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WO2001068225A1 - Focalisation isoelectrique pour micropreparation - Google Patents

Focalisation isoelectrique pour micropreparation Download PDF

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Publication number
WO2001068225A1
WO2001068225A1 PCT/EP2000/002312 EP0002312W WO0168225A1 WO 2001068225 A1 WO2001068225 A1 WO 2001068225A1 EP 0002312 W EP0002312 W EP 0002312W WO 0168225 A1 WO0168225 A1 WO 0168225A1
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WO
WIPO (PCT)
Prior art keywords
molecules
regions
buffer
volume
fixed
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/EP2000/002312
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English (en)
Inventor
Michael Cahill
Andrzey Drukier
Alfred Nordheim
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.)
ProteoSys AG
Original Assignee
ProteoSys AG
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 ProteoSys AG filed Critical ProteoSys AG
Priority to PCT/EP2000/002312 priority Critical patent/WO2001068225A1/fr
Priority to AU38120/00A priority patent/AU3812000A/en
Publication of WO2001068225A1 publication Critical patent/WO2001068225A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
    • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Definitions

  • the invention relates to a device for separation of a mixture, especially a mixture of biological molecules, by isoelectric focussing according to the preamble of claim 1.
  • electrophoresis for preparative purposes is an established technique and several types of electrophoretic apparatus exist for preparative and analytical purposes. These apparatus and their accompanying principles fall into four categories [A. T. Andrews, (1986) . Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications, Clarendon Press, Oxford.]:
  • Both isotachophoresis and disc electrophoresis utilise hydrophilic matrices, and typically exhibit high resolution, but low loading capacities. Free flow electrophoresis utilises a continuously flowing thin buffer layer which enables high throughput.
  • the method can be combined with isotachopho- retic principles in interval isotachophoresis, by which a relative concentration sample specific fractions can be combined with high loading ability [Kasic a, (1994) . J. Chromatogr. B. Bio ed. Appl. 656: 99-106].
  • Isoelectric focussing has been performed in either liquid density gradients, in gel gradients, or in multicompartment apparatus with isoelectric immobiline based separating gel medium [P. G.
  • analyte molecules do not enter the gel matrix, but remain in buffer within a specific chamber while other molecules are removed by hydraulic flow and/or electrophoresis; passage through the restrictive gel being prevented by electrophoretic migration in the counter direction, the buffering values of walls on either side of the chamber being below and above of the pH value corresponding to the isoelectric point (pi) of the analytes retained.
  • the porous filters of multicompartment isoelectric focussing apparatus are impregnated with a polyacryla ide gel matrix which contains immobilines of a defined pH buffering value.
  • the invention facilitates the task to provide a device for isoelectric focussing especially for small sample volumes, wherein material loss is minimized.
  • This problem is solved by a device comprising the features of claim 1, preferred embodiments are shown in the following claims.
  • the wording of all claims is hereby made to the content of the present speci ication by reference.
  • the device according to the invention is described in greater detail below by way of example with reference to variants. For the sake of simplicity a major part of this disclosure describes the applications for the field of protein biochemistry; the generalization to other molecules, such as lipids or nucleic acids, will be possible for those skilled in the art.
  • the principles of isoelectric focussing will be used in an inventive manner whereby a series of fluid buffered pH barrier regions are created and the analyte molecules are induced electrophoretically to approach their pi and thus concentrate into regions which will be useful for their subsequent free flow fractionation. These regions are characterized by a low buffer capacity whereby molecules can be trapped between the high pass low pass pH barriers on either side.
  • the disclosed device presents the analytes and buffer with a stratified and precisely defined medium, where within the array diverse molecules inducing different pK and buffering capacities are anchored or fixed, respectively.
  • the pK of a buffering group is normally defined to be the pH where the buffering group is 50 % ionised, and where it exerts maximum buffering potential.
  • the principle of the inventive device is based on the multicompartment isoelectric focussing, wherein instead of the known solid pH barriers fluid pH barriers, especially planar fluid pH barriers are formed, which allow a continous solution fluid and which minimize sample material loss.
  • Said pH barriers are formed by buffer molecules, which are fixed on surfaces bordering the electrophoretic volume.
  • fixed is meant, that the buffer molecules are bound or ligated to said surfaces forming a coated or semi- coated surface, wherein the immobilisation of buffering molecules according to the state of the art within a gel matrix is excluded.
  • the buffering molecules could be im- obilines covalently attached to the surface of the electrophoresis chamber without the presence of polyacr lamide, or other such buffering molecules, such as modified amino acids or oligo-peptides, or indeed any molecule containing ioni- sable groups.
  • the electrophoretic (three dimensional) volume is encased by two jux- taposed, especially opposite surfaces, broken into homogenous regions which are coated with molecules possessing a certain conductivity and buffer capacity at a given pH.
