ROTARY PLATFORM FOR CELL LYSING AND PURIFICATION AND METHOD OF USE

Abstract

Magnetic beads having cell components of interest are translated between a sequence of processing wells in a tray without need for pipetting. The circular tray contains one or more sequences of wells each interconnected by a respective channel. The tray is rotated about a central axis and a magnet, an agitator, and a heater provided external to the tray enable magnetic bead translation, mixing, and incubation, respectively. The magnet proximate a well forms a cluster of beads. Manipulation of the tray in rotation and elevation results in translation of the cluster from one well, through a channel, and into an adjacent well. The well containing a cluster may be rotationally positioned in front of the agitator, the agitator extended into contact with the well, followed by mechanical agitation. The heater, disposed beneath the tray, may accept a well lowered thereto for selective heating.

Claims

1. A method of lysing, purifying, and eluting a fluid sample, comprising: providing a rotary platform on a vertically aligned actuator, comprising a substantially circular tray having an upper surface, a lower surface, a thickness between the upper surface and the lower surface, an axis of symmetry orthogonal to the upper and lower surfaces, and an outer radial edge, a sequence of plural wells, each well having an outer surface extending from the lower surface of the tray about a periphery of the tray and having an interior volume and an opening in the tray upper surface, the opening extending into the respective well interior volume, the wells of the sequence of plural wells being disposed in an arc equidistant from the axis of symmetry, a channel formed in the tray upper surface intermediate each pair of adjacent ones of the plural wells in the sequence, each channel having a depth less than the thickness and extending between the openings of the adjacent ones of the plural wells, and a rotor mating interface coaxial with the tray axis of symmetry and in mechanical communication with an actuator, wherein the channel has inner walls and an outer wall intersecting the openings of the adjacent ones of the plural wells in the sequence, all points on each of the inner and outer walls having a respective identical radial distance from the axis of symmetry, and wherein the channel has a floor that is parallel to and intermediate the tray upper and lower surfaces; providing a magnetic member configured to selectively horizontally extend and retract a magnet with respect to a first position adjacent to the tray; disposing a lysis buffer in a primary well of the sequence; disposing wash buffers in secondary wells of the sequence; disposing an elution buffer in a tertiary well of the sequence; disposing a fluid sample into the primary well of the sequence; disposing magnetic beads into the primary well of the sequence; rotating the tray through selective actuation of the actuator to align the primary well with the magnetic member; extending the magnet, by the magnetic member, adjacent the primary well, whereby the magnetic beads are accumulated into a cluster on a wall of the primary well; lowering the tray through selective actuation of the actuator, whereby the cluster of magnetic beads, attracted by the magnet, travels up the primary well wall to the respective channel as the tray is lowered; rotating the tray through selective actuation of the actuator, whereby the cluster of magnetic beads, attracted by the magnet, travels across the respective channel to the opening of a first of the secondary wells as the tray is rotated; raising the tray through selective actuation of the actuator, whereby the cluster of magnetic beads, attracted by the magnet, travels down the well wall of the first of the secondary wells as he tray is raised; retracting the magnetic member whereby the cluster of magnetic beads is released; repeating the sequential steps of extending the magnetic member, lowering the tray, rotating the tray, raising the tray, and retracting the magnet for each subsequent secondary well including a last secondary well; extending the magnetic member whereby the magnet is adjacent the last secondary well and whereby the magnetic beads are accumulated into a cluster on a wall of the last secondary well; lowering the tray through selective actuation of the actuator, whereby the cluster of magnetic beads, attracted by the magnet, travels up the last secondary well wall to the respective channel as the tray is lowered; rotating the tray through selective actuation of the actuator, whereby the cluster of magnetic beads, attracted by the magnet, travels across the respective channel to the opening of the tertiary well as the tray is rotated; raising the tray through selective actuation of the actuator, whereby the cluster of magnetic beads, attracted by the magnet, travels down the well wall of the tertiary well as the tray is raised; and aspirating eluate from the tertiary well.

