WO2021002941A1 - Plateau multipuits pour la lyse et la purification cellulaire et procédé d'utilisation - Google Patents

Plateau multipuits pour la lyse et la purification cellulaire et procédé d'utilisation Download PDF

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Publication number
WO2021002941A1
WO2021002941A1 PCT/US2020/032415 US2020032415W WO2021002941A1 WO 2021002941 A1 WO2021002941 A1 WO 2021002941A1 US 2020032415 W US2020032415 W US 2020032415W WO 2021002941 A1 WO2021002941 A1 WO 2021002941A1
Authority
WO
WIPO (PCT)
Prior art keywords
process well
probe tip
unitary structure
platform
eluate
Prior art date
Application number
PCT/US2020/032415
Other languages
English (en)
Inventor
Dan Harris
Eric Yeaton
Original Assignee
Siemens Healthcare Diagnostics Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Siemens Healthcare Diagnostics Inc. filed Critical Siemens Healthcare Diagnostics Inc.
Publication of WO2021002941A1 publication Critical patent/WO2021002941A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50855—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using modular assemblies of strips or of individual wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/043—Hinged closures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/54—Supports specially adapted for pipettes and burettes
    • B01L9/543—Supports specially adapted for pipettes and burettes for disposable pipette tips, e.g. racks or cassettes

