads, and distributing the selected beads into a two-dimensional array. 
     A further object of the invention is to provide a bead distribution apparatus which is both simple and highly reliable. 
     This apparatus utilizes a head similar to that of a “Coulter” counter, a device used to count and size particles in a liquid. For example, it is used in the petroleum industry to assess engine wear by counting particles in lubricating oil. The principle on which the Coulter counter operates is that electrical resistance of a conductive fluid, measured by electrodes on both sides of a small aperture, increases momentarily as a solid particle passes through the aperture. The passage of particles through the aperture is detected as a electrical pulses, which can be counted electronically. The Coulter counter is described in detail in U.S. Pat. No. 2,656,508, issued Oct. 20, 1953, and the disclosure of that patent is here incorporated by reference. 
     The preferred embodiment of this invention takes advantage of the principle of the Coulter counter, but uses the principle in a different way and for a different purpose. 
     In accordance with the invention, beads, from a mixture of beads, preferably of substantially uniform size, are distributed into an array having multiple locations, so that a predetermined number of beads is deposited at each location in the array. This is carried out by forming a suspension of the mixture of beads in a carrier liquid; causing a part of the liquid to flow through an aperture of a size such that the beads can pass through the aperture only one at a time; detecting the passage of a predetermined number of the beads through the aperture; and, in response to the detection of the passage of the predetermined number of the beads through the aperture, depositing them at a predetermined location of the array. 
     In one embodiment of the invention, the selection of beads to be deposited is carried out by discontinuing the flow through the aperture upon detection of the passage of the predetermined number of the beads through the aperture. In an alternative embodiment, flow takes place continuously through the aperture, and is diverted in response to a detection signal to effect bead deposition. 
     The carrier liquid is electrically conductive, and is stirred to keep the beads in suspension. In a first embodiment, to deposit a single bead, a syringe is operated to produce a steady flow of liquid through an aperture in the side wall of a tube extending through the container for the carrier liquid. Eventually, a bead will pass through the aperture along with the liquid. When the passage of a bead is detected electrically, the operation of the syringe is discontinued and the flow of liquid through the aperture stops. This prevents other beads from passing through the aperture. After its passage through the aperture is detected, the bead is flushed out of the tube by a pumped liquid, and deposited at its location in the array, preferably into a well in a well plate. Preferably, while the syringe is causing liquid to flow into the tube through the aperture, liquid is withdrawn from the upper end of the tube by a pump at the same rate at which it flows into the tube through the aperture. This prevents liquid from passing through the lower end of the tube. Normally only one bead will be deposited at each location in the array. However, multiple beads can be deposited at each location. This is done by counting the electrical pulses corresponding to peaks in resistance. When the desired number of beads is counted, the flow of the suspension liquid is discontinued. 
     In a second embodiment, the aperture at which detection takes place is at the end of a tube through which liquid flows continuously. When a bead is detected at the aperture, a signal is produced causing the flow of liquid to be diverted so that the bead is carried to the location at which it is to be deposited. 
     In a preferred embodiment of the invention, a stack of empty well plates is initially placed in the apparatus. The lowermost well plate in the stack is automatically moved to a position underneath the head with a first row of wells positioned underneath, and parallel to a linear path of movement of the head. The head moves successively from one well to the next, depositing a bead in each well of the column. The well plates are indexed laterally to position successive rows of wells underneath the path of the head. When a plate is filled, i.e. it has one bead in each of its wells, it is moved into a new stack and the apparatus retrieves a new plate from the supply stack and begins to distribute beads to the new plate. 
    
    
     Further objects, details and advantages of the invention will be apparent from the following detailed description, when read in conjunction with the drawings. 
