WO2003028878A1 - Procedes et moyens permettant de creer des arrangements - Google Patents

Procedes et moyens permettant de creer des arrangements Download PDF

Info

Publication number
WO2003028878A1
WO2003028878A1 PCT/GB2002/004261 GB0204261W WO03028878A1 WO 2003028878 A1 WO2003028878 A1 WO 2003028878A1 GB 0204261 W GB0204261 W GB 0204261W WO 03028878 A1 WO03028878 A1 WO 03028878A1
Authority
WO
WIPO (PCT)
Prior art keywords
wells
solid support
molecules
array
open
Prior art date
Application number
PCT/GB2002/004261
Other languages
English (en)
Inventor
Anthony Joseph Brookes
Walter Mathias Howell
Magnus Jobs
Original Assignee
Dynametrix Limited
Karolinska Innovations Ab
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
Priority claimed from GB0123391A external-priority patent/GB0123391D0/en
Application filed by Dynametrix Limited, Karolinska Innovations Ab filed Critical Dynametrix Limited
Publication of WO2003028878A1 publication Critical patent/WO2003028878A1/fr

Links

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
    • B01L3/50853—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 with covers or lids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • 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
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • 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
    • B01L3/56—Labware specially adapted for transferring fluids
    • B01L3/563—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00421—Means for dispensing and evacuation of reagents using centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00641—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates to immobilization of an array of molecules on a solid surface.
  • the present invention relates to creating arrays from sample molecules of interest present within micro-formatted sample vessels (such as 1,536-well microtiter plates).
  • micro-formatted sample vessels such as 1,536-well microtiter plates.
  • the principle involves centrifugal transfer of molecules of interest onto a solid planar or flexible surface (e.g. membrane) placed over an initial sample vessel.
  • samples are transferred and immobilized upon a solid phase in order to facilitate purification and downstream processing in a high-throughput mode.
  • the solid phase may be any of a wide variety of available alternatives, for example 5 the inner surface of a reaction well, micro-beads, a planar solid surface (e.g. glass or polymer) , or a membrane.
  • [0 reactions e.g. enzyme treatments or hybridization steps
  • [0 reactions can be carried out in a highly-paralleled manner by simple immersion in suitable reagent mixtures.
  • transfer from the starting reaction vessel to the solid surface can be
  • the present invention provides a much simpler alternative to complex liquid handling systems for sample transfer and creation of arrays.
  • the inventors have shown how centrifugation principles may be utilized to create arrays on both planar solid surfaces and flexible surfaces, e.g. 0 membranes, for example starting with microtiter-plate formatted input samples ( Figure 1) .
  • Provided by the present invention are tools and protocols that enable rapid creation of desired arrays from any format of sample plate.
  • An operator of embodiments of the present may salvage any non- transferred excess materials, and may induce other reactions or sample additions prior to and/or during the transfer step.
  • Embodiments of the present invention allow for avoiding cross- contamination between features during centrifugal transfer.
  • Figure 1 illustrates in outline a use of an embodiment of the present invention.
  • An array surface is applied to a sample plate; (2) samples are transferred to the array surface by spinning in a centrifuge; (3) reagents are processed; (4) results are visualized.
  • Figure 2 illustrates a clamping plate arrangement for use in embodiments of the present invention.
  • Figure 3 illustrates layers in a clamping arrangement for centrifugal transfer of molecules from an open well plate to a flexible membrane in accordance with embodiments of the present invention.
  • Figure 4 illustrates layers in a clamping arrangement for centrifugal transfer of molecules from an open well plate to a rigid glass surface in accordance with embodiments of the present invention.
  • the present invention provides a method of creating an array of molecules on a surface of a solid support (4,7), the method comprising: providing an open well plate (6) comprising multiple open wells wherein individual wells contain a molecule to be provided within the array on the surface, securing the surface of the solid support (4,7) over the open wells, subjecting the open wells and solid support to centrifugation to transfer the molecules from the wells to the surface, creating the array.
  • Different wells may and usually do each contain a different molecular species so that an array of different molecules is created on the surface.
  • the solid support (4,7) may be secured over the open wells by clamping, and this may conveniently be achieved in accordance with embodiments of the invention by means of rigid clamping plates (1) sandwiching the open well plate.
