WO2001019520A1 - Carte haute densite, de preparation d'echantillons, coulee sur place - Google Patents

Carte haute densite, de preparation d'echantillons, coulee sur place Download PDF

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Publication number
WO2001019520A1
WO2001019520A1 PCT/US2000/024855 US0024855W WO0119520A1 WO 2001019520 A1 WO2001019520 A1 WO 2001019520A1 US 0024855 W US0024855 W US 0024855W WO 0119520 A1 WO0119520 A1 WO 0119520A1
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WIPO (PCT)
Prior art keywords
housing
sample
substrate
reservoir
sample preparation
Prior art date
Application number
PCT/US2000/024855
Other languages
English (en)
Inventor
William Kopaciewicz
Cheryl Brucato
Original Assignee
Millipore Corporation
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 Millipore Corporation filed Critical Millipore Corporation
Priority to EP00961767A priority Critical patent/EP1227888A4/fr
Publication of WO2001019520A1 publication Critical patent/WO2001019520A1/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
    • B01L3/502—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
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/141—Preventing contamination, tampering
    • 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
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum

Definitions

  • Test plates for chemical or biochemical analysis which contain a plurality of individual wells or reaction chambers are well known laboratory tools. Such devices have been employed for a broad variety of purposes and assays, and are exemplified in U.S. Patent No. 4,734,192 and • 5, 009, 780, for example. Microporous membrane filters and filtration devices containing the same have become especially useful with many of the recently developed cell culture techniques, assays, and sample preparation methods, especially in the fields of virology, immunology, genetics, drug discovery, etc. Typically, a 96-well filtration plate is used to conduct multiple sample preparations or assays simultaneously.
  • Multiscreen is a 96-well filter plate that can be loaded with adsorptive filter materials or particles.
  • Multiscreen is a 96-well filter plate that can be loaded with adsorptive filter materials or particles.
  • membrane type, well geometry and layout are important criteria in choosing the appropriate sample preparation device for a particular operation.
  • analytical techniques such as matrix assisted laser desorption ionization, time of flight, mass spectrometry (MALDI TOF MS) and 96X capillary electrophoresis systems require only a small amount of sample for analysis.
  • Traditional 96-well devices often contain far more volume than is required for these high performance analytical instruments.
  • the problems of the prior art have been overcome by the present invention, which provides a card or insert for a single or multi-well sample preparation device, and provides the mechanical apparatus containing and optionally operating the card or insert .
  • the device is a housing that is configured to receive one or more of the inserts. Preferably the inserts are readily removable from the housing, allowing interchangeability with other inserts having the same or different sample preparation characteristics from the previous insert .
  • the housing contains or is in fluid communication with a sample reservoir and a collection reservoir, and a suitable driving force such as pressure, vacuum, centrifugation, or simply capillary action may be used to effectuate the fluid movement.
  • the inserts contain the separations media, the_ properties and geometry of which may be tailored to the specific action to be carried out . High throughput sample preparation devices that can be applied to a variety of biochemical and chemical applications, for example, are thus provided.
  • the structures are monolithic and/or continuous.
  • the invention is applicable to a variety of particular sizes and configurations, and provides a means of affixing chromatographic media in a variety of volumes and layouts.
  • the invention enables the inclusion of a substantial (relative to the increase in surface area of the precipitated polymeric structure) amount of media in the polymer composite matrix.
  • the composite structures comprise particles entrapped within a porous polymeric substrate, and are cast in-place into a planar or substantially planar insert having one or more recesses, thereby providing an effective platform for high throughput micromass handling.
  • particle chemistry With the appropriate selection of particle chemistry, virtually any separation or purification operation can be conducted in multiplicity, including selective bind/elute chromatography operations, on sample mass loads less than 1 microgram in volumes of a few microliters or less, as well as larger mass loads and volumes.
  • These structures preferably are self- retaining and/or self-supporting.
  • the present invention provides for direct analysis, such as by MALDI time-of-flight (TOF) mass spectrometry, of samples without elution. This is accomplished by configuring the insert such that it is compatible with conventional mass spectrometers and can be appropriately positioned in the spectrometer for direct analysis.
  • TOF time-of-flight
  • unfilled structures which are preferably self-retaining and/or self-supporting are cast in
  • si tu into one or more wells in a suitable insert can be
  • the adsorptive properties of a non-particle filled structure can be imparted either through the native properties of the polymer (s) used or by subsequent derivatization.
  • either the filled or unfilled structures which may be self-retaining and/or self-supporting are derived from inorganic materials such as metals or ceramics .
  • Figures 1A and IB are top views of multi-well filtration cards in accordance with the present invention
  • Figure 2 is a cross-sectional view of a sample preparation device including the insert in accordance with one embodiment of the present invention
  • Figure 3A is a perspective exploded view of one embodiment of the sample preparation device of the present invention.
  • Figure 3B is a cross-sectional schematic view of a vacuum housing for the sample preparation device of Figure 3A;
  • Figure 3C is an exploded view of a spout of the underdrain of Figure 3B;
  • Figures 4A and 4B are graphs of the mass spectrum of desalted peptides in accordance with Example 4; .
  • Figures 5A and 5B are graphs of the mass spectrum of desalted peptides in accordance with Example 5;.
  • Figures 6A and 6B are graphs of the mass spectrum of desalted oligonucleotides in accordance with Example 6;
  • Figure 7 is a graph of the mass spectrum of desalted oligonucleotides in accordance with Example 7;
  • Figure 8A is a graph of the mass spectrum of peptides in accordance with Example 8.
  • Figures 8B and 8C are graphs of the mass spectrum of phosphopeptides in accordance with Example 8.
  • Figures 8D and 8E are graphs of the mass spectrum of recombinant his-tagged protein in accordance with Example 8 ;
  • Figure 9A is a graph of the mass spectrum of phosphopeptides in accordance with Example 9.
  • Figure 9B is a graph of the mass spectrum of recombinant his-tagged protein in accordance with Example 9. DETAILED DESCRIPTION OF THE INVENTION
  • membrane as used herein includes permeable and semi-permeable three dimensional structures with or without particles, having a porosity suitable for the desired application.
  • composite structure as used herein includes filled membranes.
  • Suitable adsorptive composite structures are polymer bound, particle laden adsorptive membrane structures, such as those comprised of chromatographic beads which have been adhered together with a binder.
  • a suitable particle loaded structure is comprised of about 80% w/w C18 silica and 20% w/w polysulfone binder, and is produced by Millipore Corporation.
  • Functional composite structures comprising other micron-size (e.g., 1-30 microns) resin particles derivatized with other functional groups are also beneficial, including styrenedivinyl -benzene-based media
  • silica-based media unmodified or derivatized with C 2 , C 4 , C 6 , C 8 , or C l ⁇ or ion exchange functionalities
  • C 2 , C 4 , C 6 , C 8 , or C l ⁇ or ion exchange functionalities to accommodate a variety of applications for peptides, proteins, nucleic acids, and other organic compounds.
  • selectivities e.g., hydrophobic interaction, affinity, etc.
  • particles as used herein is intended to encompass particles having ' regular (e.g., spherical) or irregular shapes, as well as shards, fibers and powders, including metal powders, plastic powders (e.g., powdered polystyrene) , silica and activated carbon.
  • metal powders e.g., metal powders, plastic powders (e.g., powdered polystyrene) , silica and activated carbon.
  • silica into a polysulfone polymer results in increased active surface area and is suitable for various applications.
  • Polysulfone sold under the name UDEL P3500 and P1700 by Amoco is particularly preferred in view of the extent of the adherence of the resulting composite structure to the housing (e.g., the walls of the apertures of the inserts) , including polypropylene, polyethylene, polystyrene, polycarbonate, etc., and mixtures thereof.
  • polystyrene and polystyrene/acrylonitrile copolymer examples include polyethersulfone, cellulose acetate, cellulose acetate butyrate, acrylonitrile PVC copolymer (sold commercially under the name “DYNEL”), polyvinylidene fluoride (PVDF, sold commercially under the name “KYNAR”), polystyrene and polystyrene/acrylonitrile copolymer, etc.
  • Any castable, membrane-forming polymer (such as nylon) can be used in the invention although viscosity may need to be adjusted and/or the use of mechanical supports may be needed to keep the material m place until precipitated.
  • Adhesion to the housing can be enhanced or an analogous effect achieved with these composite structures by means known to those skilled in the art, including etching of the housing, such. as with plasma treatment or chemical oxidation; mechanical aids such as rims inside the housing; and inclusion of additives into the housing material that promote such adhesion. Adhesion allows uniform precipitation during casting.
  • the formed structures are cast in the preferred method.
  • the membranes or composite structures formed as defined by the well geometry have an aspect "ratio (average diameter to average thickness) of less than about 20, more preferably less than about 10, especially less than 2, most preferably from about 0.5 to about 2.
  • ratio average diameter to average thickness
  • an aspect ratio within these ranges provides for suitable residence times of the sample in the composite structure during operation.
  • N-methyl-pyrolidone is a suitable solvent for polysulfones, polyethersulfones and polystyrene.
  • polystyrene pellets can be dissolved in N-methyl- pyrolidone and cast-in-place.
  • the resulting structure shows good adhesion to the walls of the wells in the insert, particularly when the insert is plastic, and has adsorption characteristics similar to polysulfone.
  • Dimethylsulfoxide (DMSO) , dimethylformamide, butyrolactone, and sulfalane are also suitable solvents.
  • N,N-dimethylacetamide is a suitable solvent for PVDF.
  • Water is the preferred precipitant. Although the water can have a wide temperature range to be effective as a precipitant, higher temperatures (about 80°C) tend to improve structure permeability.
  • a volatile solvent for the polymer binder is used. For example, in the case of cellulose acetate, acetone is a suitable volatile solvent.
  • Evaporative casting generally results in a structure which shrinks upon setting. With this method, particles in the filled structures should be at least about 30 ⁇ to allow flow through the interstitial spaces after shrinkage without having to apply higher driving force .
  • Suitable particle sizes include particles in the range of from about 100 nanometers to about 100 microns in average diameter with or without porosity.
  • Any suitable means can be used to deliver lacquer into the wells or apertures of the insert. This can occur simply due to gravity, but also can be assisted using a driving force such as pressure, or can be accomplished by directing the lacquer into the wells using a flat edge such as a knife or spatula blade. The lacquer also could be precisely dispensed into each well. In one embodiment, a sufficient amount of lacquer is simply poured onto the insert and excess removed with a straight edge.
  • the material of construction for the housing is not particularly limited, but should be made of a material which will not deleteriously react with the reagents used during the sample preparation procedure or any subsequent procedure.
  • the material also must withstand the conditions typical of the method, particularly where pressure is used as the fluid driving force. ' Suitable materials include plastics
  • polyolefins especially polyethylene and polypropylene; PVC and polystyrene
  • glass and stainless steel glass and stainless steel.
  • Polyolefins, and particularly polypropylene are preferred insert materials in view of the chemical adhesion that is created with the composite structure when the composite containing polysulfone, and in particular UDEL P3500 and P1700 polysulfones available from Amoco, , or polystyrene, in particular, STYRON 685D available from The Dow Chemical Company, is cast-in-place therein.
  • UDEL P3500 and P1700 polysulfones available from Amoco, , or polystyrene, in particular, STYRON 685D available from The Dow Chemical Company is cast-in-place therein.
  • mechanical means can be used to maintain the composite structure in the aperture.
  • adhesion to the aperture walls is the ability to "seal" the composite structure to the walls without mechanical means. Such sealing (by whatever method) prevents the sample from channeling or bypassing the composite during operation.
  • the housing 10 and number of recesses or apertures therein are not critical. Where a plurality of apertures are present, the housing forms a high- density sample array.
  • the housing shown is a 3.365" x 5.030" planar card or plate, containing 384 recesses or apertures 12, and is 0.090 inches thick.
  • the apertures 12 penetrate through the housing. Smaller or larger housings can be used, as can thicker or thinner housings.
  • the housing is planar or substantially planar.
  • the apertures 12 can be formed in the housing by any suitable means, such as drilling, punching or molding. Preferably the apertures 12 are evenly spaced.
  • the apertures 12 have a diameter of about 0.060 inches and are spaced at about 0.178" centers.
  • Figure IB shows another embodiment with 96 apertures 12, each with a diameter of 0.150 inches and spaced 0.354 inch centers. The card of Figure IB is 0.09 inches thick.
  • the preferred configuration of the recesses or apertures 12 is substantially cylindrical, as the flow vectors during operation are substantially straight, similar to chromatography, thereby minimizing or avoiding dilutional washing that might occur with non-cylindrical configurations.
  • the recesses Preferably have an open top and an open bottom opposing and spaced from the open top, and thus are coplanar with the top and bottom surfaces of the housing.
  • the composite structure contained in the recesses preferably fills each recess, but can occupy less than the total volume of each recess if desired.
  • the composite structure is preferably coterminous with both sides of the recess, but can fill less than the entire recess so as to form a distribution well for the introduction of liquid sample, for example.
  • the structures of the present invention have a final bed height of from about 0.005 to about 0.5 inches.
  • the ideal bed heights will depend upon the application and are readily determined by those skilled in the art.
  • ' Bed height is predominantly controlled by the housing thickness; the structures are preferably coterminous with the two sides of the housing that communicate with the recess or recesses.
  • the housing is planar such as a plate, the structures are preferably coplanar with the two sides of the housing that communicate with the recess or recesses.
  • the composite structures and the multi-well filtration devices of the present invention containing the composite structures have a wide variety of applications, depending upon the particle selection.
  • applications include peptide and protein sample preparation prior to analysis, peptide removal from carbohydrate samples, amino acid clean-up prior to analysis, immobilized enzymes for micro-volume reactions, immobilized ligands for micro-affinity chromatography, isolation of supercoiled and cut plasmids, clean-up of PCR and DNA products, immobilized oligo dT for RNA isolation, dye terminator removal, sample preparation for elemental analysis, etc.
  • Those skilled in the art will be able to choose the appropriate particles, polymer binder, particle chemistry and recess geometry depending upon the desired application. In some cases, a mixture of particles can be used in the same devices. Alternatively or in addition, different chemistries for separate wells can be used.
  • the structures of the present invention are not filled with particles, symmetrical or asymmetrical semi-permeable structures, or a combination of symmetrical and asymmetrical semi-permeable polymeric structures, can be formed.
  • the preferred method of formation is casting in si tu in the wells of the
  • a self-retaining, self-supporting structure suitable for separations based on size or adsorption (depending on polymer identity).
  • Recess geometry e.g., ribbed, hourglass or conical
  • Functionality can be either intrinsic or added to such a membrane to perform adsorption separations without the use of particles.
  • cellulose acetate can be treated with base to form cellulose, followed by an oxidant to render it reactive .
  • the preferred method of formation involves precipitation by means of solvent exchange, such as by introducing the casting solution into the apertures of the inserts by any suitable means, as discussed above.
  • the insert is held to the substrate by mechanical means or by adhesion (e.g., taping).
  • adhesion e.g., taping
  • the casting solution in the recesses is contacted with a liquid in which the polymer is insoluble, preferably water, so that the polymer precipitates in the recesses.
  • the inserts are placed on a flat substrate, such as a steel or glass plate, thereby forming a floor or bottom to each recess in the insert.
  • the casting solution is then applied to the insert and the recesses therein filled.
  • the insert (and substrate) is immersed in the liquid in which the polymer precipitates. Through the exchange of water for the solvent, the structure precipitates. The substrate is then removed from the insert.
  • the solvent used to prepare the casting solution and the non-solvent can contain a variety of additives.
  • the holes of the inserts • can be filled without the use of a substrate, if the casting solution has a suitably high viscosity.
  • the minimal viscosity necessary depends on the diameter and length of the hole.
  • smaller holes can hold casting solution of less viscosity than larger holes.
  • holes about 0.040 inches in diameter can hold solutions having a viscosity of at least about 500 cps, while holes having a 0.40 inch diameter require solutions with a viscosity of at least about 50,000 cps to prevent flow out of the hole.
  • suitable viscosities based upon the particular dimension of the holes without undue experimentation.
  • One particular example is a casting solution containing 30% (w/w) C18 silica solids (15 ⁇ m) in a 9% (w/w) Udel P3500 (polysulfone dissolved in N- methylpyrrolidone) solution which has a viscosity of about 800 cps.
  • This solution is suitable for use in holes of about 0.080 in diameter and about the same in length.
  • the viscosity of this solution can be increased to above about 20,000 cps by increasing the C18 solids content to about 40% (w/w) .
  • the relationship between solids content and viscosity will vary with particle type.
  • the initial semi-permeable barrier can be removed, such as by cutting or by abrading the exposed polymer on the surface of the recess. This process also removes excess polymer from the surface, thus making the card more suitable for subsequent assembly.
  • the semi-permeable barrier can be optionally left in place, especially if slightly recessed (e.g., 0.010 inches recessed) from the top surface of the recess, to carry out size-based separations with unfilled structures, as the barrier acts as a micro- or ultra-filtration membrane.
  • the cast in-place structure assumes the shape of the recesses 12 and results in a self-retaining homogeneous structure akin to a chromatographic column, providing a large surface area (e.g., when particles are included in the polymer matrix) suitable for bind/elute chromatography or for other analytical or biochemical techniques.
  • Suitable driving forces include centrifugation, gravity, capillary action, pressure or vacuum.
  • FIG. 2 illustrates one suitable device 20 that forms the sample preparation device of the present invention.
  • the device 20 includes a sample reservoir 22 and a collection reservoir 23 in fluid communication with the sample reservoir 22 through the preferably planar insert or card 10.
  • the insert or card 10 could be applied directly, or can be sealed to a housing 20 by any suitable means, such as mechanically with seal 26, by gluing, welding, such as ultrasonic welding, impulse welding or thermal welding, ensuring that all sample passes through the insert or card (via the filled recesses 12) into the collection reservoir during operation.
  • a mechanical lock down plate 25 coupled to a pair of tie rods actuated by cam lever can be used to secure the device 20 together during operation, but other mechanical means are possible and within the scope of the present invention, such as suitable lock down hardware 27 as shown.
  • the device 20 can be in communication with a source of vacuum via port 30 to provide the driving force for the filtration.
  • a rigid underdrain 32 is provided for support and direction of flow.
  • Figure 3A illustrates a design that can be bonded together as a single unit that can fit into a conventional vacuum housing ( Figure 3B) used for 96 or 384 well filter plates.
  • the upper sample chamber array 30, which contains a plurality of wells 33 (which for a 96X device, can be spaced by 0.36 inch centers; for a 384X device, can be spaced by 0.178 inch centers, etc.) is bonded to the insert 31 along an underdrain assembly 32.
  • the underdrain 32 is an array of spouts 34 (preferably in a single part) that is bonded to the underside of the insert 31 and serves to direct the filtrate liquid into a collection vessel or collection reservoir 23.
  • the spouts 34 are sized according to the size of the wells in the insert.
  • suitable means e.g., glue, such as cyanomethacrylate, UV curable glue, epoxy, silicone, etc., thermal or ultrasonic welding, etc.
  • glue such as cyanomethacrylate, UV curable glue, epoxy, silicone, etc., thermal or ultrasonic welding, etc.
  • the three pieces now form a disposable housing that when placed in a suitable vacuum manifold (with a collection plate 35) will operate as a multiwell (96, 384, 1536X, etc.) sample preparation device ( Figure 3B) .
  • the upper chamber and lower spout assemblies can be comprised of a variety of plastics, including polypropylene, polycarbonate, polystyrene, etc.
  • FIG. 3B the vacuum chamber for operating the plate assembly unit of Figure 3A is shown in Figure 3B.
  • a lower chamber 52 is provided which houses the collection plate 35 as shown.
  • a removable lid 53 is configured to mate with the side walls 54 of the lower chamber 52, and includes elastomeric seals 55 to seal against the side walls and against which the plate assembly unit seals. Once the plate assembly unit is sealed in the device, vacuum or other driving force can be applied to effectuate flow.
  • the device of Figure 3A may be assembled by turning the upper housing 30 upside down and precisely dispensing an adhesive onto the partition using a computer controlled liquid delivery device. Onto this, the insert 31 is laid, while taking care to maintain the proper orientation. Once the adhesive in this sub-assembly cures, additional adhesive is applied to the partitions on the side of the insert opposite that adhered to the upper housing 30. The underdrain 32 is set upon this additional adhesive and carefully oriented for axial alignment of recesses. The result after curing is a disposable, high throughput, sample preparation device ready to fit into current state-of-the-art robotics.
  • the device 20 can be adapted to accommodate more than a single insert at any given time.
  • two or more inserts can be aligned so that their respective recesses are in fluid communication with one another.
  • one or more holes in a single insert can be filled with media having different chemistries.
  • the insert or card 10 can be used by itself or can be bonded or coupled to other elements to provide a wider range of function. Solutes that have been treated by the card 10 can be collected and transferred to analytical equipment for further analysis. Alternatively, once solute is bound to the card 10, the card itself can be inserted into an analytical instrument such as a MALDI TOF mass spectrometer, and analyzed directly by laser interrogation, for example, thereby eliminating a transfer step.
  • an analytical instrument such as a MALDI TOF mass spectrometer
  • a desorbing matrix can be applied to the sample bound to the structure in the insert, such as a matrix including acetonitrile, which desorbs the sample for MALDI TOF mass spectrometry, and also helps mediate the effect of the laser so that the sample is not destroyed.
  • the unit was removed and a razor blade was used to remove excess polymer.
  • the card was then removed from the glass plate and was re-immersed in the water for an additional 0.5 hours to complete solvent exchange.
  • the insert was then removed from the water and allowed to air dry.
  • a polymer solution (about 25 ml) of polysulfone is prepared in N-methylpyrrolidone to about 9% w/w in polymer solids in an about 100 ml beaker.
  • particles are added (which may or may not have adsorptive properties) while constantly mixing until the lacquor is of the consistency of plaster (approximately 40% solids and 25,000 cps).
  • the lacquor is spread into the holes of a predrilled card (same as Example 1) which is being held by the outer edges. Once the holes are filled, the excess on the exterior of both sides is removed such as by scraping, and the card is immersed in a water bath at 80C.
  • the card After the structure is precipitated and desolvenated (about 1 hour) , the card is removed and the excess polymer above the plane of the card surface is removed such as with a razor blade or wiping with a cloth. The card is then re- immersed and agitated in a water bath for about 5 minutes to remove particulate matter, and is then removed and allowed to dry.
  • Example 3
  • the device of Figure 3 is placed into the top (spouts facing down) of a common vacuum housing 40 as shown in Figure 4. It is sealed to a removable lid 42 such as by elastomeric seals 43.
  • a collection tray 35 is underneath within the lower chamber 45.
  • a conditioning solution is added to the upper sample chambers and the vacuum turned on to pull the solution through. Sample is then applied. As the vacuum pulls the sample solution through, the components of interest adsorb to the polymeric structure within the insert, while the contaminants flow into the collection vessel 35. "
  • a wash solution is then added to the upper chamber 30 (with the vacuum on) to rinse away trace contaminants. The vacuum is turned off and the collection vessel 35 is replaced with a clean one.
  • the unit is re-assembled and a suitable desorption solution is added to the upper sample chambers. As this solution flows through the insert, it desorbs the components of interest, which are captured by the clean collection vessel 35.
  • the plastic was then immersed into an ambient temperature water bath for 1 hour to precipitate the polymer. After this period, the plastic substrate was removed and polymer precipitated on the exterior surfaces was cut off with a sharp razor blade.
  • the cast-in-place structure was then placed on top of a vacuum flask and washed liberally with water (under vacuum) to ensure that the "cast in place" polymer structure was completely solvent exchanged.
  • the structure was then washed with 10 ⁇ l of water containing 0.1% (v/v) TFA to remove GuHCl . This was performed by overlaying the structure with the liquid followed by forcing it through with positive pressure using a 1 cc syringe containing a gasket that formed a seal around recess .
  • oligonucleotides were desalted using a ZipTip ⁇ C18 ⁇ and eluted from tip using 2 ⁇ l of
  • Example 4 Using the device described in Example 4, 2 ⁇ l of methanol were deposited directly onto the free side of the structure and allowed to pass into the plug by capillary action. This step was followed by the addition of 5 ⁇ l of 50 mM triethylamine acetate buffer, pH 7.0, which entered the plug in the same way. Onto the equilibrated structure, 3 ⁇ l of 50 mM triethylamine acetate buffer, pH 7.0, containing 5 pmole each of three oligonucleotides (20, 25 and 29 mer) was added and allowed to absorb into the plug by capillary action for about 1 min. Any remaining sample on top of the plug was forced through using positive pressure.
  • the structure was liberally washed with water using positive pressure (see Example 4) . Once washed and air dried, 1.0 ⁇ l of matrix (45 mg/ml hydroxypiccolinic acid, 5 mg/ml ammonium citrate, 45% acetonitrile) was carefully deposited onto the structure and again allowed to air dry. The plastic substrate was then snapped into a PE BioSystems (Framingham, MA) disposable MALDI sample plate holder (P/N V700314) in the same orientation. The carriage was inserted in a PE BioSystems VoyagerTM MALDI-TOF MS mass spectrometer with delayed extraction and the matrix spot was analyzed directly. ( Figure 7) .
  • matrix 45 mg/ml hydroxypiccolinic acid, 5 mg/ml ammonium citrate, 45% acetonitrile
  • a 0.02" cylindrical hole was bored into the center of a 1.75 X 1.75 X 0.04" piece of polypropylene plastic using a sharp point.
  • lacquer was added consisting of 10% (w/w) polystyrene (Dow Chemical, Styron 685D) : 90% (w/w) N-methylpyrrolidone containing 30% (w/w) of imidodiacetic acid coated-200A-15 ⁇ m spherical silica particles using the tip of a spatula.
  • the plastic was then immersed into an ambient temperature water bath for 1 hour to precipitate the polymer. After this period, the plastic substrate was removed and polymer precipitated on the exterior surface was cut off with a sharp razor blade.
  • the cast-in- place structure was then placed on top of a vacuum flask and washed liberally with water (under vacuum) to ensure that the polymer structure was solvent exchanged. Following washing, the structure was contacted tightly on one side with an absorbent paper towel .
  • To charge the metal chelate resin with copper ions 2 ⁇ l of methanol were deposited directly onto the free side of the structure and allowed to pass into the plug by capillary action onto an absorbent paper towel, followed by 5 ⁇ l of 50% acetonitrile, 5 ⁇ l of 200 mM cupric sulfate
  • the structure was then washed liberally with 10 mM ammonium bicarbonate buffer containing 20% acetonitrile (pH « 8.0) and 5 mM MES, pH 6.5 buffer, respectively, to remove contaminants. Once washed, 3 ⁇ l of 5 % acetic acid was carefully deposited onto the structure. A slight positive pressure was applied sufficient to drive the solution through the structure forming a drop on the underside. The drop containing recombinant protein (recDHFR) was then collected, spotted onto a MALDI-TOF MS target, overlaid with matrix (10 mg/ml sinnapinic acid, 60% acetonitrile, 0.3% TFA) and analyzed (Figure 8D) .