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a diagrammatic front view of a bead distribution apparatus, showing a movable bead distribution head, and mechanisms for transporting well plates from a supply stack to a location underneath the distribution head, and from the distribution head to a second stack; 
     FIG. 2 is a diagrammatic top plan view of the bead distribution apparatus; 
     FIG. 3 is a vertical section through the bead distribution head; 
     FIG. 4 is a fragmentary sectional view, showing details of the bead selection aperture in the bead distribution head; 
     FIG. 5 is a top plan view of the cover of the bead distribution head; 
     FIG. 6 is a section taken on plane  5 — 5  in FIG. 4; and 
     FIG. 7 is a section taken on plane  6 — 6  in FIG. 4; 
     FIG. 8 is a schematic diagram showing the fluid paths and controls of the apparatus, and illustrating the manner in which a bead is selected from a suspension of beads in a liquid; 
     FIG. 9 is a schematic diagram illustrating the movement of well plates from the supply stack to a location underneath the distribution head, and from the distribution head to the second stack; 
     FIG. 10 is a typical plot of electrical voltage versus time across the aperture of the bead distribution apparatus; and 
     FIG. 11 is a schematic diagram showing a distribution head assembly in accordance with an alternative embodiment of the invention. 
    
    
     DETAILED DESCRIPTION 
     Referring to FIGS. 1 and 2, the bead distribution apparatus  10  comprises a loading guide  12  in which empty well plates, of a standard, commercially available type, may be stacked. The empty well plates are unloaded from the bottom of guide  12  by an unloader  14  into a conveyor  18 , and are transferred individually by the conveyor to a position underneath a distribution head  16 . Each well plate, e.g. well plate  19  in FIG. 2, has a rectangular array of wells disposed in rows and columns. While a well plate is underneath the distribution head, the distribution head moves from well to well in a row (perpendicular to the plane of FIG.  1 ), depositing a bead in each well. When the distribution head has traversed a row of wells, the conveyor  18  indexes the well plate to position a next row underneath the path of the head. This is repeated until beads are deposited in all of the wells. Then the conveyor moves the well plate to a position underneath a stacking guide  20 , and the well plate is loaded into the stacking guide by a stacker  22 . 
     The unloader  14  and the stacker  22  are elevators with platforms which engage the lowermost well plates in the guides and move vertically to load and unload the conveyor. Catches (not shown in FIGS. 1 and 2) are provided at the lower end of the loading guide  12  for supporting the stack of well plates in the loading guide when the unloader  14  is retracted. These catches are electrically operated, and microprocessor controlled so that they cooperate with the unloader  14 , allowing the unloader to receive a well plate from the loading guide  12  and deposit the well plate onto the conveyor. Ratchet-type catches (not shown in FIGS. 1 and 2) are provided at the lower end of the stacking guide  20  to support well plates in the stacking guide while allowing the stacker  22  to deposit the well plates therein. 
     While the well plate is underneath the distribution head  16 , a bead is deposited in each well, the distribution head being indexed from well to well in each row of wells along a supporting arm  24 . The distribution head can also be moved to a position just above a waste collector  25 , which, as shown in FIG. 2, is located behind the path of movement of the well plates. 
     The distribution head supporting arm  24  is itself movable vertically so that the distribution head can be moved up and down. All of the fluid conducting lines and electrical leads to the distribution head  16  are flexible, and preferably bundled together in a single flexible sheath  15  (FIG.  1 ), so the that the distribution head can move freely. 
     The distribution head, shown in detail in FIG. 3, comprises a cup-shaped vessel  26  having a cover  28  secured to a flange  30  of the vessel by a threaded ring  32 . A sleeve  34  extends through the center of the cover, and a pipette  36  is held in the sleeve by seals  38  and  40 . The pipette extends through vessel  26 , its upper end  42  being located above the cover  28 , and its lower end  44  being below the bottom of the vessel. The pipette extends through a seal  46  at the bottom of the vessel. 
     The outside wall of the pipette is cylindrical throughout most of its length. However, a flat area  48  is formed on the outer wall of the pipette at a location within the interior of the vessel  26  just below the lower end of sleeve  34 . As shown in FIG. 4, an opening  50  is formed in the wall of the pipette within the flat area  48 , and a small watch jewel  52 , having an accurately machined aperture  54  is secured to the flat area by an adhesive, with aperture  54  in register with opening  50 . The aperture  54  is typically 500 mm in diameter, but can be larger or smaller, depending on the size of the beads to be distributed. In general, the diameter of the aperture should be less than twice the diameter of the beads. Alternatively, the aperture can be formed directly in the wall of the pipette, obviating the use of the jewel. 