Definitions

  • the disclosure herein relates generally to the field of cell lysing and purification. More particularly, the present disclosure relates to a multi-well tray having particular utility in the field of cell lysis in molecular diagnostics.
  • a sample is moved by a pipette system to a well within a multi-well plate along with a cell lysis buffer and by a quantity of magnetic beads.
  • a succession of magnetic bead separation, supernatant aspiration, and dilution/washing steps are repeated with respect to the well.
  • Heating of the multi-well plate may also be employed to facilitate lysis and/or binding.
  • the sample transfer, washing, and elution steps require separate aspiration and dispensing tips to avoid cross-contamination.
  • An alternative system and technique involves the use of a magnet disposed within a sealed probe.
  • the probe is selectively disposed within a respective well to allow the magnetic particles to be attracted to the probe by the magnet located within.
  • the probe may be removed from one well and inserted into fluid within another well. The magnet may then be extracted from within the probe, thus releasing the magnetic particles to be released from the probe surface. Further processing may then follow.
  • the present disclosure provides a disposable unitary structure having a series of open fluid wells.
  • a first well is a working well where all of the cell processing occurs.
  • Other wells serve as liquid storage wells.
  • the working well is provided with a facility to enable sonication without unintended fluid dispersion. Provision is also made for selective, customizable direct heating of the working well to enhance incubation and lysing, if desired.
  • a particular point of distinction associated with the presently disclosed system and method includes provision of a receptacle for a sample tip for use in transferring the respective sample and for transferring other fluids from the storage wells into the working well, thus reducing costs associated with the provision of redundant disposables.
  • Elution of the processed sample requires a purpose-specific eluate probe to avoid contamination.
  • the present system and method enable the provision of a tray with a preloaded eluate probe tip, thereby speeding up the overall lysis process.
  • multiple trays may be provided in bulk, in a stacked configuration, with each tray having a respective preloaded eluate probe tip.
  • the tray is preferably usable within a process flow that enables discrete sample, wash, and eluate pipettors for greater efficiency.
  • FIG. 1 is a front, left, top perspective view of the unitary structure according to the present disclosure
  • FIG. 2 is top view of the unitary structure of Fig. 1;
  • FIG. 3 is a left side view of the unitary structure of Fig. 1;
  • Fig. 4 is a bottom view of the unitary structure of Fig. 1;
  • Fig. 5 is a right side view of the unitary structure of Fig. 1;
  • FIG. 6 is a front view of the unitary structure of Fig. 1;
  • Fig. 7 is a rear view of the unitary structure of Fig. 1;
  • Fig. 8 is a side section view of multiple unitary structures of Fig. 1 is a vertically stacked configuration
  • Fig. 9 is a flowchart of a method of sample lysis, purification, and elution.
  • Disclosed herein is a multi-well disposable tray for use in cell lysing and purification. Use of the presently disclosed and described tray enables simplified and faster cell lysis as compared to currently practiced methods.
  • a unitary structure comprised of a substantially planar and rectangular platform having a process well and plural storage wells enables all steps of a cell lysis process to be carried out under optimal conditions prior to extraction of eluate and disposal of the structure.
  • the storage wells are configured to retain all process materials required to carry out the lysis including wash buffer, elution buffer, and magnetic beads.
  • Overall process costs are reduced through the provision of a sample probe tip retaining feature which enables a sample probe tip to be used multiple times during the process, thus freeing up a pipetting system during time periods when the sample probe tip is not in use and obviating the need for a new sample probe tip when the pipetting system is again needed for aspiration and dispensing buffer within the process.
  • Figs. 1, 2, and 3 illustrate an embodiment of a unitary structure 100 according to the present disclosure.
  • the structure is comprised of a base unit 102 having a substantially planar platform 104 surrounded by a partially peripheral wall 106.
  • the wall may help inhibit the unintended flow of working fluid off the platform.
  • a storage tip retaining feature 120 is provided as a substantially circular aperture through the platform 106 at the second end 109 of the platform 104.
  • Plural elution probe tip retaining features 122 are also each provided as a substantially circular aperture through the platform.
  • the elution probe tip retaining features are located intermediate the process well 110 and the storage wells 112 and are preferably alternating on opposite side of the longitudinal axis 132 of the platform.
  • two elution probe tip retaining features are centered on one side of the longitudinal axis while a third, intermediate elution probe tip retaining feature is centered on the opposite side of the longitudinal axis, as best viewed in Fig. 2.
  • the process well 110 and the storage wells 112 each have a tapered lower extent.
  • This enables multiple unitary structures 100 to be vertically stacked, as shown in Fig. 8, whereby the outer surface of a process well of a first unitary structure is received within the process well of a lower, second unitary structure.
  • the outer surfaces of the storage wells of the first unitary structure are each received within a respective storage well of the lower, second unitary structure.
  • the sample probe tip retaining feature 120 of the first unitary structure is vertically aligned or coaxial with a sample probe tip retaining feature of a lower, second unitary structure.
  • each of the eluate probe tip retaining features 122 of the first unitary structure is vertically aligned or coaxial with a respective eluate probe tip retaining feature of the lower, second unitary structure.
  • the unitary structure 100 further comprises a sonication cap 116 at the first end 108 of the platform 104 of the base unit 102, proximate the process well 110.
  • the sonication cap includes a hinge 128, such as a living hinge, that enables the selective positioning of the sonication cap between a relaxed or open position, as shown in Fig. 1, and a closed position, in which the sonication cap engages with and covers the upper extent 114 of the process well at the platform.
  • the sonication cap and the process well may mechanically cooperate or engage with each other such as through interference fit or through the mutual engagement of cooperative physical features such as a peripheral ridge about the upper extent of the process well and inwardly extending projections within the sonication cap. This engagement thus allows for selective, releasable sealing of the process well and inhibits the discharge of fluid within the process well during a sonication process.
  • the sonication cap 116 may be provided with a tab 118, as shown in Figs. 1 and 3.
  • This tab which may project from an outer surface of the sonication cap, provides a surface against which an external mechanical translating mechanism may bear for forcing the cap into communication with the process well and for disengaging the tab therefrom, thereby implementing the selective, releasable sealing.
  • a sample probe tip for pipetting applications comprises a tapered shaft having an aperture at a distal end for aspirating and dispensing fluid therethrough and a mating socket, connected to an upper end of the shaft, for engagement with a pipettor.
  • the mating socket is typically cylindrical or frustoconical with a minimum outer diameter that is greater than the maximum width of the shaft.
  • the sample probe tip retaining feature 120 is selected to have a diameter greater than the maximum width of the sample probe tapered shaft but less than the minimum width of the mating socket.
  • a pipettor may eject a sample probe tip into the sample probe tip retaining feature whereby the tapered shaft extends downwardly through the retaining feature while a lower extent of the mating socket rests on an upper surface of the platform 104 about the retaining feature.
  • eluate probe tips are known in the art to have a similar structure, with a tapered shaft having an aperture at a distal end thereof and a mating socket that is cylindrical or frustoconical.
  • Each eluate probe tip retaining feature 122 formed in the platform 104 has a diameter that is greater than the maximum diameter of the eluate probe tip tapered shaft but less than the minimum diameter of the mating socket, whereby the eluate probe tip shaft may be ejected into the respective retaining feature while the mating socket rests on the platform surface adjacent the respective retaining feature.
  • the unitary structure further comprises a reinforcing feature or member 124 disposed on an underside surface of the platform 104
  • the reinforcing feature is a wall-like structure that extends from near the second end 109 of the base unit 102 to and intermediate the downwardly projecting storage wells 112 These portions of the reinforcing feature are substantially planar.
  • the reinforcing feature 124 also extends from one of the downwardly projecting storage wells 112 to an inner side of the downwardly projecting process well 110, orthogonal to a lower surface of the platform and underneath the eluate probe tip retaining features 122 Rather than continuing to extend underneath the platform 104 and the eluate probe tip retaining features in a planar fashion, the reinforcing feature is undulating or serpentine from a side of the proximate storage well 122, around an inner side of the first eluate probe tip retaining feature, between the first and second eluate probe tip retaining features, between the second and third eluate probe tip retaining features, and around an inner side of the third eluate probe tip retaining feature to a side of the process well.
  • the reinforcing feature enhances the rigidity of the platform and resists its deflection such as when a pipettor presses down against a sample probe tip or an eluate probe tip retained within the unitary structure 100.
  • the undulating pattern underneath the plural eluate probe tip retaining features is particularly effective at enhancing the rigidity of the platform and physically isolating the areas beneath the eluate probe tip retaining features, as discussed below.
  • the reinforcing feature also enforces a degree of vertical separation between unitary structures when stacked, as shown in Fig. 8, where a lower extent of the reinforcing feature rests on an upper platform surface of the underlying unitary structure.
  • each unitary structure 100 when unitary structures 100 are vertically stacked, eluate probe tip retaining features are mutually vertically aligned.
  • each unitary structure it is preferable for each unitary structure to be provided with a respective eluate probe tip. This enhances the efficiency of the elution process by obviating the need for a pipettor to translate to a remote carrier of eluate probe tips.
  • disposable eluate probe tips used for molecular diagnostics are typically provided with an internal filter. Thus, these probe tips cannot be deeply nested within each other.
  • each unitary structure in a stack has an eluate probe tip in an eluate probe tip retaining feature that is not occupied by a probe tip in the unitary structure above or below.
  • the process well 110 of the unitary structure 100 may be subjected to heating or sonication, depending upon the lysis process implemented therewith.
  • the outer, tapered surface of the process well may be configured to be received within a heater or a sonicator external to the unitary structure.
  • a heater or sonicator may swing into position from beneath the unitary structure, receiving the outer, tapered surface of the process well therewithin for a required or desired time period.
  • the unitary structure 100 of the present disclosure may be utilized in conjunction with an external magnet that may be selectively translated to and away from a side wall of the process well 110 in order to attract and release, respectively, magnetic beads disposed within the process well.
  • the unitary structure 100 may be provided with retention features 126 proximate the base unit 102. These retention features may be provided as lateral projections extending from the base unit on either side of the base unit. As seen in Figs. 1, 2, and 3, the retention features may be shelf-like extensions. During processes such as heating and sonication, when external devices move relative to the lower extent of the process well, the retention features may be selectively engaged by external gripping mechanisms, thereby maintaining the unitary structure in a fixed position relative to the external devices. The retention features may also be of use during sample probe tip and eluate probe tip retrieval as a pipetting system presses down on the respective tip in order to effect mechanical engagement.
  • Figs. 4, 5, 6, and 7 provide alternative views of the unitary structure according to the present disclosure.
  • step 202 wash and elution buffers are bulk loaded into the storage wells 112, and in step 204, lysis buffer is bulk loaded into the process well 110.
  • a sample is then loaded into the process well.
  • the sample may be aspirated by a pipetting system using a sample probe tip from a sample source then dispensed into the process well. After the sample has been dispensed, the sample probe tip may be inserted into the sample probe tip retaining feature 120
  • Magnetic beads are also inserted into the process well 110, as at step 208. While this illustrated process depicts a certain order of loading the process well, other orders may be employed, such as disposing the magnetic beads into the process well prior to adding the sample.
  • the contents of the process well 110 may be heated and/or agitated, such as through sonication, prior to the illustrated step 210 of applying a magnetic field to an exterior surface of the process well.
  • the process well liquid is then aspirated 212 by the pipetting system using the sample probe tip retrieved from the sample probe tip retaining feature 120.
  • the aspirated liquid is dispensed to a waste receptacle.
  • the unitary structure is processed in a position with a waste receptacle immediately below the sample probe tip retaining feature.
  • the liquid aspirated from the process well may be dispensed from the sample probe tip to waste in more or less the same location as where the sample probe tip may be disengaged by the pipetting system.
  • the magnetic field is then removed from the process well 214.
  • Wash buffer is next aspirated from one of the storage wells 112 by the pipetting system using the sample probe tip and dispensed 216 into the process well.
  • the steps of applying 210 a magnetic field to the process well 110, aspirating and disposing of 212 the aspirated liquid, removing the magnetic field 214, and aspirating and dispensing 216 wash buffer into the process well may be repeated 218 a desired number of times. Heating and agitation, such as through sonication, may also be implemented after wash buffer is reintroduced into the process well.
  • a magnetic field is reapplied 220 to the process well 110 and the liquid is aspirated and disposed of 222.
  • the sample probe tip is used by the pipetting system to aspirate 224 a quantity of elution buffer from one of the storage wells 112 and to dispense it into the process well 110.
  • agitating of the process well may again occur.
  • a magnetic field is again applied 226 to the process well 110.
  • a pipettor system then engages an elution probe tip from one of the elution probe tip retaining features 122 and is used to extract 228 the eluate from the process well.
  • the unitary structure may then be transferred to a waste receptacle.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