  • recDHFR recombinant protein
  • protein was enriched using a ZipTip Mc " charged with copper ions.
  • the protein was eluted from the tip using 3 ⁇ l of 5% acetic acid directly onto MALDI-TOF MS sample plate and overlaid with matrix (Figure 8E) .
  • Example 8 Using the device described in Example 8 charged with copper ions, 2 ⁇ l of methanol were deposited directly onto the free side of the structure and allowed to pass into the plug by capillary action. After this step, 5 ⁇ l of 50 mM 2- (N- morpholino) -ethanesulfonic acid, pH 5.5 ⁇ (MES buffer) containing 10% acetonitrile was deposited directly onto the free side of the structure and allowed to pass into the plug by capillary action. Onto the equilibrated structure, 2 ⁇ l of MES buffer, pH 5.5 containing 10% acetonitrile and 2.5 picomoles of beta casein tryptic peptides was added and allowed to absorb into the plug by capillary action for about 1 min.
  • Example 8 Using the device described in Example 8 charged with copper ions, 2 ⁇ l of methanol were deposited directly onto the free side of the structure and allowed to pass into the plug by capillary action. Then, 5 ⁇ l of 8 M Urea, 0.1 M NaH 2 P0 4 ,

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Abstract

L'invention concerne une pièce rapportée (31) à placer dans un dispositif de filtration à alvéoles de microtitration, qui présente des ouvertures remplies d'une matrice de filtration. Ladite matrice contient éventuellement des particules d'adsorption.
PCT/US2000/024855 1999-09-13 2000-09-11 Carte haute densite, de preparation d'echantillons, coulee sur place WO2001019520A1 (fr)

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EP00961767A EP1227888A4 (fr) 1999-09-13 2000-09-11 Carte haute densite, de preparation d'echantillons, coulee sur place

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US15360699P 1999-09-13 1999-09-13
US60/153,606 1999-09-13
US19578000P 2000-04-10 2000-04-10
US60/195,780 2000-04-10

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003064041A1 (fr) * 2002-01-31 2003-08-07 Varian, Inc. Mat d'etancheite a commande d'ecoulement unidirectionnel pour plaque a puits
EP1390539A2 (fr) * 2001-05-25 2004-02-25 Waters Investments Limited Plaques de desorption-ionisation par impact laser assiste par matrice (maldi) de concentration d'echantillons pour la spectrometrie de masse maldi
EP1398614A2 (fr) * 2002-09-13 2004-03-17 Millipore Corporation Appareil et méthode pour la preparation d'echantillons et le spotting direct d'éluant sur cibles maldi-tof
EP1477792A2 (fr) * 2003-05-13 2004-11-17 Becton, Dickinson and Company Procédé et dispositif pour purifier et dessaler des échantillons biologiques
EP1477812A2 (fr) * 2003-05-13 2004-11-17 Becton, Dickinson and Company Procédé et dispositif pour le traitement d'échantillons biologiques et chimiques
WO2005008244A1 (fr) * 2003-07-15 2005-01-27 Ekstroem Simon Dispositif et procede d'analyse d'echantillons utilisant un traitement d'echantillon combine et un dispositif de support d'echantillon
US6952011B2 (en) 2001-08-17 2005-10-04 Micromass Uk Limited MALDI sample plate
EP1591164A1 (fr) * 2004-04-23 2005-11-02 Millipore Corporation Plaque à cavités multiples avec volume de rétention réduit
US7053366B2 (en) 2001-05-25 2006-05-30 Waters Investments Limited Desalting plate for MALDI mass spectrometry
EP1854542A1 (fr) * 2006-05-12 2007-11-14 F. Hoffmann-La Roche AG Dispositif filtrant multipuits
EP2027925A3 (fr) * 2003-06-24 2011-01-05 Millipore Corporation Ensemble collecteur
US7867452B2 (en) * 2006-05-12 2011-01-11 F. Hoffmann-La Roche Ag Multi-well filtration device
EP3289856A1 (fr) * 2016-09-05 2018-03-07 Deutsche Saatveredelung AG Dispositif de culture et de prélèvement d'échantillon pour plantes
EP3784368A4 (fr) * 2018-04-25 2022-01-26 Optofluidics, Inc. Collecteur à vide pour microscopie à filtration

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US3888770A (en) * 1971-10-21 1975-06-10 Shlomo Avital Plural-sample filter device
US4948564A (en) * 1986-10-28 1990-08-14 Costar Corporation Multi-well filter strip and composite assemblies
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
US5401466A (en) * 1993-06-01 1995-03-28 Miles Inc. Device for the direct measurement of low density lipoprotein cholesterol

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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1390539A2 (fr) * 2001-05-25 2004-02-25 Waters Investments Limited Plaques de desorption-ionisation par impact laser assiste par matrice (maldi) de concentration d'echantillons pour la spectrometrie de masse maldi
EP1390539A4 (fr) * 2001-05-25 2007-06-27 Waters Investments Ltd Plaques de desorption-ionisation par impact laser assiste par matrice (maldi) de concentration d'echantillons pour la spectrometrie de masse maldi
US7053366B2 (en) 2001-05-25 2006-05-30 Waters Investments Limited Desalting plate for MALDI mass spectrometry
US6952011B2 (en) 2001-08-17 2005-10-04 Micromass Uk Limited MALDI sample plate
US7294831B2 (en) 2001-08-17 2007-11-13 Micromass Uk Limited MALDI sample plate
WO2003064041A1 (fr) * 2002-01-31 2003-08-07 Varian, Inc. Mat d'etancheite a commande d'ecoulement unidirectionnel pour plaque a puits
EP1398614A2 (fr) * 2002-09-13 2004-03-17 Millipore Corporation Appareil et méthode pour la preparation d'echantillons et le spotting direct d'éluant sur cibles maldi-tof
EP1398614A3 (fr) * 2002-09-13 2004-06-23 Millipore Corporation Appareil et méthode pour la preparation d'echantillons et le spotting direct d'éluant sur cibles maldi-tof
EP1477792A2 (fr) * 2003-05-13 2004-11-17 Becton, Dickinson and Company Procédé et dispositif pour purifier et dessaler des échantillons biologiques
EP1477792A3 (fr) * 2003-05-13 2005-02-09 Becton, Dickinson and Company Procédé et dispositif pour purifier et dessaler des échantillons biologiques
EP1477812A3 (fr) * 2003-05-13 2005-02-09 Becton, Dickinson and Company Procédé et dispositif pour le traitement d'échantillons biologiques et chimiques
JP2004340971A (ja) * 2003-05-13 2004-12-02 Becton Dickinson & Co 生物学的サンプルを精製および脱塩する方法並びに装置
EP1477812A2 (fr) * 2003-05-13 2004-11-17 Becton, Dickinson and Company Procédé et dispositif pour le traitement d'échantillons biologiques et chimiques
JP4575708B2 (ja) * 2003-05-13 2010-11-04 ベクトン・ディキンソン・アンド・カンパニー 生物学的サンプルを精製および脱塩する方法並びに装置
US7405083B2 (en) 2003-05-13 2008-07-29 Becton, Dickinson And Company Method and apparatus for purifying and desalting biological samples
EP1972919A1 (fr) * 2003-05-13 2008-09-24 Becton, Dickinson and Company Procédé et appareil de traitement biologique et échantillons chimiques
AU2004202002B2 (en) * 2003-05-13 2009-04-09 Corning Incorporated Method and apparatus for processing biological and chemical samples
US8007743B2 (en) 2003-06-24 2011-08-30 Millipore Corporation Multifunctional vacuum manifold
EP2027925A3 (fr) * 2003-06-24 2011-01-05 Millipore Corporation Ensemble collecteur
WO2005008244A1 (fr) * 2003-07-15 2005-01-27 Ekstroem Simon Dispositif et procede d'analyse d'echantillons utilisant un traitement d'echantillon combine et un dispositif de support d'echantillon
EP1591164A1 (fr) * 2004-04-23 2005-11-02 Millipore Corporation Plaque à cavités multiples avec volume de rétention réduit
EP1854542A1 (fr) * 2006-05-12 2007-11-14 F. Hoffmann-La Roche AG Dispositif filtrant multipuits
US7867452B2 (en) * 2006-05-12 2011-01-11 F. Hoffmann-La Roche Ag Multi-well filtration device
EP3289856A1 (fr) * 2016-09-05 2018-03-07 Deutsche Saatveredelung AG Dispositif de culture et de prélèvement d'échantillon pour plantes
WO2018041308A1 (fr) * 2016-09-05 2018-03-08 Deutsche Saatveredelung Ag Dispositif de culture et d'échantillonnage de plantes
CN109688801A (zh) * 2016-09-05 2019-04-26 德国萨特维尔德伦股份公司 栽培和取样装置
US11199477B2 (en) 2016-09-05 2021-12-14 Deutsche Saatveredelung Ag Cultivation and sampling device for plants
CN109688801B (zh) * 2016-09-05 2022-01-04 德国萨特维尔德伦股份公司 栽培和取样装置
EP3784368A4 (fr) * 2018-04-25 2022-01-26 Optofluidics, Inc. Collecteur à vide pour microscopie à filtration

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EP1227888A1 (fr) 2002-08-07

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