2. The method of claim 1, further comprising; providing an agitating member configured to selectively horizontally extend and retract an agitator with respect to a second position adjacent to the tray; and rotating the tray through selective actuation of the actuator, whereby the well containing the magnetic beads is aligned with the agitating member; extending the agitating member whereby the agitator is in mechanical communication with the well containing the magnetic beads; agitating the well containing the magnetic beads; and retracting the agitating member.

3. The method of claim 1, further comprising: providing a heater at a position radially offset from the axis of symmetry substantially equal to the radial offset of each well in the sequence of wells; rotating the tray through selective actuation of the actuator, whereby a well to be heated is disposed above the heater; lowering the tray through selective actuation of the actuator, whereby the well to be heated is disposed within the heater; actuating the heater to heat the well to be heated to a desired temperature or temperature profile for a desired time period; and raising the tray through selective actuation of the actuator, whereby the well to be heated is retracted from the heater.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Illustrative embodiments of the disclosed technology are described in detail below with reference to the attached drawing figures, which are incorporated by reference herein and wherein:

[0014] FIG. 1 is a perspective view of a rotary platform having plural well sequences each interconnected by a respective channel according to the present disclosure;

[0015] FIG. 2 is a top view of the rotary platform of FIG. 1;

[0016] FIG. 3 is front elevation view of the rotary platform of FIG. 1;

[0017] FIG. 4 is a rear elevation view of the rotary platform of FIG. 1;

[0018] FIG. 5 is a right side elevation view of the rotary platform of FIG. 1;

[0019] FIG. 6 is a left side elevation view of the rotary platform of FIG. 1;

[0020] FIG. 7 is a bottom view of the rotary platform of FIG. 1;

[0021] FIG. 8 is a bottom perspective view of the rotary platform of FIG. 1;

[0022] FIG. 9 is a system for cell lysing and purification incorporating the rotary platform of FIG. 1 according to the present disclosure; and

[0023] FIG. 10 is a flowchart of a presently disclosed process for cell lysis, purification, and elution employing the system of FIG. 9.

DETAILED DESCRIPTION

[0024] Disclosed herein is a rotary platform for cell lysing, sample purification, and elution. Use of the platform enables simplified movement of magnetic particles or beads and associated sample fragments such as nucleic acids through a multi-stage cell lysis and purification protocol without the requirement for external pipetting systems and associated pipetting tips.

[0025] A rotary tray having at least one sequence of processing wells distributed along a periphery thereof is mounted for selective rotation and elevation relative to an extendable and retractable magnet. The magnet enables selective attraction of magnetic beads into a cluster within a well. Coordinated movement of the tray in rotation and elevation enables the magnetic bead cluster to be moved from well to well without the use of a pipetting system or associated disposable tips and while the magnet remains stationary. The system may also include an extendable and retractable agitator for selective agitation of the contents of a well, such as for purposes of washing. In addition, a heater may be located beneath the tray and offset from an axis of symmetry of the tray such that a well may be aligned above the heater, the tray lowered, and the aligned well received within the heater for a desired period of heating, such as for lysing or elution.

[0026] With regard to FIG. 1, a rotary platform 100 for cell lysing, purification, and elution is shown. The platform comprises a substantially circular tray 102 having an upper surface 104 and an opposite lower surface 106 (FIG. 7), the upper and lower surfaces defining a thickness therebetween. A vertical axis of symmetry 110 passes through the center of the tray, orthogonal to the upper and lower surfaces thereof. The tray outer extent is defined by an outer radial edge 112. The tray further includes a rotor mating interface 140 (FIGS. 2 and 7) coaxial with the axis of symmetry for mechanically mating to an actuator 302 via a rotor 304 configured to selectively rotate and adjust the elevation of the tray, as will be discussed subsequently.

[0027] Disposed in conjunction with the tray 102 is at least one sequence 120 each comprising plural wells 122. In the figures, there are two such sequences per tray, symmetrically distributed about the axis of symmetry 110. As can be seen in FIG. 1, each well has an outer surface that extends from the lower surface 106 of the tray about a periphery of the tray in an arc equidistant from the axis of symmetry. The outer surface as illustrated is frustoconical and extends substantially orthogonal to the tray lower surface, narrowing as the well extends distal from the tray.

[0028] Each well 122 has an interior volume 124 and an associated opening 126 in the tray upper surface 104. The opening of each well extends into the respective interior volume. Each opening is substantially circular and coplanar with the tray upper surface 104, except where it is intersected by a channel 130 of the respective well sequence 120.

[0029] The channel 130 is formed in the tray upper surface 104. The channel connects each adjacent pair of wells 122 within a sequence 120 of wells. Thus, as there are two sequences of wells in the presently illustrated embodiment, there are two channels, one for each sequence.

[0030] The depth of each channel 130 is less than the thickness of the tray 102 between the upper 104 and lower 106 surfaces. Intermediate each pair of wells, the channel has inner walls 132 and an outer wall 134. The inner and outer walls intersect the openings 126 of the adjacent wells.

[0031] All points on the inner walls 132 have the same radial distance from the tray 102 axis of symmetry 110. This radial distance is the same as the radial distance between the axis of symmetry and the center point of each circular opening 126 associated with each well 122. Similarly, all points on the outer wall 134 have the same radial distance from the axis of symmetry. The outer wall radial distance is greater than that of the inner walls. The radial distance from the axis of symmetry for points on the outer wall is the same as the maximum radial distance from the axis of symmetry to each well opening 126. Each channel 130 also includes a floor surface 136 that is substantially parallel to and intermediate the upper 104 and lower 106 surfaces of the tray 102.

[0032] The rotary platform 100 as described in the foregoing may be used in a system 300 for cell lysing, purification, and elution. As seen in FIG. 9, in addition to the previously described platform, the system includes an actuator 302 having a rotor 304. The actuator enables the selective rotation and adjustment in elevation of the platform. Specifically, the rotor 304 is configured to mate with a tray 102 mating interface 140 (FIGS. 2 and 7) that is coaxial with the tray axis of symmetry 110. The actuator, under control of a controller as known to one skilled in the art, is capable of selectively rotating the tray and adjusting its height.

[0033] The system 300 also includes a magnetic member 306 adapted to selectively extend and retract a magnet 308 with respect to a first position adjacent the tray 102 in a horizontal plane. In addition, an agitating member 310 is adapted to selectively extend and retract an agitating tip (also referred to herein as an agitator) 312 with respect to a second position adjacent the tray 102 in a horizontal plane. The agitating member is also capable of imparting agitating motion in the agitating tip. The magnetic member and the agitating member may also be under control of the same or distinct programmable controllers. In the illustrated embodiment, the first and second positions are separated by 180 degrees about the tray.

[0034] The system may further comprise a heater 316 disposed beneath the tray 102 and, in particular, offset from the tray 102 axis of symmetry 110 the same distance as the wells 122.

[0035] Thus, through coordinated movement of the rotary platform 100, the actuator 302, magnetic member 306, agitating member 310, and heater 316, a method of lysing, purifying, and eluting a sample may be carried out. This method is described with respect to FIG. 10.

[0036] Lysis buffer is disposed 202 in a first well 122 of a sequence of wells 120, wash buffer is disposed in one or more second wells, and elution buffer is disposed in a third well. Ideally, a bulk liquid loader (not illustrated) may be used for this purpose in order to expedite the testing method.

[0037] A quantity of fluid sample may then be disposed 204 into the first well 122, previously loaded with lysis buffer, through the use of a pipetting system (not illustrated). The actuator 302 is then activated to align 206 the first well with the agitating member. The agitating tip 312 is extended 208 laterally towards the first well, and the agitating tip agitates the first well exterior surface. Such agitation may, for example, by ultrasonic. After agitation, the agitating tip is retracted.

[0038] Next, the tray 102 is again rotated 210 by the actuator 302 via the rotor 304 to align the first well 122 with the heater 316. The tray is lowered 212 through adjustment in tray height via the actuator, by which the first well is lowered into the heater. A desired temperature or temperature profile is applied to the well for a desired temporal period. The tray is the elevated by the actuator.

[0039] Magnetic beads are then disposed 214 within the first well 122, and the tray 102 is rotated 216 by the actuator 304 to align the target well with the magnetic member 306. The magnet 308 is extended by the magnetic member to a position proximate the target well, allowing magnetic beads to cluster on the well inner wall near the magnet.

[0040] With the magnet remaining extended, the actuator 302 lowers 218 the tray. Since the cluster of magnetic beads is held in vertical position by the magnet, the cluster is effectively dragged into the channel 130 as the tray descends. Next, the actuator rotates 220 the tray 102 thereby moving the relative location of the cluster through the channel into the upper extent of the second well 122. The actuator then raises 222 the tray, thereby positioning the cluster into the lower extent of the second well. The magnetic member 306 then retracts 224 the magnet 308, releasing the cluster of magnetic beads from the well side wall.

[0041] To enhance the dispersion of the magnetic beads, with sample components of interest attached thereto, the tray is again rotated 226 about the axis of symmetry 110 by the actuator 302 to align the target well 122 with the agitating member 310. The agitating tip 312 is extended 228 and the well is agitated, following which the agitating tip is retracted.

[0042] Optionally, the tray 102 is once again rotated 230 to the heater 316, the tray is lowered 232, disposing the target well into the heater, the well is heated as desired, and the tray is elevated.

[0043] The cycle of clustering the magnetic beads, moving them from one well into the channel and into the next well, retracting the magnet, agitating the well contents, and optionally heating the well contents may be repeated 234 for each subsequent second well having wash buffer therein.

[0044] Once the last wash iteration has been completed, the actuator 302 aligns 236 the target second well having magnetic beads with sample components associated therewith with the magnetic member 306, and the magnet 308 is extended into proximity with the target well. The tray 102 is lowered 238 by the actuator 302, thereby moving the cluster up the well wall to the channel 130. The tray is again rotated 240 to move the cluster through the channel to the third well, which is the elution well. The tray is raised 242 to locate the cluster at a lower extent of the respective well, and the magnet is retracted 244. The magnetic beads are again dispersed by rotating 246 the tray to align the target well with the agitating member 310, which extends 248 the agitating tip 312 to the well, agitates the contents thereof, then retracts the agitating tip.

[0045] The tray is again rotated 250 to align the target well with the magnetic member 306 and the magnet 308 is extended to attract the magnetic beads from which the components of interest have been released by the elution buffer and agitation. The eluate is then aspirated 252. While not illustrated, the method may further include rotating the target well to the heater for incubation after agitation and prior to attracting the magnetic beads and removing the eluate.

[0046] The tray 102 may then be removed from the mating interface 140 and transferred to a waste receptacle, following which a new tray may be provided.

[0047] The foregoing description has been directed to particular embodiments. However, other variations and modifications may be made to the described embodiments, with the attainment of some or all of their advantages. It will be further appreciated by those of ordinary skill in the art that modifications to the above-described systems and methods may be made without departing from the concepts disclosed herein. Accordingly, the invention should not be viewed as limited by the disclosed embodiments. Furthermore, various features of the described embodiments may be used without the corresponding use of other features. Thus, this description should be read as merely illustrative of various principles, and not in limitation of the invention.

[0048] Many changes in the details, materials, and arrangement of parts and steps, herein described and illustrated, can be made by those skilled in the art in light of teachings contained hereinabove. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub combinations and are contemplated within the scope of the claims. Accordingly, it will be understood that the following claims are not to be limited to the embodiments disclosed herein and can include practices other than those specifically described, and are to be interpreted as broadly as allowed under the law. Additionally, not all steps listed in the various figures need be carried out in the specific order described.