  • clamping plates For whole plate transfer such plates need to be of sufficient planar dimension to cover the whole area of solid support on which the array is to be created.
  • Clamping plates may be secured together and tightened to force the solid support surface against the open wells using any suitable arrangement. This may be by using a simple nut and bolt arrangement (such as for example is illustrated in Figure 2) .
  • One or more deformable compression pads (5) may be used to abut the solid support (4,7) and/or open well plate (6), assisting in application of firm and uniform pressure across the solid support surface, pushing it against the open wells. This may used to reduce and preferably eliminate leakage between wells and running together of spots on the support surface.
  • the solid support (4) may be deformable, e.g. a membrane, or it may be rigid, e.g. with a glass surface (7) .
  • a deformable e.g. rubber gasket (8) may be placed between the open wells (6) and the surface of the solid support (7) . Holes in the gasket line up with the open wells of the plate and expose areas of the solid support surface to molecules transferred from the wells under centrifugation. Extending hole dimensions or merging holes that sit above separate wells may be used to provoke mixing of samples during 5 array creation, and/or used to create array formats different from that of the underlying plate .
  • a gasket may be used to reduce and preferably eliminate leakage between wells and running between spots on the surface.
  • providing the open wells with deformable lips can be used to create a seal between the open wells and discrete regions or spots on the surface used to provide the array.
  • the open well plate (6) used may be of any suitable kind, and 15 may comprise 96 wells, 384 or 1,536 wells in conformity with readily available materials, but without limitation in the number of wells, given suitable apparatus. Higher well densities are available and may be used in embodiments of the present invention >0
  • the molecules transferred to the surface of the solid support may be immobilized upon a suitably prepared surface, and this may be by any available chemical, ionic or molecular biological interaction, e.g. biotin-streptavidin binding, DNA- .5 nitrocellulose desiccation.
  • Excess transfer molecules that cannot (e.g. due to capacity limitations) or are not intended, (e.g. specific chemical variants) to have become bound to the surface of the solid 50 support may be returned to the wells by centrifugation. This may be achieved by simply inverting the complete array assembly after immobilization has occurred and performing a second similar centrifugal transfer step that moves reagents into the wells and away from the array surface. In some cases it may be desirable to add further reagents or chemical reactants to the samples or the surfaces that will comprise the centrifugal array.
  • Such reagents may include one or more pH changing solutions, salt mixtures, chemical stabilizers/destabilizers, signal generating molecules, buffers, catalysts and enzymes. Addition of these may be achieved in one or more of several ways in accordance with embodiments of the present invention.
  • the immobilization surface may be immersed in a reagent before creating the centrifugal array.
  • membranes are typically soaked in some buffer before the centrifuge procedure. This is illustrate experimentally below by pre-wetting of the utilized membranes in water.
  • a sheet of absorbent material e..g. tissue paper
  • tissue paper e.g. tissue paper
  • a flexible gel e.g. agarose matrix
  • Such a gel may also be created directly on the immobilization surface or even be the immobilization surface itself.
  • a mist of the new reagent solution may be sprayed over the open plate followed by "pre-spin” arranged so that it forces droplets of reagent on the well-walls down into the wells.
  • Plate and/or sealing gasket designs may be arranged to provoke mixing of reactants in different wells (containing array molecules and new additives respectively) during array creation by centrifugation.
  • the centrifugation used to transfer molecules from the open wells to the surface may apply a G-force suitable for moving the reagents as desired. This is usually of the order of about 200-400G, e.g. 270G, although larger forces may be used where surface tension is relatively high, e.g. for smaller volumes, and also where samples are transferred through a gel or other material on route to the immobilization surface.
  • the array of molecules on the solid surface (4,7) may be subject to chemical or biological reaction or other experimentation, as desired. For instance where nucleic acid molecules are provided in an array, these may be contacted with a sample that may contain a nucleic acid of interest and potentially complementary to one or more molecules within the array. Following exposure of the array molecules to a test sample, the presence or absence of binding of a molecule within the test sample to one or more array molecules may be determined.
  • hybridization target oligonucleotide in the membrane experiments was named JTN6G-01T and the complementary Bodipy TMR labeled probe oligonucleotide was named JTN6G+02P.