     As shown in FIG. 4, a first platinum electrode  56  is located within the pipette, with its tip adjacent to opening  50 . Electrode  56  extends through a seal  60  in the wall of the pipette and is connected to a flexible, multistrand lead  58 , which is isolated from the suspension in vessel  26  by sleeve  34 . Another platinum electrode  62  has its tip located adjacent to the outer end of aperture  54 , and extends through seal  40  into the space between the pipette and the inner wall of sleeve  34 , where it is connected to a flexible lead  64 . 
     Returning to FIG. 3, the top of the pipette is provided with a fitting  66  for connection to a pump. The pump (not shown in FIG. 3) is used to withdraw liquid from the upper end of the pipette as it flows into the pipette through aperture  54 . The pump is also used to deliver liquid for flushing selected beads out through the lower end of the pipette. 
     FIGS. 5,  6  and  7  show that the cover  28  has three additional openings besides its central opening through which sleeve  34  extends. The first opening,  68 , is an opening for introducing beads into the vessel, and is closable by a removable threaded plug  70  (FIG.  3 ). The second opening,  72 , is an inlet for connection to a syringe used to produce flow of liquid into vessel  26 . The third opening,  74 , is a vent opening. As shown in FIGS. 3,  6  and  7 , the underside of the cover  28  has an annular groove  76  which varies in depth, with its deepest point being at the location of vent opening  74 . The groove becomes continuously shallower in both directions from the vent opening toward a shallowest point  78  (FIG. 7) opposite the vent opening so that air can be exhausted completely from the vessel. A porous filter  80  is provided in the vent opening. 
     FIG. 8 shows vessel  26  filled with a suspension consisting of a liquid  82  and beads  84 , held in suspension by an external magnetic stirring mechanism  86  cooperation with an agitator  88  inside the vessel. The density of the beads  84  is preferably greater than that of the liquid, so that they sink in the liquid. To prevent surface tension from causing the beads to float, they are pre-treated so that they are wetted by the liquid. 
     An example of an ideal liquid for use with polystyrene beads is a solution of ammonium acetate (2% w/w), and ammonium carbonate (2% w/w). 
     The liquid  82  is supplied to vessel  26  from a supply container  90  through a peristaltic pump  92 , which is controlled by a control unit  94 , and a check valve  93 . A syringe  96  is used to control the flow of the liquid after the vessel  26  is filled. The syringe is operated by an actuator  98 , controlled by the control unit. When the plunger of the syringe is withdrawn, the syringe draws liquid from supply container  90  through a check valve  100 . Forward movement of the plunger causes the liquid to flow through check valve  101  into vessel  26 . 
     A reversible peristaltic pump  104  is provided to withdraw liquid from the upper end of the pipette  36  as it flows into the pipette through aperture  54 , and to pump liquid through the pipette from container  102  for the purpose of flushing beads out of the pipette either to the wells, or to the waste collector  25 . Pump  104  is also under the control of unit  94 . 
     In the operation of the system, the beads are introduced into the vessel  26  through opening  6   8  (FIGS.  5  and  6 ), which is then closed by plug  70  (FIG.  3 ). The control unit then operates pump  92  to fill the vessel with liquid from supply container  90 . The operation of the pump  92  is discontinued when the vessel is filled with liquid. In the meanwhile, a well plate  106  is removed from the supply stack in guide  12  (FIG. 1) and moved into position underneath the vessel  26 , which is a part of the distribution head. 
     The well plate  106 , as shown in FIG. 1, comprises a two-dimensional rectangular array of wells  108  (FIG.  8 ), each of which is preferably closed at its bottom. 
     With the distribution head positioned so that the pipette is over the waste collector  25 , the syringe  96  is operated to initiate a controlled flow of liquid at a constant rate into vessel  26  through check valve  101 . While other kinds of pumps can be used to carry out this operation, the syringe is desirable because it is capable of producing a steady flow of liquid at a very slow rate. While the syringe  96  is causing liquid to flow into the vessel  26  and through the aperture  54  into the pipette  36 , pump  104  is operated to withdraw liquid from the upper end of the pipette  36 . The pump withdraws liquid at the same rate at which it is being introduced into the pipette by the operation of the syringe  96 . The result is that liquid is prevented from being forced out the tip of the pipette, no matter how long it takes for a bead to pass through the aperture. This prevents unnecessary flow of liquid into the waste collector, and also prevents loss of beads through the tip of the pipette when multiple beads are being collected in the pipette for deposit into a well. 
