CA2238982A1 - Partitioning device - Google Patents

Abstract

A device for precisely arraying small objects into a plurality of containers (9) which comprises a vessel (1) containing said objects in a fluid suspension, a tube (4), a small-objects detector (6), a drop detector (8), a means for controlling flow of said suspension (10) through said tube (4) by pressurizing said vessel (1), means for discriminating between those signals from said small-objects detector (6) caused by said small objects and those signals caused by other events, means for comparing signals from said smallobjects detector (6) caused by said small objects with signals from said drop detector (8), and means for determining the number of said objects deposited into said containers (9).

Description

CA 02238982 1998-0~-28 W 097/30897 PCTrUS97/02735 TITLE OF THE INVENTION

PARTITIONING DE~VICE

BACKGROUND OF TH~ INVENTION

There is interest in methods for the synthesis of large numbers of diverse compounds that can be screened for various possible physiological or other activities. Techniques have been developed in which one adds individual units sequentially as part of the chemical synthesis to produce all or a substantial number of the possible compounds which can result from all the different choices possible at 3LO each sequential stage of the synthesis. WO 93/06121, April 1, 1993, teaches methods for solid support-based synthesis of random oligomers wherein identification tags on the solid supports are used to facilitate identification of the oligomer sequence synthesized. A detachable tagging system is described in Still et al., WO 94/08051, April 14, 1994, which teaches the synthesis of large combinatorial libraries of compounds attached to solid supports.
In order to screen the compounds produced by these new synthetic methods, it is desirable to partition from a pool cont~ining very large (up to the range 106-109) numbers of solid supports into collection plates with wells, typically 96 wells cont~inin~ 1-30 supports per well.
These solid supports, commercially available as beads, are generally 50-1000 ~m in diameter. The number of beads per well is a matter of choice, but should be at least one and not greater than 200, constrained by screening statistics, solubility factors, or size. Furthermore, the number per well should be consistent. Therefore, no well should be empty. However, an average variation of +5 beads per well is acceptable for wells cont~ining 20 beads. When single beads are to be screened in W 097/30897 PCT~US97/0273S

each well, it is desirable to minimi7e empty wells while avoiding multiple beads per well in order to avoid the need for rescreening these beads.
Various devices are known in the art which sort particles S from liquid suspensions. For example, U.S.P. 3,710,933, Fulwyler et al., describes a particle sorter applicable to the sorting of biological cells which analyzes cells in a flow chamber and then produces celI-cont~ining droplets via a piezoelectric crystal. Emerging droplets are sorted into two receptacles. Droplets cont~ining selected cells are 10 electromcally charged and then deflected by a static electric field into one receptacle. Unselected cells drop into the other receptacle. U.S.P.
4,173,415, Wyatt, describes an apparatus for charact~ri~in~; organic cells such as leukocytes which creates an aerosol from a cell suspension to produce a series of droplets which are then divided into cell-containing 15 and non-cell-cont~ining streams. The cell-contz~inin~ stream is then air-dried and the cells finally analyzed by monochromatic light. U.S.P.
4,606,631~ Anno et aL, describes a particle counter which utilizes a flowing sheath solution to surround the sample solution which contains the particles, typically blood corpuscles, to be counted. U.S.P.
2Q 4,680,977, Conero et al., teaches an apparatus for sensing the flow of a liquid by detecting and measuring drops through an optical drip chamber.
U.S.P. 4,G55,265, Duteurtre et al., describes an apparatus for the batch transfer of brittle particles, specifically yeast-cont~;ning ~l~in~te beads, from a suspension into fermentation containers. U.S.P. 5,142,140, 2~ Y~m~7.~ki et al., describes an apparatus which uses a polarized beam splitter for counting particles, typically leukocytes, suspended in a iluid.
U.S.P. 5,166,537, Horiuchi et al., describes ~n improved Coulter Counter device which utilizes a light detection method in combination with an eiectric impedance method and compares the signals therefrom to CA 02238982 1998-0~-28 W O 97/30897 PCTrUS97/02735 exclude false data obtained from multiple particles being present simultaneously. U.S.P. 5,286,452, ~Iansen, teaches a method for analyzing multiple analytes in a single fluid sample and a sheath-type flow cell for performing said method. None of these methods and 5 teachings are suita~le for accurately counting a number of small objects, including beads in the size range mentioned above removed from a fluid reservoir, and depositing all or a pre-selected number of them into one or multiple containers in the form of droplets.