  • oligonucleotide was named JPSN2+04F and was used pre- hybridized to the complementary Cy3 labeled oligonucleotide named JPSN2+05P.
  • This pre-hybridization was accomplished by mixing the oligonucleotides together at lpmol/ ⁇ L in HEN buffer (0.1M HEPES, 0.05M NaCl and 0.01M EDTA) at room temperature for 1 hour. All oligonucleotides were synthesized by Thermo- Hybaid, Interactiva Division (Germany) .
  • the membrane was pre-wet in ddH 2 0, briefly drained, and incorporated directly into the microtiter plate plus compression pad plus clamp arrangement. After array creation by centrifugation, the membrane was air- dried and the samples were cross-linked to the membrane by UV treatment for 5 min on a regular UV transilluminator.
  • the membrane was rehydrated in 2xSSC, and then immersed in APH solution, comprising 5xSSC, BLOTTO (fat free powder milk 5% and sodium azide 0.02%) and 0.5% SDS, for 30 min at 60°C.
  • APH solution comprising 5xSSC, BLOTTO (fat free powder milk 5% and sodium azide 0.02%) and 0.5% SDS, for 30 min at 60°C.
  • the Bodipy TMR labeled oligonucleotide probe JTN6G+02P was diluted to lpmol/ ⁇ L in APH, and hybridized to the surface-bound target JTN6G-01T by placing the membrane on a glass plate, pipeting an even layer of probe solution over the membrane ( ⁇ 4 ⁇ L/cm 2 ) and covering with another glass plate.
  • the sandwiched membrane was then placed on a heating block at 85°C for 5 min and air-cooled to room temperature. Unhybridized probe was removed by three room temperature washes of 5 minutes each in 2xS
  • a silylated glass surface (CSS-25, Cel Associates INC, Texas) was employed for experiments described herein. For array creation, this was incorporated directly into the microtiter plate plus compression pad plus sealing gasket plus clamp arrangement. After array creation by centrifugation, the glass plate was briefly rinsed in HEN buffer in a buffer trough, prior to signal visualization.
  • Clamps were made out of 6mm Plexiglas measuring 144 x 105mm. Eight holes, in diameter for screws were drilled three on each long side, 45 mm apart and one centered on each short side. All holes were drilled 6 mm from the edge of the Plexiglas plate.
  • One clamp consists of two such plates with nylon screws and nuts holding them together ( Figure 2) .
  • Sample plates contained 2 ⁇ L liquid sample per well.
  • the membrane was placed directly on top of the sample plate ( Figure 3) .
  • a rubber gasket (with holes aligned to the sample plate wells) was placed between the solid surface and the sample plate ( Figure 4) .
  • These assembled components were inserted into the pressure clamp device, with compression pads (HB-TD-SFOAM, Hybaid) positioned immediately internal to each Plexiglas plate of the clamp. The clamp was tightened by hand, and the assembly placed in an ordinary microtiter plate centrifuge (B4i Jouan, Inc) .
  • Centrifugation was conducted at 1500 rpm (rotor s20) for 30 sec. For both the glass and membrane studies reported here, surface binding occurred very rapidly. Therefore, as soon as the centrifuge had slowed to a stop binding was considered to be complete, and the clamp assembly was turned around so that excess reagents and liquid could be spun back into the microtiter plate wells (same speed and duration) . Once the centrifuge had again come to a standstill, the clamps were disassembled and the glass plate or membrane removed for analysis of the created array.
  • Membranes were examined by placing in an Illumtool LT-9500 instrument (Lightool research) inside a GelDoc2000 gel imager (Biorad) .
  • the membrane was epi-illuminated on the Illumatool board with 540nm light and imaged through a 590nm viewing glass.
  • Glass slides were analyzed in a GMS 418 Array Scanner (Genetic Microsystems) using the 532 nm laser for excitation.
  • centrifugal forces were used to move liquid phase molecules of interest from a multiple-well formatted plate to bring them into contact with a solid surface positioned over that plate, thereby precisely maintaining the 2-D sample arrangement present with the initial plate.
  • a suitable reaction captures the sample molecules as an array, and any excess molecules plus the complete liquid volume may then be transferred back into the sample plate (and this may be by centrifugal transfer) .
  • the inventors have identified advantageous centrif ⁇ gation conditions for quick but efficient transfer, and have established a means to ensure that no cross-contamination of samples occurred.
  • Different embodiments of the present invention may employ different solid supports; this disclosure contains experimental demonstration of use of embodiments of the present invention upon two types of solid support.
  • the inventors have created high precision derivative arrays on a filter membrane and on a glass plate, without the need for any liquid-handling tools or robotics.
  • the centrifugal transfer step needs to be brief (for high- throughput application) , sufficiently forceful (to overcome surface tension effects that tend to retain small volume samples in wells) , but not so severe that reagents cross- contaminate each other by escaping from the junction between the upper rim of the starting vessel and the destination solid surface.