     As seen in FIG. 8, a bead  110  in the suspension will eventually pass through aperture  54  into the interior of the pipette. The passage of the bead is detected by the control unit  94  as a change in the resistance measured between platinum electrodes  56  and  62 . When the control unit detects the passage of a single bead, it causes actuator  98  to stop pushing the plunger of syringe  96  and simultaneously stops pump  104 . As a result, the liquid flow through the aperture is discontinued, and only a single bead passes into the pipette. The control unit moves the distribution head to the next well in sequence, and after a predetermined delay, during which the bead inside the pipette settles by gravity to the tip of the pipette, the control unit activates pump  104  in the opposite direction for a short interval just sufficient to wash the bead out of the pipette  36  into the well. 
     The control unit then causes the distribution head to return to the waste collection point, and the bead depositing operation is repeated for each well in the row. After beads are deposited in each well in a row, the well plate is indexed to position another row of wells underneath the path of the distribution head. The bead depositing operation continues until beads are deposited in each well in the well plate, whereupon, the well plate is transferred to a position underneath the stacking guide  20 , and elevated into the stacking guide. The movement of the well plates is depicted in FIG.  9 . 
     FIG. 9 also depicts laterally-slidable catches  111 , which are operated by actuators (not shown) under the control of the control unit, for supporting the stack of well plates in the loading guide. Also shown are the ratchet-type catches  113 , which support well plates in the stacking guide while allowing well plates to be raised into the stacking guide  20  by stacker  22 . 
     The passage of beads through the aperture  54  in the pipette of the distribution head is detected by measuring changes in the electrical resistance across the aperture between electrodes  56  and  62 . Preferably this is achieved by applying a current to the aperture by means of a constant current source  126 , as shown in FIG. 8, and monitoring the voltage across the aperture. Alternating current is preferred in order to avoid the effects of polarization. The passage of a bead through the aperture results in an increase in the resistance across the aperture manifested by an increase in the voltage measured across the aperture. The voltage variation is depicted in FIG. 10, in which the voltage level remains essentially constant except when a bead passes through the aperture, at which time a voltage peak  128  appears. The peak is detected in the control unit and used to produce a signal to stop the operation of actuator  98  and pump  104 . 
     As will be apparent from the description, the apparatus reliably selects individual beads from the mixture and deposits them in wells in the well plates. 
     Although the flow of liquid through aperture  54  is stopped almost immediately when the passage of a bead through the aperture is detected, occasionally, more than one bead will pass through the aperture into the pipette. The accidental passage of multiple beads through the aperture will be detected by the control system as a series of voltage peaks, and the control system responds by causing the distribution head to remain over the waste collector  25  while the beads are flushed out of the pipette. These beads can be reintroduced into the cup-shaped vessel  26  for later distribution. Waste liquid is delivered to a closed container  122 , and a vacuum is drawn continuously on the waste collector  25 , through container  122 , by pump  120 . 
     In the alternative embodiment shown in FIG. 11, a suspension of beads  130  is established in a vessel  132 . The vessel comprises a cylinder  134  having a top closure  136  and a bottom closure  138 . The top closure has a bottom face  140  in the form of a symmetrical cone. A fluid inlet is provided at  142 , and an air outlet  144  at the peak of the cone has a filter  146 . The bottom closure  138  has its top face  150  in the form of an asymmetric cone with an emptying port  152  provided with a valve  154 . 
     A metal tube  158  extends upward through the bottom closure  138  to a location within the cone defined by bottom face  140  of the top closure. The tube is coaxial with that cone and has an opening  160  at its upper end for receiving beads along with liquid from the suspension  130 . The beads are maintained in suspension by a magnetic flee  162  operated by an external magnetic stirrer  163 . 
     The cone tends to concentrate beads at the location of the end opening  160  of tube  158 , and beads enter the tube  158  along with liquid. 
     The lower part of tube  158  extends through the bottom closure  138  of the vessel  132 , and into an insert  164  of PTFE or other similar material which is not electrically conductive. The insert fits into the upper end of a passage  166  in a metal block  168 , and tube  158  extends into the insert to a location near, but spaced from the lower end of the insert. The lower end of the insert has an opening  170 , having a diameter equal to the internal diameter of the tube  158  so that the tube and opening  170  form a continuous, smooth passage for the flow of liquid and beads. 