L'invention concerne un système et un procédé de lyse cellulaire automatisée et de traitement de purification comprenant un plateau ayant une série de puits de fluide ouverts. Un premier puits est un puits de traitement pour le traitement de cellules et des puits supplémentaires sont destinés au stockage tampon. Un capuchon de sonication est associé au puits de traitement. L'invention concerne également le chauffage et la sonication direct du puits de traitement. Le stockage pour une pointe de sonde d'échantillon respective permet une réutilisation de la pointe, tandis qu'un système de stockage de pointe d'éluat flexible permet la pré-population de pointes d'éluat avant le déploiement.
PCT/US2020/032415 2019-07-03 2020-05-12 Plateau multipuits pour la lyse et la purification cellulaire et procédé d'utilisation WO2021002941A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962870093P 2019-07-03 2019-07-03
US62/870,093 2019-07-03

Publications (1)

Publication Number Publication Date
WO2021002941A1 true WO2021002941A1 (fr) 2021-01-07

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090130745A1 (en) * 2007-07-13 2009-05-21 Handylab, Inc. Integrated Apparatus for Performing Nucleic Acid Extraction and Diagnostic Testing on Multiple Biological Samples
US8703492B2 (en) * 2007-04-06 2014-04-22 Qiagen Gaithersburg, Inc. Open platform hybrid manual-automated sample processing system
US9040288B2 (en) * 2006-03-24 2015-05-26 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using the same
US9519000B2 (en) * 2010-07-23 2016-12-13 Beckman Coulter, Inc. Reagent cartridge

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9040288B2 (en) * 2006-03-24 2015-05-26 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using the same
US8703492B2 (en) * 2007-04-06 2014-04-22 Qiagen Gaithersburg, Inc. Open platform hybrid manual-automated sample processing system
US20090130745A1 (en) * 2007-07-13 2009-05-21 Handylab, Inc. Integrated Apparatus for Performing Nucleic Acid Extraction and Diagnostic Testing on Multiple Biological Samples
US9519000B2 (en) * 2010-07-23 2016-12-13 Beckman Coulter, Inc. Reagent cartridge

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