  • the inventors have found that surface tension effects made it necessary to employ rather high relative centrifugal forces (G) compared to those typically used for activities such as simply aggregating liquid volumes in Eppendorph tubes ( ⁇ 100 G) .
  • G-forces of between 200 and 400 G were used regardless of type of solid surface, although values outside this range also work adequately.
  • centrifuge (B4i Jouan, Inc) at 1500 rp , imposing about 270 G, a preferred centrifugal force in certain embodiments of the invention.
  • the duration of the transfer is less critical. Once the full G- force is reached, sample transfer is usually complete, and so the centrifuge may be stopped immediately. This allows for very fast execution, conducive to high-throughput screening. Keeping the centrifuge running beyond this, however, does no harm as far as the inventors are aware from their various trials .
  • Clamps for use in embodiments of the present invention may be comprised of two Plexiglas slabs (1) with eight nylon screws (2) distributed around the edges (see Figure 2) .
  • nylon nuts (3) are used to tightening the clamps just finger tight.
  • compression pads (5) e.g. made of rubber
  • the clamps (1,2,3) and outer pressure or compression pads (5) may not be sufficient to seal effectively between the sample plate (6) and the transfer surface (7) .
  • the inventors found it preferable also to add some additional sealing at the junction of the surface and well-rim.
  • a viscous hydrophobic matrix e.g. waxes, petroleum jelly
  • a deformable (e.g. rubber) gasket (8) may be employed between the solid surface (7) and the sample plate
  • sample plates comprise a deformable lip or ⁇ crush' zones around each rim (e.g. made of rubber), specifically to achieve sealing against an applied planar surface.
  • the inventors transferred 2 ⁇ L aliquots of a solution of unlabeled oligonucleotide molecules (JTN6G-01T, at most lpmol/ ⁇ L) from a 1,536-well microtiter plate.
  • the resulting array was hybridized with Bodipy TMR labeled oligonucleotide JTN6G+02P, showing availability of the transferred molecules for subsequent reactions .
  • Three duplicate columns of samples were transferred, with the outer two column pairs representing identical step-wise dilution series (diluting each sequential sample by 50%) . In • the central column pair, every second sample was omitted (empty wells) in order to show that no leakage was occurring between wells.
  • the hybridization signals on the created array features were complete and even representations of the sample layout in the initial microtiter plate, the transferred molecules were fully available for hybridization, and there was no evidence of any cross-contamination between the wells.
  • the inventors transferred 2 ⁇ L aliquots of a solution of 5'- aminated oligonucleotide JPSN2+04F that had been pre- hybridized to an equimolar amount of Cy3 labeled oligonucleotide JPSN2+05P (1 pmol/ ⁇ L each) .
  • the starting plastic-ware was again a 1, 536-well microtiter plate, and the glass surface was silylated allowing it to bind JPSN2+04F.
  • a 3x6 array of samples was transferred, amongst which 2 wells were left empty (row 2, columns 4 and 5) .
  • a sealing gasket was used to ensure no leakage between adjacent wells.
  • the glass surface was rinsed briefly in HEN buffer and analyzed in a microscope slide laser scanner. The created array features were valid representations of the sample layout in the initial microtiter plate, and there was no evidence of any cross-contamination between the wells.
  • the inventors have provided and demonstrated efficacy for creation of arrays by centrifugation. This allows for facile, low cost, and very fast transfer of materials, onto any appropriate solid support.
  • the experiments described herein employ two different common types of solid supports, and transfer of arrays to these solid supports from a 1,536-well microtiter plate.
  • sample transfer principle entailed no complex liquid- handling manipulations or expensive robotic devices. Importantly the same concept is applicable to any number of different starting vessel and destination surface combinations, of almost any scale or density, and based upon a whole range of binding chemistries.
  • centrifugal-array approach of the present invention has the potential to help research studies to be applied on ever- greater scales with simple and affordable .machinery.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Clinical Laboratory Science (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne des procédés et des moyens permettant d'immobiliser des arrangements de molécules échantillons étudiées présentes dans des récipients d'échantillons micro-formattés (tels que des plaques micro-titre à 1,536 puits (6) sur une surface solide (4). Les molécules étudiées sont immobilisées par transfert centrifuge sur une surface solide plane ou souple (par exemple, une membrane) (4) placée sur un premier récipient d'échantillons (6).
PCT/GB2002/004261 2001-09-28 2002-09-18 Procedes et moyens permettant de creer des arrangements WO2003028878A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US32569401P 2001-09-28 2001-09-28
GB0123391A GB0123391D0 (en) 2001-09-28 2001-09-28 Methods and means for creating arrays
GB0123391.5 2001-09-28
US60/325,694 2001-09-28