SUMMAR~ OF T~E INVENTION

A device has now been made which identifies and counts small solid objects, and which can precisely array said small objects into one or more containers. The device comprises a vessel cont~ining said objects in a lluid suspension; a tube, the proximal end of which is immersed in said suspension and the distal end of which is connected to 15 a member which directs gravitationally-formed fluid drops from said distal end downward into a container positioned below said distal end; a small-objects detector, for detecting said objects in the fluid in the tube, disposed near said distal end; a drop detector connected to said member below said distal end; means for controlling flow of said suspension 2~ through said tube; means for discrimin~ting between those signals from said small-objects detector caused by said small objects and those signals caused by other events, i.e., not caused by said small objects; means for comparing signals from said small-objects detector caused by said small objects with signals from said drop detector; and means for determining 25 the number of said objects deposited into said container. Preferably the ~ proximal end of said tube is positioned substantially at the bottom of said vessel. Also, preferably the means for controlling flow of said suspension through said tube is by pressurizing said vessel. The device further comprises positioning means for re-positioning the distal end of said tube from one said container to another said container, with timing derived from said small-objects and drop detectors. The positioning means comprises an X-Y transport which is either 1) a movable member 5 which moves the distal end of said tube and said detectors or 2) an X-Y
stage which moves the containers, with said tube and said detectors attached to a stationary member. Another embodiment of the invention enables removal of drops as they fall from the tube to said containers.
This assures that selected drops contain a single bead without debris or 10 fragments. This modification also avoids delivery of multiple-object cont:~ining drops in the single object/container application. Evaluation of a drop for acceptability is deterrnined electronically while the drop is in flight.

BR~EF DESCRIPTION OF THE DRAWINGS

.
FlgUre 1 IS a general drawlng of the mventlon mdlcatmg ItS
principal components with movable X-Y arm or platform.
Figure 2 is a drawing of the smalI-objects and drop detectors.
Figure 3 is a schematic representation of the small object detector, fraction collector, and discnmin~tor logic.
Figure 4 contains electrical bead disturbance wave forms captured from an oscilloscope. These wave forms correspond to Signal S2 in Figure 3. Threshold levels and event window time periods are shown superimposed.
Figure 5 is a schematic representation of a preferred system shown with drop deflector and additional deflector logic.

CA 02238982 1998-0~-28 W O 97/30897 PCTrUS97/02735 DESCRIPTION OF THE PREFERRE~D EMBODIMENTS

An embodiment of the invention is an apparatus for precisely arraying small solid objects into a plurality of containers, which comprises:
a vessel cont~ining small solid objects in a fluid suspension;

a tube, the proximal end of which is immersed in said suspension and preferably is positioned substantially at the bottom of said vessel, said tube being adapted for transport of fluid from said vessel;

a member, connected to the distal end of said tube, adapted for directing gravitationally-formed fluid drops from said distal end downward to be deposited into a plurality of containers positioned below said distal end;

a small-objects detector, adapted for detecting said objects in the fluid in said tube, disposed near said distal end;
a drop detector attached to said member below said distal end;
means for controlling flow of said suspension through said tube, preferably by pres.~nri7.ing said vessel;

means for discrimin~tin~ between signals from said small-objects detector caused by said objects and sign~l.s not caused by said objects;

means for comparing signals from said small-objects detector caused by said small objects with signals from said drop detector;

CA 02238982 1998-0~-28 W 097/3~897 PCTAUS97/02735 means for deterrninin~ the number of said objects deposited into each of said containers;

means for re-positioning the distal end of said tube from one said container to another said container; and means for timing said re-positioning with respect to signals from said drop detector, said small-objects detector, or the combination thereof.