     The exterior of the lower end of insert  164  is narrower than the portion of passage  166  surrounding it, thereby providing an annular space  172  for the flow of liquid received through a passage  174 . The liquid introduced through the passage  174  flows past the tip of insert  164 , downwardly through a tapered part  176  of passage  166 , and outwardly through an exit opening  178 . The tip of the insert and the passage  166  are gradually tapered and shaped so that the flow past the tip of the insert is laminar. 
     The flow of liquid through passage  174  draws beads individually from the tip of insert  164  and delivers them into the upper part of passage  178 , which serves as a collection chamber, from which they can be deposited in an array through opening  180 . A solenoid-operated valve  182 , located above opening  180 , is movable in a direction perpendicular to the plane of the drawing to permit or block flow through opening  180 . This valve is shown in its closed condition. A branch  184 , communicating with passage  178 , is provided with a similar solenoid-operated valve  186 , which is shown in its opened condition. The valves  182  and  186  are provided with hollow internal passages through which liquid can be caused to flow for washing the passages  178  and  184 . 
     In the apparatus of FIG. 11, the tube  158  serves as one of two electrodes, and block  168  serves as the other electrode. Passage of a bead through opening  170  effects a change in the electrical resistance measurable between tube  158  and block  168  in the same manner in which a bead passing though aperture  54  in FIG. 4 affects the resistance measured between electrodes  56  and  62 . In the absence of an electrical signal produced by the passage of a bead through opening  170 , valve  182  is closed and valve  186  is open, allowing liquid entering the block  168  through passage  174  to flow to waste through branch  184 . The electrical signal produced in response to the passage of a bead controls the operation of valves  182  and  186  in such a way that valve  182  opens momentarily and valve  186  closes. The time delay between the detection signal and the operation of the valves is set in relation to velocity of movement of beads in the space below the tip of tube  158 , so that valve  182  opens and valve  186  closes precisely at the time that the detected bead is in close proximity to the connection of the branch  184  to passage  178 . The operation of the valves allows beads to be deposited individually into a suitable array, for example into wells in a microtitre plate. 
     In the apparatus of FIG. 11, the fluid introduced through passage  174  serves as a sheath fluid, and may be the same as the suspension fluid passing downwardly through tube  158  from vessel  132 . The sheath fluid flows in the same direction in which the beads move through tube  158 . Preferably, the suspension fluid and the sheath fluid flow through passage  178  in laminar flow, i.e. in substantially non-mixing layers respectively of the suspension fluid and the sheath fluid. Such laminar flow helps the beads to flow through passage  178  along a substantially straight path. the sheath fluid also helps to even out the flow of beads and assists in achieving suitable serial separation of beads. The sheath flow may be controlled to optimize the flow of beads through passage  178 . 
     Various modifications can be made to the apparatus and process described. For example, by incorporating an electronic counter in the control unit, it is possible to count electrical peaks and disable the syringe actuator only after a predetermined number of peaks is counted. In this way, if desired, a preselected number of beads can be deposited in each well. The control system can be programmed to cause the beads in the pipette to be flushed into the waste collector if the number of beads passing through the aperture into the pipette exceeds the preselected number. 
     The pipette (with its aperture  54 ) can be readily removed for cleaning, or for replacement by another pipette having a different aperture. It is possible to eliminate the jewel  52  altogether, and thereby avoid the potential problems resulting from detachment of the jewel from the pipette. This can be done by forming the aperture directly in the wall of the pipette, provided that the wall thickness is sufficiently small. 
     In still another modification of the apparatus, openings are provided at the bottoms of the wells in the well plates, and filters are situated in the openings. A vacuum head is situated underneath the path of the distribution head and engageable with the undersides of the well plates. A vacuum is drawn continuously though the vacuum head, and is used to remove liquid form the wells. This keeps the wells from overflowing, and is an alternative to the previously described withdrawal of liquid from the upper end of the pipette at the same rate at which it enters the pipette through aperture  54 . 
     In the embodiment of FIG. 11, various alternative valves and flow passage configurations can be used, and fluidic control can be utilized to divert the flow of liquid from one passage to another. 
     Still other modifications can be made to the apparatus and process without departing from the scope of the invention as defined in the following claims.