Publications (1)

Publication Number Publication Date
WO2003028878A1 true WO2003028878A1 (fr) 2003-04-10

Family

ID=26246591

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2002/004261 WO2003028878A1 (fr) 2001-09-28 2002-09-18 Procedes et moyens permettant de creer des arrangements

Country Status (1)

Country Link
WO (1) WO2003028878A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE112006000361B4 (de) * 2005-02-18 2012-06-06 National University Corporation Saitama University Verfahren zur Einführung und Überführung einer Vielzahl kleinster Probenmengen
JP2016508025A (ja) * 2012-11-08 2016-03-17 ウエハージェン インコーポレイテッド ウェルからの拘束液体の抽出

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999051349A1 (fr) * 1998-04-06 1999-10-14 Pharmacopeia, Inc. Procedes et systeme offrant de grandes capacites de transfert plaque a plaque ou plaque a membrane
WO2000066267A1 (fr) * 1999-04-30 2000-11-09 Gentra Systems, Inc. Systeme empechant l'intercontamination dans une plaque multipuits
US6193642B1 (en) * 2000-01-28 2001-02-27 Pharmacopeia, Inc. Multiple-axis centrifugation bucket for centrifugal transfer between microwell plates
WO2001033211A1 (fr) * 1999-10-29 2001-05-10 Avery Dennison Corporation Appareil destine a la production a haut rendement de reseaux de materiaux de revetement, et procedes analytiques utilisant ces reseaux