Another embodiment of the invention is a method for precisely arraying small solid objects into a plurality of containers, which comprises:
providing said objects in a fluid suspension;

transporting said fluid suspension, at a controlled rate, to a small-objects detector;

discrimin~ting between signals from said smaIl-objects detector caused by said objects and ~ign~ not caused by said objects;

allowing discrete fluid drops to forrn due to action of gravitational forces upon said fluid suspension;

allowing said discrete drops to fall, due to action of said gravitational forces;
providing a drop detector in the path of the falling drops;

comparing signals from said drop detector with ~i~n~l~ from said small-objects detector caused by said objects, so as to determine the number of said objects in each drop;

.. _ CA 02238982 1998-0~-28 W O 97/30897 PCTrUS97102735 directing said drops into a container;

redirecting said drops into a different container, based on signals from said drop detector, signals from said small-objects detector, or signals from both such detectors indicating that a desired number of said small objects have been directed into the previous container; and repeating said redirecting step.

Referring to FIG. 1, a sealable vessel 1 cont~ining an isobuoyant suspension of the small solid objects (e.g., beads) 2 to be 10 sorted is placed on an orbital shaker 3. The inside, lower surface of the vessel is preferably concave or has a depressed area to facilitate removal of the maximum number of beads. The beads generally are those suitable for combinatorial libraries, i.e., the combinatorial synthesis of diverse compounds, as described, for example, in Still et al., WO
1~ 94/08051. Such combinatorial libraries may comprise very large numbers (0.01-1.5 million) of different members each represented in a redundancy of up to 1000.
Generally, the beads are about 50-1000 ,um in diameter and have a rigid or semi-rigid surface. They include cellulose beads, controlled 20 pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, poly-acryla~nide beads, latex beads, dimethyl-acrylamide beads optionally cross-linked with N,N'-bis-acryloyl ethylene 25 ~ mine, glass particles coated with hydrophobic polymer, etc.
Preferably, the beads are divinylbenzene-cross-linked, polyethyleneglycol-grafted polystyrene beads optionally functionalized W O 9713~897 PC~US97/02735 --8--with amino groups (for example, TentaGel~) S NH2, Rapp PoIymere).
The beads in any batch are of approximately equal size, i.e., having a range no greater than 2x difference in diameter. Typically, the beads have been through a process of combinatorial synthesis such as described in Still et al. (supra); i.e., the beads may have attached to them the synthesized compounds, the tagging molecules, or both.
Ideally, the beads are m~int~ined in a unii~orm random suspension in the vessel so that any volume sampled by the tube contains the same statistical distribution of bead count. Aggregated beads are especially undesirable when arraying plates in a single-bead-per-well format. To keep the beads separated from one another and to ensure an isobuoyant suspension and proper flow of the suspension through the tube and, ultimately, proper drop formation, the beads are suspended by gentle agitation in a liquid which is chemically inert, non-corrosive to tube 4, and has a room temperature viscosity in the range û.500-4.000 mPa-sec, a surface energy in the range 15-65 mJ/m2, and a density in the range of 1.00-1.50 g/cm3. Typical such suspending liquids are trichloroethane/isopropanol (TCE/IPA) mixtures in proportions ranging from about 70-90:30-10 by volume and water/KE~r/isopropanol mixtures in proportions ranging from about 35-45:35-45:15-25 by weight. In these mixtures the IPA is a surfactant additive which helps avoid bead clumping or aggregation to further ensure a uniform random sampling of beads. The volatile TCE/IPA mixture is useful when the container is to be rapidly dried after bead sorting. However, the choice of volatiles as suspending liquids is constrained because of their possible residues and incompatibilities with the container materials (e.g., plastics). The waterfKBr/IPA mixture is useful when a liquid compatible with most E}lastics is desired. The water/KBr/IPA mixture can be rinsed through the CA 02238982 1998-0~-28 WO 97/30897 PCT~US97/Q273 _9_ containers if the containers have porous bottoms that retain beads. A
two-step cle~nin~ process involves rinsing the containers with 2-5 water wash steps followed by a single IPA wash step. The end result should be a residue-free collection of beads in each container.
Agitation of the suspension can be elimin~f d if its viscosity is sufficiently high. ~or example, an agitated suspension (not necessarily isobuoyant) of beads in an aqueous solution of agarose (ca. 0.05%) will become quite viscous when cooled to room temperature from about 40~C. The viscosity of the cooled suspension is so high that the beads 11~ will remain in their uniformly random state without further agitation during sampling. Other commercially available thickening agents such as alginate and xanthan gum may also be useful. It is important in most cases to remove the additive that causes the viscosity by filtering, as through the porous bottoms described above. This may require some reversibility in viscosity. For example, the containers can be heated to 40~C to allow the agarose to become thin enough to wash out. In other cases dilution, possibly with acid or base treatment, may cause the solution to become more readily removed. If the need for agitation is elimin~ted, then the suspension vessel 1 can be attached directly to and above arm 5. In this case tube 4 is subst~nti~?lly shorter than the 1.00-2.00 m described below.
A single, seamless, thin-wall, flexible, optically clear tube 4, the proximal end of which is positioned substantially at the bottom of vessel 1 to facilitate removal of the ma~imllm amount of suspension from the vessel, connects the vessel to an arm or member 5. By "optically clear" is meant that the wall of the tube readily permits tr~n~nli.ssion of radiation at the wavelength utilized by the small-objects detector. The tube is composed of an inert or non-stick material such as WO 97130897 PCT~US97/02735 Teflon (polytetrafluoroethylene), has an inside diameter of 200-4000 llm, and a length of 1.0-2.0 m, of which approxTm~tely 24 rmm (i.e., the length of the tube which would accommodate one drop of fluid) extends through and beyond optical bead detector 6. The inside diameter of the 5 tube is chosen to be approximately twice the diameter of the largest bead.
There are no joints, kinks, or valves from the point the beads enter the tube within the vessel to the point they exit the tube at the distal end in the forrn of drops. This avoids any lodging of beads, bead fragments7 or foreign matter that could interrupt the flow of beads. The drops of this 10 invention form by gravitational forces; i.e., unlike those of, for example, U.S.P 3,710,933, which are produced by a piezoelectric transducer, the present drops have essentially zero velocity at the time of their release from tube 4. As used herein, by "gravitational forces" is meant any method whereby the size of a drop is comparable to that formed by the 15 force of gravity alone; e.g., vibrational release of a nearly formed drop.
The arm 5 may be movable, in which case it is activated by an X-Y transport which positions the distal end of tube 4, the bead detector 6, and the drop detector 8 over a plurality of stationary collection containers 9. A suitable collection device for this embodiment 20 is the commercially available Gilson FC204 fraction collector (Gilson Medical Electronics, Inc.). Alternatively, the containers can be moved under a stationary arm 5 by means of a movable X-Y stage 7. Such containers may be bottles, tubes, vials, reaction vessels, the individual wells of a multi-well collector, such as a commercially available 96-well 25 plate (Millipore or NUNC), or other containers suitable for collecting beads. The 96-well plates have solid or porous bottoms (e.g., Millipore Multi ~creen BV plates with 1.2 ,urn low-binding PVDF membranes).

CA 02238982 1998-0~-28 W O 97/30897 PCT~US97/02735 Drop detector 8 is attached to arm 5 below the distal end of tube 4 (and therefore below the bead detector 6) such that the drop detector is in the path of the drops as they fall, due to gravitational forces, off the distal end of the tube 4. The X-Y transport 7 centers the 5 individual containers beneath the falling drops, with the plane of X-Y
motion and the surface that supports the containers substantially perpendicular to the drop path.
Positive pressure is m~int~ined above the isobuoyant suspension from a source of compressed air 10, preferably an inert gas 1~ such as N2, by a regulator and valve 11. Alternative methods of maintaining a satisfactory flow rate are also possible. For example, if vessel 1 has flexible walls or a movable wall member, flow can be controlled by reducing its volume; i.e., by controllably squeezing the flexible walls of the vessel or driving said movable wall as by a piston.
The pressure is determined empirically, such that a llow rate of about 60 drops~min is achieved at the distal end of tube 4. Such a relatively slow flow rate permits time to count the drops, move the X-Y transport when necessary, and deflect non-bead-containing drops. Ideally, the pressure is sufficiently above atmospheric pressure so that changes in ambient pressure over an eight-hour period do not significantly affect the flow rate. Note that flow rate of the suspension is inversely related to its viscosity. Drop volumes are in the range 5-40 ,uL per drop. This allows a pl~rality of drops to be deposited into each container before it is necessary to redirect the drops to the next container. For example, a 96-well plate with 200 ,uL wells allows 1-40 drops per well.
Referring to FIG 2, an optical bead detector 6 is shown which comprises a collimator block 12 which is subst~nti~lly optically opaque, said block cont~ining two bores intersecting at right angles. The CA 02238982 l998-0~-28 W O 97130897 PCTrUS97/02735 diameter of the first, subsf~nti~lly perpendicular bore 13, is such as to snugly allow passage of tube 4. The diameter of the second bore 14 is subst~nti~lly identical to the inside diameter of tube 4. One side of bore 14 contains a radiation source 15 and the other side a suitable radiation 5 detector 16. The radiation source may be any convenient source, preferably an infra-red light-emitting diode. When an optical bead detector is used, tube 4 is preferably optically clear at the detection wavelength.
Alternative bead detectors are also within the scope of the 10 invention. The functional requirement is for the detector to generate a distinct signal when a bead has moved past the detection area, which is a s~ort (~3 mm) segrnent of the tube near its distal end. The detector must not create obstructions that would slow or trap beads. Some alternatives to optical detectors are:

L5 Ultrasonic detection methods, such as are employed in cornmercial bubble detectors (ZEVEX, Mass.~ for critical small i:luid paths (e.g., intravenous drip lines in hospitals), can be used. These devices employ an ultrasonic sound transducer that transmits vibrations across the tube to a detector on the other side of the tube. Any object that crosses between the detector and the transmitter that has sound tr~nsm;~sion properties significantly different from those of the suspending liquid (e.g., different compressibility, viscosity, etc.) will create a transient signal in the detector.

~5 ~lectrical resistance methods may be used which utilize two electrodes fabricated searnlessly in the walls of the tube. The electrical resistance to AC voltage signals between the two CA 02238982 l998-0~-28 WO 97/3~897 PCTrUS97/02735 -13-electrodes is modulated when a non-conducting bead displaces the conducting salt solution as it travels between the electrodes.
Circuitry analogous to that shown for the optical bead detector in Fig. 3 generates an electrical signal during this event. This - 5 technology was used in early Coulter Counters (e.g., U.S.P.
5, 166,537).

The drop detector 8 is disposed below bead detector 6 and the distal end of tube 4, and comprises a light source 17 and a suitable light detector 18 located opposite each other and such that drops 36 from tube 4 will intersect a light beam traveling between the two. The light source may be any convenient source, preferably infra-red or laser.
Referring to FI~. 3, generally, bead and non-bead events in the bead detector assembly generate sign~ls which are interpreted by the discrimin~tion algorithm and correlated with signals from a drop detector. The comparison of these signals determines the repositioning of the X-Y transport, which moves the distal end of tube 4 relative to collection containers 9. Specifically, servo amp 19 regulates current through light source 15 to m~int~in a constant signal in I/V amp 20 from the current in light detector 16. When beads in tube 4 intersect light from source 15, a transient increase in current through light source 15 is caused in order to maintain constant I/V from detector 16. This causes a disturbance in voltage across sense resistor, R s~ 21. Collimator hole 14 maximizes the relative change in disturbance signal by maximi7ing the relative area blocked by the bead while assuring that no bead can be missed.
The disturbance signal S1 is amplified and filtered by differentiator 22 to create an analog voltage signal S2. (See W 097130897 PCT~US97/02735 FIG. 4 and the detailed description of signal S2 below.) The filtering selects frequencies in a range typical of bead-caused disturbances for a given bead velocity and size. Bead velocity is deterrnined by drop rate and tube inner diameter. A filtering frequency range of 0.1 to 1 kHz is 5 typical for drop rates of 60 drops/min, average bead diameters of 200 ,um, and tube inner diameter of 500 ,um. When signal S2 reaches -Vreft at trigger comparator 23 it initiates an event window signal S3 from window generator 24. This window of time, typically 20 to 50 msec, represents the maximum time a bead takes to travel through collimator - 10 14 in its path to the distal end of the tube. Signature comparator 25, counter 26, and decoder 27 comprise a discrimin~tor circuit that allows confinn~tion that signal S2 represents a true bead signal. In that event, a bead pulse S4 is generated at the end of window signal S3.
An effective bead discrimin~tion algorithm has been developed that counts signature threshold crossings by S2 within an event window after trigger threshold. This bead discrimin~tion algorithm is implemented using discrete logic components. When the initial negative going portion of the waveform reaches the trigger threshold (-Vreft~ at trigger comparator 23, an event window signal S3 is generated by window generator 24. The duration of this window is set to be about the longest time that a disturbance signal would last if caused by a bead.
