Chemistry: analytical and immunological testing – Automated chemical analysis – With aspirator of claimed structure
Reexamination Certificate
1998-12-17
2001-07-03
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Automated chemical analysis
With aspirator of claimed structure
C422S063000, C422S065000, C422S105000, C436S043000, C436S047000, C436S049000, C436S180000
Reexamination Certificate
active
06255116
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the handling of beads and more particularly to an improved process and apparatus for depositing such beads in two-dimensional arrays.
BACKGROUND OF THE INVENTION
Synthesis of combinatorial compound libraries on bead supports is a well-established method for generating chemical diversity for screening against targets of pharmacological relevance. Such libraries may be synthesized as bulk populations or discrete sublibraries with or without identifier tags for deconvolution. In most cases each bead carries a single unique compound and is present in a mixture of beads containing other compounds. To test the beads for activity against a pharmacological target, it is desirable to separate the beads so that each bead may be individually assayed against one or more targets. The beads carrying active compounds can be identified and the structures of the compounds elucidated. It is also desirable to separate the beads into arrays that are geometrically compatible with robotic screening systems, for example 8×12 arrays or other formats such as 384 or 896-well configurations.
Current methods of bead arraying include manual picking and hydrodynamic sorting in which beads are allowed to flow though an aperture and once detected are deposited into assay wells. Manual picking is slow and tedious. Hydrodynamic methods heretofore in use have been slow and the equipment is prone to clogging. Buoyancy variations within the bead library are also known to cause problems in hydrodynamic sorting. A problem common to the prior methods is that, because the beads are extremely small, typically 300
m(0.3 mm) or smaller, there is a tendency for two or more beads, carrying different compounds, to be deposited occasionally at a single location in the array. Still another problem encountered in bead arraying is that the beads tend to be fragile, and can be broken up into fragments especially when mechanically agitated.
These problems have recently been addressed in a bead picking apparatus by which beads are picked individually from a suspension of beads in a liquid. The suspension can be established by releasing gas bubbles from orifices at the bottom of a vessel to keep the liquid in motion. Alternatively, two immiscible liquids can be used, one having a density greater than that of the beads and the other having a density lower than that of the beads. The beads are suspended at the interface of the two liquids.
The bead picking apparatus comprises a set of hollow needles. Liquid is drawn from the suspension into the internal passages of the needles through openings at an ends of the internal passages, each opening being of a size smaller than any one of the beads and shaped so that it can be substantially closed off by a bead. When a bead closes off the opening, a pressure differential is established such that the external pressure exerted on the bead closing off the opening is greater than the pressure within the passage, and the bead is thereby held in engagement with the opening. The set of needles is then withdrawn from the vessel with the beads in engagement with the openings, and the beads can then be released and deposited into wells at a remote location.
The procedure just described has been used successfully, but has certain drawbacks. It is difficult to use gas bubbles to prevent settling of beads. Elaborate measures must be taken in order for gas bubbles to keep beads in suspension. For example, the vessel for containing the suspension of beads may be constructed with separate compartments, one for each needle in the picking apparatus. The compartments have sloping walls, and each compartment has its own gas-releasing orifice. This arrangement does not lend itself readily to use with a bead picker in which the needles are very close to one another. Therefore, in the case in which beads were to be deposited in a conventional well plate having eight columns of wells, it was necessary to use a picker comprising four needles spaced from one another so that they were alignable with every other well in a row of eight wells, and to index the picker laterally in order to deposit beads in odd-numbered wells of a row in a first step, and thereafter deposit beads in the even-numbered wells of the same row.
In the case of immiscible liquids it is difficult to achieve a satisfactory spatial distribution of beads at the liquid-liquid interface so that beads readily become attached to every needle of the bead picker.
Another difficulty encountered with the prior bead picking apparatus is that excess beads adhering to the needles are jarred loose by stopping the picker suddenly, or by bringing the needles into contact with a stop. Using this method, the excess beads not readily dislodged without a significant risk of dislodging the desired beads attached to the openings of the needles.
Still another difficulty with the prior bead picking apparatus is the clogging that can occur especially in the case in which a needle picks up a broken bead.
SUMMARY OF THE INVENTION
The principal object of this invention is to provide a bead arraying apparatus having improved reliability. Further objects of the invention include avoiding the deposition of unwanted beads into wells, the avoidance of empty wells, and the avoidance of clogging of the picker needles, especially by broken beads.
The bead arraying apparatus in accordance with the invention comprises a bead supply vessel containing a mixture comprising beads in a liquid, and a paddle within the vessel and immersed in the liquid therein. An air cylinder, motor, or other suitable device effects reciprocation of the paddle within the liquid so that the beads are maintained in suspension in the liquid by movement of the liquid induced by the paddle as it reciprocates.
Preferably, the horizontal projection of the paddle has an area sufficient in comparison to the horizontal cross-sections of the vessel that movement of the paddle within the liquid in a substantially vertical path induces a substantial vertical movement of the liquid. The means for effecting reciprocating movement of the paddle preferably moves the paddle in a substantially vertical path, whereby the beads are maintained in suspension in the liquid by the substantial vertical movement of the liquid induced by the paddle as it reciprocates.
The apparatus also includes an array of suitable hollow elements, preferably elongated needles spaced from one another. Each hollow element has an internal passage and an opening at an end of the internal passage. Each of the openings is circular in shape so that it can be substantially closed off by a spherical bead. The array is insertable into the vessel so that all of the openings of the elements are immersed in the liquid within the vessel. A pump or other suitable means is provided for drawing liquid from the vessel into the internal passages of the hollow elements through their openings to establish a pressure differential across each opening such that the external pressure exerted on a bead closing off the opening of each hollow element is greater than the pressure within the passage of the same hollow element. The pressure difference holds the bead in engagement with the opening at least while the opening is immersed in liquid. A robot arm or other suitable means is provided for withdrawing the array of hollow elements from the vessel with beads in engagement with the openings thereof and for moving the array to a remote location for deposition of the beads.
In the case of elongated needles spaced from one another, the needles preferably extend parallel to one another, and the paddle preferably comprises at least one opening through which the needles can extend when the needles are inserted into the vessel. The opening allows the reciprocating movement of the paddle to continue as the needles are immersed in the vessel. The reciprocating movement of the paddle is preferably in a path parallel to the elongation of the needles.
In a preferred embodiment of the invention, the needles are supported
Leber Jack Dale
Lock Brian E.
SmithKline Beecham Corporation
Soderquist Arlen
Venetainer S.
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