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999051349A1 (fr) * 1998-04-06 1999-10-14 Pharmacopeia, Inc. Procedes et systeme offrant de grandes capacites de transfert plaque a plaque ou plaque a membrane
WO2000066267A1 (fr) * 1999-04-30 2000-11-09 Gentra Systems, Inc. Systeme empechant l'intercontamination dans une plaque multipuits
WO2001033211A1 (fr) * 1999-10-29 2001-05-10 Avery Dennison Corporation Appareil destine a la production a haut rendement de reseaux de materiaux de revetement, et procedes analytiques utilisant ces reseaux
US6193642B1 (en) * 2000-01-28 2001-02-27 Pharmacopeia, Inc. Multiple-axis centrifugation bucket for centrifugal transfer between microwell plates

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE112006000361B4 (de) * 2005-02-18 2012-06-06 National University Corporation Saitama University Verfahren zur Einführung und Überführung einer Vielzahl kleinster Probenmengen
US8664005B2 (en) 2005-02-18 2014-03-04 National University Corporation Saitama University Method for introducing and transferring multiple minute quantity samples
JP2016508025A (ja) * 2012-11-08 2016-03-17 ウエハージェン インコーポレイテッド ウェルからの拘束液体の抽出
EP2916953A4 (fr) * 2012-11-08 2016-06-29 Wafergen Inc Extraction de liquide retenu à partir de puits
US9447925B2 (en) 2012-11-08 2016-09-20 Wafergen, Inc. Extraction of restrained liquid from wells
US10500585B2 (en) 2012-11-08 2019-12-10 Takara Bio Usa, Inc. Extraction of restrained liquid from wells

Similar Documents

Publication Publication Date Title
DK1339495T3 (en) METHOD OF BIOCHEMICAL ANALYSIS AND RELATED DEVICE
US9428800B2 (en) Thermal cycling apparatus and method
US6677131B2 (en) Well frame including connectors for biological fluids
CA2500936C (fr) Chargement en parallele de reseaux
EP1372848A1 (fr) Procede et systeme d'interfa age microfluidique avec des reseaux
EP1567638A1 (fr) Dispositifs et procedes servant a traiter les caracteristiques de surface d'un substrat
EP1694279A2 (fr) Dispositif de preparation de multiples echantillons de dosage a l'aide de multiples surfaces de dosage
WO2005016532A2 (fr) Systeme automatise de chambre de reaction pour essais biologiques
CN213327664U (zh) 旋转阀、液路系统及核酸序列测定系统
WO2014053237A1 (fr) Dispositif microfluidique multicouche et procédé de dosage
EP2294214A2 (fr) Compositions et procédés pour fournir des substances à une matrice et provenant de celle-ci
EP1746168B1 (fr) Appareil de micro-réseaux avec une membrane microporeuse et ensemble des chambres de incubation
US20230100295A1 (en) System and method for rapid multiplexed sample processing with applications for nucleic acid amplification assays
US20050106607A1 (en) Biochip containing reaction wells and method for producing same and use thereof
WO2011014357A1 (fr) Système de colonne de centrifugation et procédés connexes
US20220280941A1 (en) Systems and methods for generating droplets and performing digital analyses
US20240017262A1 (en) Flow path selection value, system and method, storage medium, and application
CN213655273U (zh) 液路系统和检测系统
WO2003028878A1 (fr) Procedes et moyens permettant de creer des arrangements
JP2006519384A (ja) 極小の高さのリアクターを持つ高集積解析チップとその応用
Jobs et al. Creating arrays by centrifugation
CN114321036A (zh) 液路系统、检测系统和应用
CN114317703A (zh) 核酸序列测定方法、系统、存储介质和计算机程序产品
US7341865B1 (en) Liquid delivery devices and methods
US12000842B2 (en) Systems and methods for generating droplets and performing digital analyses

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VC VN YU ZA ZM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP