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WO2001007164A1 - Tampon de filtration a vide - Google Patents

Tampon de filtration a vide Download PDF

Info

Publication number
WO2001007164A1
WO2001007164A1 PCT/US2000/020419 US0020419W WO0107164A1 WO 2001007164 A1 WO2001007164 A1 WO 2001007164A1 US 0020419 W US0020419 W US 0020419W WO 0107164 A1 WO0107164 A1 WO 0107164A1
Authority
WO
WIPO (PCT)
Prior art keywords
gasket
filter plate
gasket sheet
multiwell
cells
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US2000/020419
Other languages
English (en)
Inventor
Masato Mitsuhashi
Steve Disper
Teva C. Rothwell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Resonac Corp
Resonac America Inc
Original Assignee
Hitachi Chemical Co Ltd
Hitachi Chemical Research Center 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 Hitachi Chemical Co Ltd, Hitachi Chemical Research Center Inc filed Critical Hitachi Chemical Co Ltd
Publication of WO2001007164A1 publication Critical patent/WO2001007164A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • B01L3/502—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
    • B01L3/50255—Multi-well filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/18—Apparatus therefor

Definitions

  • the Field of the Invention relates generally to vacuum pads, seals and gaskets, particularly to a vacuum pad for use with a high throughput filter plate for use with biological and chemical samples.
  • Microplates have become the preferred platform for automated analysis due to their easy handling by commercially available robotic equipment.
  • Microplates which incorporate a membrane filter at the base of the wells are known as filter plates.
  • Filter plates are becoming more widely used as research tools as well. Filter plates allow separation of liquid and solid material quickly and easily and are compatible with microplate robotic equipment.
  • Commonly used applications of filter plates in molecular biological research include trapping of cells. The trapped cells may be either washed or lysed directly on the filter plate. If the cells are lysed, the filter plate facilitates separation of cellular debris from cell lysates. Filter plates may also be used to collect pass-through fractions individually if desired.
  • the present invention is drawn to a porous gasket sheet having intercommunicating pores in a longitudinal direction, said intercommunicating pores having a size effective to permit air to flow therethrough.
  • the gasket sheet has an edge adapted to be connected to a vacuum source.
  • the gasket sheet is adapted to be secured between a multiwell filter plate and a receiving plate and the porous gasket has holes corresponding to the wells of the multiwell filter plate.
  • the gasket sheet may be made of any appropriate material known to the skilled artisan. Preferred materials are selected from the group consisting of polypropylene, polyethylene, polyvinylidene fluoride, polytetrafiuoroethylene, nylon, polyethersulfone, and ethyl vinyl acetate. Preferably, the gasket sheet has a thickness of 1 / 16 to V. inch. More preferably, the gasket thickness is inch to inch.
  • the disclosed gasket sheet has 6 to 384 holes. More preferably, the gasket sheet has 96 holes.
  • the gasket sheet may have an edge adapted to be connected to a vacuum source, wherein each hole has an inner peripheral edge through which air can be absorbed when the vacuum source is activated.
  • the gasket has a range of average pore size of 7 to 250 microns. More preferred is a gasket with a range of average pore size of 45-90 microns. A mean pore size of 70 microns is most preferred.
  • each well of the multiwell filter plate comprises a nozzle for application of a vacuum.
  • each well of the multiwell filter plate has a membrane filter at its bottom, and the gasket sheet has an edge adapted to be connected to a vacuum source, wherein the pressure below the membrane filter can be reduced by absorbing air through an inner peripheral edge of each hole when activating the vacuum source.
  • an elution system comprising a multiwell filter plate with a gasket sheet as set forth above, and a receiving plate, wherein the gasket sheet is secured between the multiwell filter plate and the receiving plate, each well has a membrane filter at its bottom, and the gasket sheet has an edge adapted to be connected to a vacuum source, wherein the pressure below the membrane filter can be reduced by absorbing air through an inner peripheral edge of each hole when activating the vacuum source.
  • the solution used for treating the cells in step (b) is a washing buffer or a lysis buffer. In an alternate embodiment, if the solution used for treating cells in step (b) is a washing buffer, then after step (d), steps
  • the method will further comprise collecting the lysate from the receiving plate.
  • the above-described method is used for obtaining mRNA from the cells.
  • the multiwell filter plate in combination with the gasket described above is used to obtain mRNA in a method comprising the steps of: a) applying ceils to the filter plate; b) washing the cells with washing buffer; c) iysing the cells with lysis buffer; and d) collecting the lystate to obtain mRNA.
  • Figure 1 shows a filter plate containing the gasket of the present invention.
  • the gasket is placed between the filter plate which has a membrane filter and a receiving plate.
  • Figure 2 shows a filter plate assembly containing the gasket of the present invention with the application of a vacuum through the porous gasket to facilitate movement of liquid through the filter plate.
  • FIG. 3 shows an alternate embodiment of the presently claimed invention in which the filter plates may be stacked, each filter plate containing a gasket of the presently claimed invention.
  • a device which is designed to eliminate potential cross-contamination between the nozzles and/or undersides of commercially-available or custom-produced filter plates.
  • the device is a porous die-cut or similarly fashioned gasket containing openings corresponding to the nozzles of a filter plate.