During the window signal, counter 26 is enabled and signature comparator 25 outputs a pulse whenever a positive going transition of the waveform reaches the signature threshold (Vrefs ). The pulses from signature comparator 25 increment counter 26. Decoder 27 polls the output of counter 26 for a "2" or a "3," which would be indicative of the signature of a bead (see FIG. 4(c)). If this condition is satisfied, then CA 02238982 l998-0~-28 W O 97130897 PCTrUS97/0273 bead flip-flop 28 is enabled. In this case, on the falling edge of the event window S3, bead pulse S4 is initiated and then ended by reset pulse generator 29. This reset pulse also clears counter 26.
The signature logic can be disabled by perrnanently enabling S bead flip-flop 28. ~n this way all events detected by trigger comparator 23 generate bead detection pulses S4 independently of the signature, i.e., regardless of the characteristics of each event. This is effectively a pulse height discrimin~tion algorithm, as compared with the algorithm based on pulse height and shape described above. Bead counter 30 is 10 incremented by bead pulses S4. User-selectable thumbwheel switch 31 programs magnitude comparator 32 to generate an n-bead pulse S5 each time the desired count is reached.
Once the desired number of beads has been counted, it is the function of state machine 34 (which is the collective term for devices 35, 37, 38, 39, and 40) to synchronize the movement of the X-Y transport with the fall of the correct drop. For example, if no more than l0 beads per container are to be permitted, the X-Y transport moves so as to direct drops into a new container immediately after the drop cont~ining the tenth bead has fallen into the previous container. Selector switch 33 20 allows the user to trigger state machine 34 by bead pulses S4 or n-bead pulses S5, respectively, for single-bead-per-container or multiple-beads-per-container settings. State machine trigger signal S6 passes through gate 38 and triggers bead delay 35. This delay period, typically 50 to 50~ msec, corresponds to the time re~uired for the bead causing the 25 trigger event at collimator hole 14 to travel to the end of tube 4 to be contained in a forming drop 36, such time being determined by flow rate, inner diameter and tube length beyond the collimator. At the end of this CA 022389X2 l998-05-28 W O 97/30897 PCTrUS97/02735 delay, drop flip-flop 37 is toggled, disabling future triggers through gate 38. Drop flip-flop 37 also enables drop signal S7 from drop detector 8 through drop gate 39 to toggle reset flip-flop 40 and reposition the end of tube 4 over the next container by activating the X-Y transport ~or either the movable arm 5 or movable platform 7. This assures relative motion only when there is no danger of ~h~king a drop off tube 4. Reset flLip-flop 40 resets state machine 34 to be prepared for the next trigger event.
Upon completion of its movement, the X-Y transport creates a reset signal S8 to clear bead counter 30.
F~G. 4 is a compilation of various S2 signals (voltage vs.
time) generated by both bead and non-bead (i.e., bubble, debris, or bead ~ragment) events that generate an event window signal S3 through comparator 23. FIG. 4(c) is a typical waveform of a single bead with trigger threshold (-Vreft) and signature threshold (Vrefs) levels 15 superimposed. The S2 signal for bubbles (F~. 4(b)), ~lbers (FIG. 4(e)), and bead fragments (~G 4(a)) are clearly qualitatively different from typical bead signals. The method of discrimin~ting bead from non-bead events focuses on the number of major oscillations typical for these signals. Using FIG 4(c) as a reference, bead event ~ign~ typically 20 cross the signature threshold three times. In fact, occasional bead event signals (e.g., FIG. 4(d)) are asymmetrically offset, resulting in only two crossings. Typical bubble events result in only one crossing. Bead fragments and ~lbers create variable signals, with fewer than two or more than three crossings, respectively. The variable low levels associated 25 with these signals can be further discrimin~ted by setting the signature threshold suitably high. This level can be determined by observing typical bead signals and setting the threshold accordingly.

CA 02238982 l998-0~-28 W O 97130897 PCTrUS97/02735 ~ eferring to F~G. 5, another embodiment of the invention is shown that enables drops to be re-directed from their path to the container 9 by a deflector 41. These unwanted drops are caught by a collection trap 42 where the fluid and debris are removed through a S vacuum sump pump 43. Thus, only drops cont~ining beads, but not debris or bead fragments (or multiple beads, in the single-bead-per-container setting), are allowed to accumulate in a given container. This is advantageous in the single-bead-per-container setting by avoiding cases where multiple beads (see FIG. 4(d)) are delivered to a container or ~0 where the container is filled with liquid before any bead is delivered. In either multiple- or single-bead-per-container settings, this embodiment is advantageous by removing instances of debris or bead fragment-cont~ining drops in a container. Where a drop is deflected due to the undesirable presence of multiple beads therein, the present invention 15 contemplates that such multiple-bead-contz~ining drops may be collected and recycled to vessel 1.
The decision to deflect or deliver a given drop to a container must be made after the drop has been released from the end of tube 4 and detected in-flight by drop detector ~, since the information necessary to 20 decide is not complete until that time. Bead threshold electronics (23, 247 28, 29) initi~tP the event of detecting a bead as described above.
Bead signature electronics (25, 26, 27, 28, 29) discrimin~te between bead and non-bead events as described above. Deflector logic 44 is alerted by any event triggered by the bead threshold electronics and is prepared to 25 advance the X-Y transport upon completion of the upcoming drop event.
Bead authenticity information with respect to the alerting event and all subsequent events is also provided to the deflector logic 44 by the bead signature electronics. The state machine 34 provides a timing pulse to CA 02238982 l998-05-28 W 097/30897 PCT~US97/02735 the deflector logic 44 when the drop Cont~ining the initi~fin~ bead is in flight. Deflector logic 44 can now determine whether that drop is acceptable based on the timely information described above. If the drop in question is acceptable (i.e., is determined to contain only beads or a S single bead), then the deflector logic activates the solenoid drive 45 to move the deflector 41 out of the path of the drop, so as to complete the drop event ~he time taken to accept the drop and activate the solenoid is in the range of 2 to 15 msec, based on the ffight time of the drop and the position of the deflector. This is easily achieved by standard digital lO electronics and electromechanical actuators of the a~lo~liate size and weight. Device 46 is a rotary solenoid-based actuator that moves deflectors by rotation caused by electrical current.
Although the invention has been disclosed with reference to the embodiments depicted herein, it will be apparent to one of ordinary skill 15 in the art that various modifications and substitutions may be made to such embodiments. Any such modifications and/or substitutions are intended to be within the scope of the invention as defined by the following claims.

Claims (25)

WHAT IS CLAIMED IS:

1. An apparatus for precisely arraying small solid objects into a plurality of containers, which comprises:

a vessel containing small solid objects in a fluid suspension;

a tube, the proximal end of which is immersed in said suspension, said tube being adapted for transport of fluid from said vessel;

a member, connected to the distal end of said tube, adapted for directing gravitationally-formed fluid drops from said distal end downward to be deposited into a plurality of containers positioned below said distal end;

a small-objects detector, adapted for detecting said objects in the fluid in said tube, disposed near said distal end;

a drop detector attached to said member below said distal end;

means for controlling flow of said suspension through said tube;

means for discriminating between signals from said small-objects detector caused by said objects and signals not caused by said objects, means for comparing signals from said small-objects detector caused by said small objects with signals from said drop detector;

means for determining the number of said objects deposited into each of said containers;

means for re-positioning the distal end of said tube from one said container to another said container; and means for timing said re-positioning with respect to signals from said drop detector, said small-objects detector, or the combination thereof.

2. An apparatus of Claim 1 wherein the objects in said vessel are in an in agitated, isobuoyant fluid suspension.

3. An apparatus of Claim 1 wherein said tube is flexible and optically clear at the wavelength of the radiation utilized by said small-objects detector.

4. An apparatus of Claim 1 wherein the means for controlling flow of said suspension through said tube include pressurizing said vessel.

5. An apparatus of Claim 1 wherein the proximal end of said tube is positioned substantially at the bottom of said vessel.

6. An apparatus of Claim 1 wherein said small-objects detector comprises an optical detector and a collimator block.

7. An apparatus of Claim 1 wherein said objects are about 50-1000 µm in diameter and are cellulose beads, controlled pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, poly-acrylamide beads, latex beads, dimethyl-acrylamide beads optionally cross-linked with N,N'-bis-acryloyl ethylene diamine, or glass particles coated with hydrophobic polymer.

8. An apparatus of Claim 2 wherein said isobuoyant fluid suspension has a room temperature viscosity in the range 0.500-4.000 mPa-sec, a surface energy in the range 15-65 mJ/m2, and a density in the range of 1.00-1.50 g/cm3.

9. An apparatus of Claim 8 wherein said isobuoyant fluid is trichloroethane/isopropanol (TCE/IPA) in the range 70-90:30-10 by volume or water/KBr/isopropanol in the range 35-45:35-45:15-25 by weight.

10. An apparatus of Claim 3 wherein said tube has an inside diameter of 200-4000 µm, and a length of 1.0-2.0 m.

11. An apparatus of Claim 4 wherein said means for controlling flow is compressed gas.

12. An apparatus of Claim 1 wherein said means for re-positioning the distal end of said tube comprises an X-Y transport which is either (i) a movable member which moves the distal end of said tube and said detectors; or (ii) an X-Y stage which moves the containers, wherein said tube and said detectors are attached to a stationary member.

13. An apparatus of Claim 1 further comprising a drop deflector for removal of drops as they fall from said tube to said containers.

14. A method for precisely arraying small solid objects into a plurality of containers, which comprises:

providing said objects in a fluid suspension;

transporting said fluid suspension, at a controlled rate, to a small-objects detector;

discriminating between signals from said small-objects detector caused by said objects and signals not caused by said objects, allowing discrete fluid drops to form due to action of gravitational forces upon said fluid suspension;

allowing said discrete drops to fall, due to action of said gravitational forces;
providing a drop detector in the path of the falling drops;

comparing signals from said drop detector with signals from said small-objects detector caused by said objects, so as to determine the number of said objects in each drop;
directing said drops into a container;

redirecting said drops into a different container, based on signals from said drop detector, signals from said small-objects detector, or signals from both such detectors indicating that a desired number of said small objects have been directed into the previous container; and repeating said redirecting step.

15. A method of Claim 14 wherein said fluid suspension is an agitated, isobuoyant fluid suspension.

16. A method of Claim 14 wherein said transporting of said fluid suspension at a controlled rate is achieved by pressurizing said suspension.

17. A method of Claim 14 wherein said small-objects detector comprises an optical detector and a collimator block.

18. A method of Claim 14 wherein said objects are about 50-1000 µm in diameter and are cellulose beads, controlled pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, poly-acrylamide beads, latex beads, dimethyl-acrylamide beads optionally cross-linked with N,N'-bis-acryloyl ethylene diamine, or glass particles coated with hydrophobic polymer.

19. A method of Claim 15 wherein said isobuoyant fluid suspension has a room temperature viscosity in the range 0.500-4.000 mPa-sec, a surface energy in the range 15-65 mJ/m2, and a density in the range of 1.00-1.50 g/cm3.

20. A method of Claim 19 wherein said isobuoyant fluid is trichloroethane/isopropanol (TCE/IPA) in the range 70-90:30-10 by volume or water/KBr/isopropanol in the range 35-45:35-45:15-25 by weight.

21. A method of Claim 16 wherein said pressurizing is achieved with compressed gas.

22. A method of Claim 14 wherein the controlled rate is about 60 drops/min., and wherein drop volumes are in the range 5-40 µL
per drop.

23. A method of Claim 14 wherein redirecting said drops is effected with an X-Y transport.

24. A method of Claim 14 further comprising deflecting certain drops as they fall, so that the drops are not directed into a container.

25. A method of Claim 24 wherein the deflecting occurs after comparing signals from said drop detector with signals from said small-objects detector.