  • the gasket 3 is placed on the underside of a filter plate forming a "sandwich" between the filter plate 1 and the receiving plate 2 (see Figure 1 ).
  • the filter plate contains a membrane filter 4 and wells containing samples in solution 7.
  • the solution passes through the membrane filter 4 and nozzle 5 into the receiving plate 2.
  • the gasket 3 will absorb any aerosol or splashing that may occur thus preventing any cross-contamination to adjacent wells 7 of the receiving plate 2 .
  • Movement of the solution may be facilitated by application of either a positive or negative pressure.
  • a nozzle 6 may be provided for attachment to a vacuum source.
  • the porous nature of the gasket 3 also allows a vacuum to be applied directly through the gasket. This permits a tight sealing "sandwich" to be formed between the filter plate 1 , gasket 3, and receiving plate 2, which further reduces potential cross-contamination.
  • the disclosed gasket comprises a continuous porous plastic membrane, with dimensions similar to a regular microtiter plate, with corresponding holes.
  • the thickness of the porous membrane is '/, 6 inch to 1 /z inch. More preferably, the porous membrane thickness is inch to % inch.
  • the disclosed gasket is manufactured from porous polymeric plastic material including, but not limited to, any of the following materials: polypropylene, polyethylene, glass fiber, polyvinylidene fluoride, nylon, nylon-6, pol ⁇ ethersulfone, polytetrafluoroeth ⁇ lene, ethyl vinyl acetate, or other similar materials.
  • Porous polymeric plastic such as hydrophobic polyethylene with median porosity range 45-90 micron materials for the practice of the claimed invention are commercially available from a variety of sources including POREXTM (Fairbum, GA) and FluoroLogic LLCTM (Los
  • the controlled pore structure within the gasket can have any predetermined average pore size.
  • a range of average pore size from 7-250 microns is preferred. More preferred is a range of average pore size of 45-90 micron.
  • a mean pore size of about 70 micron Most preferred is a mean pore size of about 70 micron.
  • a preferred void volume is at least 35%.
  • a most preferred void volume is in the range of 35-65%.
  • the gasket material may be prepared using a two-step die-cutting process.
  • the virgin gasket material is cut to fit the X-Y dimensions of a microplate footprint.
  • the die has rounded corners which produce the corresponding rounded corners on the gasket. This ensures that no excess gasket is exposed to the surrounding air.
  • the gasket can remain unsealed to allow greater absorptive ability should unexpected aerosol or droplet contamination occur.
  • a second die is used to punch a desired number of holes in each rectangular die cut piece.
  • 96 holes are punched. For both steps, the excess material which is cut away from the gasket is removed and discarded.
  • the gasket can be formed by injection molding with the holes already in place.
  • Injection molding offers potential advantages over die cutting.
  • Injection molding potentially provides a consistently flat surface throughout the gasket to ensure a tight seal, eliminating local areas of gasket compression.
  • die cutting can introduce particulate matter onto the surface of the gasket, thus requiring detailed inspection of each part prior to affixing the gasket onto the filter plate.
  • Molding parts can theoretically be accomplished in a Clean Room environment, thus minimizing and potentially eliminating extraneous dust and dirt that is typically found in machine shop or tooling environments. Additionally, molding is more precise than die cutting if the mold is properly constructed. Thus, a much tighter tolerance in variation is achieved from one gasket to the next.
  • 96 holes are punched out for a 96-well microplate.
  • the gasket can be manually sandwiched in between the filter plate and the receiving plate.
  • Pores are evenly distributed, consistently sized and form an open-cell tortuous patch throughout the material.
  • a cassette is then used to hold the "sandwich" together while the vacuum is being applied. Once the liquid contents of the filter plate have been passed through to the receiving plate, the vacuum is turned off and the sandwich can then be removed from the cassette.
  • the gasket can be applied to the underside of the filter plate using an adhesive compound. Note that prior to die cutting, the adhesive sheet material is first applied to the gasket. This prevents any misalignment and/or registration problems were the gasket and adhesive sheet die cut separately.
  • One kind of adhesive comprises a sheet of clear flexible plastic material that is sticky on both sides (ECHOtape TM, DC-XS301, 24 inches x 60 yards, Edelstein Diversified, Chino, CA).
  • the sticky sheet is applied to the gasket material and then die cut as described above.
  • a removable sheet is provided on the other side of the sticky sheet and is removed just prior to placing the gasket onto the filter plate.
  • This embodiment provides added convenience for the user in that only two components need to be handled instead of three. That is, filter plate/gasket combination and receiving plate. A cassette would still be used to ensure a tight seal between the gasket and the receiving plate.
  • the porous nature of the gasket material allows a vacuum to be applied directly through the gasket itself.
  • a hollow tube is adhered to the side of the gasket along its entire cross section perimeter. An opening in the tube allows connection to a vacuum source. When a vacuum is applied, the force of the vacuum permeates and penetrates through the gasket 3 and into the airspace surrounding each nozzle 5 of the filter plate. This facilitates the movement of liquid from the topside of the filter plate 1, through the nozzles 5, past the gasket 3, and into the receiving plate 2 (see Figure 2).
  • the porous nature of the gasket provides sufficient vacuum to facilitate liquid movement, but not overly strong so as to misdirect the liquid flow away from the receiving plate and into the gasket itself.
  • the thickness of the gasket can vary depending on the nature of the filter plate and length of the nozzles underneath. In one embodiment, the thickness of the gasket varies from 1 /, 6 inch to % inch. In a most preferred embodiment, the thickness of the gasket varies from between inch to % inch.
  • a regulator will be provided to control the amount of vacuum applied to the filter plate.
  • a perforated plastic seal is provided for the user to place on the underside of the gasket. The purpose of the seal is to protect the gasket from inadvertent contamination such as placement on a contaminated benchtop.
  • the plastic seal is perforated in either columns or rows allowing the user to expose a subset of the nozzles at any given time. This allows for multiple uses of the filter plate and gasket so that it is not necessary to use all the wells at one time. For example, 8 nozzles of a 96 well filter plate could be used.
  • the disclosed gasket may be used in combination with a multiwell filter plate, for solid phase synthesis, membrane-based immunological, biological and molecular biological assays, cell harvesting and counting, robotic manipulations, as well as use with a column packing such as silica or polymeric resins, gel filtration beads, and the like.
  • a column packing such as silica or polymeric resins, gel filtration beads, and the like.
  • the use of the gasket minimizes and potentially eliminates cross contamination from nozzle to nozzle.
  • the porosity of the gasket allows a vacuum to be applied directly through the gasket itself.
  • Applications for this device include, but are not limited to, molecular biology research, nucleic acid research (including DNA and RNA research), drug discovery research, combinatorial chemistry research, and other research projects involving the use of microplates and filter plates.
  • this invention provides for a multiple vertically-stacked assembly.
  • a tower can be constructed consisting of a collection plate at the base of the tower and alternating assemblies of the disclosed gasket 3 and filter plate (see Figure 3).
  • This configuration provides the user with greater flexibility in introducing different compounds via different filter plates, at the desired time, quickly and easily, simply by turning on the vacuum supply to the appropriate filter plate. As a result, this allows researchers in fields such as combinatorial chemistry to conduct complex meaningful experiments in a rapid and cost-effective manner.
  • BHK-21 and COS 7 cell lines Two adherent cell lines, BHK-21 (newborn hamster kidney fibroblasts) and C0S-7 (African green monkey kidney fibroblasts) were processed using the mRNA Express Kit from RNAture. Between 10,000 and one million cells for each cell line were applied to a multiwell filter plate in combination with a gasket of median porosity hydrophobic polyethylene (mean pore size 70 micron, range 45-90 micron POREXTM). The gasket dimensions were 2 x 4 % x inch. The gasket contained 96 holes. Each hole had a diameter of inch. Cells were counted with a hemac ⁇ tometer and were applied in a maximum volume of 200 I.
  • PBS was added to make the sample volumes equal.
  • the cells were washed twice with 100 I of PBS, lysed with Lysis Buffer, and transferred to a GenePlate® following the standard mRNA Express centrifugation protocol. Final lysate volumes transferred to the GenePlate® were 75 I.
  • the mRNA was hybridized to the GenePlate® for 90 minutes at room temperature. Elution was performed by adding 80 I of Elution Buffer to the wells and incubating at 65°C for 5 minutes. The eluted mRNA was transferred to a black, clear bottom microplate and qua ⁇ titated with the fluorescent RiboGree ⁇ TM RNA Quantitation Kit (Molecular Probes) using ribosomal RNA as a standard.
  • RT-PCR Competitive reverse transcription PCR
  • Messenger RNA was isolated from HS587T human breast cancer cells with the mRNA Express Kit. To begin, 4500 HS587T cells were applied to each of six wells on a multiwell filter plate in combination with a gasket of median porosity hydrophobic polyethylene (mean pore size 70 micron, range 45-90 micron POREXTM). The gasket dimensions were 2 x 4 % x inch. The gasket contained 96 holes. Each hole had a diameter of inch. The cells were washed with PBS by centrif ugation. 50 I of Lysis Buffer containing between 1.3 x 10 6 and 3.3 x 10 s competitor molecules were applied to the appropriate wells and incubated at room temperature for 5 minutes.
  • the lysate was transferred to the GenePlate® by centrif ugation (670 x g for 5 minutes) and allowed to hybridize for 15 minutes at room temperature. Following three washes with Wash Buffer, a two-step RT-PCR reaction was performed.
  • the reverse transcription reaction was conducted at 42°C for 50 minutes in 20 I reaction volumes using 100 units of M-MLV reverse transcriptase per reaction.
  • the wells were washed twice with 10 mM Tris, pH 7.5, then subjected to 25 cycles of amplification using a single set of -actin specific primers in 20 I reaction volumes.
  • PCR products were analyzed by electrophoresis through a 2% agarose gel pre-stained with ethidium bromide. At the completion of electrophoresis, the gel was photographed with Polaroid Black and White Film Type 665 under UV light. Scanning de ⁇ sitometry was performed using a Personal Densitometer SI from Molecular Dynamics. The ratios of the densities of the competitor and wild type -actin bands were determined. The logs of these values were plotted against the logs of the number of -actin competitor molecules originally applied to the wells on the multiwell filter plate.
  • Messenger RNA isolated by the mRNA Express Kit was used successfully with a competitive, quantitative RT-PCR methodology to quantitate the expression levels of human -actin in HS587T cells.
  • About 4400 copies of • actin occur in each HS587T cell under the culture conditions employed for this example. This number falls within the reported range of message copies expected for an abundant message in mammalian cells.

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

Abstract

L'invention concerne un joint destiné à être utilisé avec une microplaque à cupules multiples. Ledit joint, fait dans un matériau poreux, présente des ouvertures correspondant aux buses d'une plaque filtrante. Le matériau poreux facilite le déplacement de solutions par l'application d'une pression positive ou négative. Le joint de l'invention permet de résoudre le problème de la contamination croisée susceptible de se produire entre des buses adjacentes situées sur la face inférieure d'une plaque filtrante.
PCT/US2000/020419 1999-07-27 2000-07-27 Tampon de filtration a vide Ceased WO2001007164A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14587799P 1999-07-27 1999-07-27
US60/145,877 1999-07-27

Publications (1)

Publication Number Publication Date
WO2001007164A1 true WO2001007164A1 (fr) 2001-02-01

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PCT/US2000/020419 Ceased WO2001007164A1 (fr) 1999-07-27 2000-07-27 Tampon de filtration a vide

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1366818A1 (fr) * 2002-05-31 2003-12-03 Tecan Trading AG Dispositif et méthode d'aspiration de liquides dans des plaques d'extraction en phase solide
US7244598B2 (en) * 2000-08-14 2007-07-17 Surface Logix, Inc. Biomolecule arrays
WO2007086990A2 (fr) 2005-11-17 2007-08-02 Board Of Regents, The University Of Texas System Modulation de l’expression genetique par des oligomeres cibles vers l’adn chromosomique
WO2025242707A1 (fr) * 2024-05-21 2025-11-27 Fundación Tekniker Dispositif de filtration d'echantillons de fluide et procédé de détection de concentration de particules dans les échantillons de fluide par filtration

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5108704A (en) * 1988-09-16 1992-04-28 W. R. Grace & Co.-Conn. Microfiltration apparatus with radially spaced nozzles

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5108704A (en) * 1988-09-16 1992-04-28 W. R. Grace & Co.-Conn. Microfiltration apparatus with radially spaced nozzles

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7244598B2 (en) * 2000-08-14 2007-07-17 Surface Logix, Inc. Biomolecule arrays
EP1366818A1 (fr) * 2002-05-31 2003-12-03 Tecan Trading AG Dispositif et méthode d'aspiration de liquides dans des plaques d'extraction en phase solide
WO2007086990A2 (fr) 2005-11-17 2007-08-02 Board Of Regents, The University Of Texas System Modulation de l’expression genetique par des oligomeres cibles vers l’adn chromosomique
EP2431467A2 (fr) 2005-11-17 2012-03-21 Board Of Regents, The University Of Texas Modulation de l'expression génétique par oligomères ciblés à l'ADN chromosomique
EP2641970A1 (fr) 2005-11-17 2013-09-25 The Board Of Regents, The University Of Texas System Modulation de l'expression génétique par oligomères ciblés à l'ADN chromosomique
WO2025242707A1 (fr) * 2024-05-21 2025-11-27 Fundación Tekniker Dispositif de filtration d'echantillons de fluide et procédé de détection de concentration de particules dans les échantillons de fluide par filtration

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