  • One or both said surfaces may comprise a coating of buffering molecules.
  • the said surfaces of the inventive device are for example the surfaces of two opposite silica or plastic chips, not truly planar or (especially) planar-, with certain opposite regions being coated with molecules which impart defined buffering and conductivity properties to aqueous buffer within the electrophoretic volume between said surfaces.
  • chip is meant a micro-apparatus of the type where electrical circuitry or microfluidic channels can be created by modern methods such as photoetching, and which will be apparent to those expert in the art. Between said surfaces there is no contact other than at the edges of the surfaces, where the volume or chamber is physically sealed by non buffering ad- hesives and/or physical spacers. The distance between said surfaces is typically between 10 and 1000 nm, especially between 10 and 500 nm. Extremely small distances between two planar surfaces can be maintained by columns of approximately 10 to 30 nm diameter which are created when the surrounded surface is removed by microetching.
  • Such columns occupy a negligible volume relative to the volume of that region of the electrophoretic volume or chamber, and exert negligible effects on electrical conductivity, fluid flow and micro- turbulences.
  • the generation of these extremely small distances permits the pH of the aqueous medium to be determined by buffering molecules immobilised on the surface of the chamber.
  • the miniaturisation of isoelectric focussing permits the creation of restrictive buffer regions somewhat similar to those generated in multicompartment isoelectric focussing apparatus.
  • the device disclosed differs in two critical respects. Due to the miniaturisation obtained, the restrictive medium does not physically span the electrophoretic volume or chamber from wall to wall. Indeed a direct and unbroken buffer channel exists between all regions of the device. This is possible since the microscale allows buffer molecules on the walls of the electrophoretic volume itself to determine the pH of -the buffer within. The buffer molecules need not to be immobilines, which is the second important difference to the known multicompartment isoelectric focussing apparatus.
  • self assembly monolayer a system where a surface is functionalized to allow covalent bonding to one or more types of specific substrate molecules, such that the surface binds substrate in a specific manner, in the ideal case forming a closely packed monolayer.
  • An innovative multi-channel fluidic system is formed by high spatial resolution dense patterns of grooves and miniaturised vessels, provided by e.g. modern photoetching methods, especially applicable to silica but not restricted thereto, and electric field activated and/or computer controlled fluidic switches.
  • Such fluidic systems can be produced not only in silicon dioxide, but also in plastic, especially in teflon, and a plurality of other solid state materials. It is pointed out that for the proposed application the main challenge besides the channel's definition is mainly the quality of surface which should be prepared so as to make negligible the adherance of components from heterogenous mixtures of molecules, such as proteins.
  • teflon as material, especially as coating material, for the fluidic system is especially preferred, because due to the nature of this material hydrophobic interactions with the sample material are as far as possible excluded and thereby material loss is minimized.
  • the body of the device could be made of one material, e.g. (but not restricted to) silica, tungsten, suitable nitrides and carbides, diamond, or plastic, while the surface to which buffering and conducting molecules are attached, and which is in contact with analyte molecules during electrophoresis, could be made of another material, e.g. (but not restricted to) teflon.
  • hybrid system a system in which separate systems such as microcapillaries of HP C (high performance liquid chromatography) columns are connected through a series of external pipings.
  • in-silico fluidic systems permits handling of very small sample volumes. If in the classical capillary systems up to 500 nl of fluid has to be loaded, in the in-silico systems only a few tens of nl can be loaded. This draws attention to the detection method, because often sub fmol amounts of the material of interest are to be detected.
  • maximum sensitivity detectors coupled with the in-silico fluidics is disclosed as preferred implementation of the proposed method of fractionation of small sample volumes.
  • a preferred implementation involves an array of miniaturised prior art isotope detectors such as scintillation detectors or Multiple Photon Detection (MPD) detectors.
  • MPD Multiple Photon Detection
  • MPD (e.g. US Patent Number 5532122) is a technique for the - detection and measurement of certain radioisotopes at activity levels which are much below the naturally occurring background. This is made possible by the nearly perfect rejection of background events which is achieved through sophisticated signal processing and analysis. Certain iso- topes emit both X-rays as electrons move to lower orbitals, and gamma rays as neutrons decompose upon radioactive decay. The high degree of sensitivity of MPD is obtained by considering only signals with coincident X- and gamma-rays, and where the energy profile of the measured photons corresponds to those expected for the isotope in question.
  • the isotopes compatible with MPD come from two broad families of radio- isotopes which together include over 100 members appropriate for use as tags. In particular, they include the isotope iodine-125 ( 125 I) , one of the most commonly used isotopes in biomedical diagnostics.
  • MPD is able to reduce the number of background events to less than one per week. MPD permits multi-labeling (up to 16 co-resident labels) , good spatial resolution (about 0.3 mm) and very high dynamic range (linearity over nine orders of magnitude in label down to 10 ⁇ 21 mole) .
  • Figure 1 first embodiment of the inventive device
  • FIG. 1 and 2 One implementation of the device according to the present invention is presented in Fig. 1 and 2. This pattern, and variations thereof, like in Fig. 3 (but not restricted thereto) , define the arena in which separations according to this principle and device, and other variations thereof, will be performed.
  • a device as shown in Fig. 1 and 2, consists of an electrophoretic volume, here formed as a channel-like chamber (no reference number) , with anode 1 at the end of the most extreme acid pH buffered barrier, and cathode 2 at the opposite end of the chamber with the most extreme basic pH buffered barrier.
  • the channel-like chamber has a very small dimension, the distance between the opposite surfaces 12 is especially less than 10 ⁇ m and may be as small as 500 nm to 10 nm.
  • the length of the chamber is shown in a shortened manner indicated by the diagonal lines (obliques) , which are used in all figures.
  • Distinct pH barriers 3 and 4 which are built up by buffer molecules fixed to the two opposite surfaces 12 or to surfaces attached to these surfaces 12 bordering the electrophoretic chamber, subdivide the chamber into regions with defined pH value, called barrier regions, e.g. pH 6 in region 3 and pH 8 in region 4, and collection regions 6, 7, and 8 surrounding the pH barriers.
  • barrier regions e.g. pH 6 in region 3 and pH 8 in region 4
  • collection regions 6, 7, and 8 surrounding the pH barriers e.g. pH 6 in region 3 and pH 8 in region 4
  • Inlet channels 5, 13, 14, 15, and 16 lead towards the electrophoretic chamber or the corresponding regions, respectively, and outlet channels 9, 10, and 11, lead away.
  • Analyte molecules are induced to migrate electrophoretically or are carried by bulk fluid movement through the system.
  • the electrophoretic chamber contains pH barrier regions 3 and 4 of buffered pH, e.g. pH 6 and pH 8 in Fig. 1, which define the charge and therefore the electrophoretic migration of the analytes. Voltage applied across the isoelectric focussing electrodes 1 and 2 results in electophoresis of the analytes towards their isoelectric point.
  • analytes migrate until they- arrive at a region where they are close to being neutrally charged, such that movement in either direction requires traversing an impassable electrophoretic barrier, e.g. a molecule with pi of 7 entering the chamber from channel 14 into pH barrier 3 (pH 6) is positively charged and migrates towards cathode 2.
  • an impassable electrophoretic barrier e.g. a molecule with pi of 7 entering the chamber from channel 14 into pH barrier 3 (pH 6) is positively charged and migrates towards cathode 2.
  • pH barrier 3 (pH 6) When it moves from pH barrier 3 (pH 6) into collection region 7, it could not successfully re-enter pH barrier 3 (pH 6) since at this pH the molecule would be negatively charged and migrate towards cathode 2.
  • pH barrier 4 pH barrier 8
  • Analyte molecules from collection regions 7 and 8 could be removed via channels 10 and 11 respectively, experimental design permitting.
  • the channels referred to here can be capillaries, but could be any vessel, pipe, tube or microchannel permitting (preferably controlled) fluid flow, including icrochan- nels on a chip or a chip based fluid filter. All channels can be connected to receptacles/ reservoirs, graphically indicated in Fig. 1 as circular entities, but not designated by reference numbers.
  • fluid is introduced to the electrophoretic volume, realized as a channel-like chamber, by hydraulic force from channel 13 at the anode or channel 16 at the cathode region, and/or by channels 14 and 15 which discharge into buffered regions 3 and 4 respectively, and/or by channel 5 which discharges into collection region 7.
  • Fluid can be removed from the electrophoretic chamber via channels 9 and 11 after the fluid has been induced to traverse at least part of the chamber. Note that if fluid was introduced to the electrophoretic chamber via channel 13 or via channel 14, it would normally be induced to exit the chamber via channel 11. The same would apply for entry from channels 16 or 15, with exit via channel 9. If fluid entered via channel 5 it could be induced to flow out of the chamber via channels 9 and/or 11, as desired.
  • the required fluid pumping force could be supplied by an external mechanical or electroendoosmotic pump, whereby fluid hydrostatic cohesion would pull fluid, containing analytes, through the whole device as described above.
  • the transport of fluids containing analytes is achieved either with the aid of pumps and valves, and/or by the application of electric fields to suitable portions of the system. Note that for fluid pumping by an electroosmotic pump it may be advantageous to alternate between an electric field across 1 and 2 which induces isoelectric focussing between buffer regions, and one or more electric fields which introduce and expel
  • an electric field transporting analytes into the electrophoretic volume or chamber is applied in addition to the external pumping force, e.g. a mechanical or electroendoosmotic pump.
  • the external pumping force e.g. a mechanical or electroendoosmotic pump.
  • This embodiment provides the important advantage, that the analytes are charged at the moment of entering the electrophoretic volume or chamber.
  • the isoelectric focussing of analytes starts instantly and is much faster, resulting in a shortened separation time compared to apparatus of the prior art.
  • the device can be transversed by fluid flow in a controlled manner from the inlet and outlet sources concurrently with isoelectric focussing rather than intermittently.
  • fluids could be introduced from channels 13 or 16 leading from a reservoir containing analyte molecules at a given end, and also removed from the same end via channels 9 or 11.
  • This fluid removal system facilitates removal of small, highly mobile ions which originate from the sample.
  • these ions are removed from the plane of analysis of less mobile analytes, thus reducing joule heating and allowing subsequent application of higher voltages across the region of the array containing analyte molecules, and thereby decreasing separation time.
  • the fluid flow through the inlet channels and outlet channels renews the electrolyte solutions and sample buffer in specified regions to the electrophoretic volume or chamber, such as the cathodic and anodic zones.
  • the described process reduces the complexity of the analysed sample within a particular fraction, while increasing the concentration, and thereby improves the ability to detect low abundance components in a concentration dependant manner in this and subsequent analyses.
  • the device provides additionally the advantage that migration through a channel or capillary during subsequent analysis will not be perturbed by overloading leading to difficulties in controlling artifacts. This reduction of complexity in order to facilitate sub- sequent analysis of complex mixtures of molecules is also innovative when applied to such a pH multiplexer.
  • immobilised anions are unable to migrate electrophoretically to the cathode, and instead induce a bulk fluid flow towards the anode. Such effects will be minimised in the disclosed device, and could be compensated for by removal or replacement of fluid at an appropriate rate from the capillary pumps described above.
  • analyte molecules After analyte molecules have been concentrated in the collection regions between pH barriers, they are removed by the aid of further channels which induce fluid flow in another direction to the isoelectric fractionation by use of hydrau- lie forces, or by the application of electric fields to suitable portions of the system as described above.
  • These channels lead simply to a collection vessel (reservoir) , or preferentially are themselves part of a further fractionation device such as microbore HPLC (high performance liquid chromatography) or CE (capillary electrophoresis) .
  • outlet channels are connected to subsequent further fractionation steps, such as discrimination by density sedimentation, discrimination by particle size, melting point, mechanical properties, polarity (electric moments) , isoelectric point, affinity, spectro- scopic properties, chromatography or electrophoresis.
  • the electropho- retic volume is fitted with e.g. at least one of the following devices: a device for keeping the electrophoretic volume at a controlled temperature, a device for pH measurement and control, a device for conductivity monitoring, a biosensors detection system, an immunoelectrophoretic device, a laser excited fluorescence detection system, a device for radioiso- top monitoring, preferably a MPD detection system, a roboti- cally coupled system, a device for monitoring the sample solution in the UV or visible light or by fluorescence observation, or a device for measuring the dielectric con- stant of the sample solution.
  • a device for keeping the electrophoretic volume at a controlled temperature e.g. at least one of the following devices: a device for keeping the electrophoretic volume at a controlled temperature, a device for pH measurement and control, a device for conductivity monitoring, a biosensors detection system, an immunoelectrophoretic device, a laser excited fluorescence detection system, a device for radioiso- top monitoring
  • the inventive immobilised buffer array uses immobilised buffering molecules. According to the invention there is no direct physical contact of molecules from one wall of the electrophoretic volume or chamber with molecules on the other wall, with the resulting uninterrupted buffer channel fundamentally distinguishing this device from prior art multi- chamber isoelectrophoresis apparatus.
  • Any buffering molecule in principle can fulfill the buffering function, and indeed will be preferable without the structural and chemical constraints imposed by the acrylamide-like backbone of immobilines.
  • the array is created using suitable precursors and microcontact printing, self assembly reactions, or photo-activated chip technology where reactions take place in photo-activated regions, or other prior art methods. However the principle and device variants would be functional over a large range of physical dimensions.
  • outlet channels 9, 10, or 11 of the multiplex may be connected to a receptacle tube which could provide the required pump action.
  • pH multiplexers are incorporated, such that analyte molecules are partitioned into several different analytical compartments.
  • a preferred embodiment of the invention arranges several such pH multiplexers in a tree-like series, one example of which is depicted in Fig. 3.
  • the possible number of arrangements of such configurations is unlimited, in principle.
  • the components of the sample are fractionated in a pH multiplexer 21 as essentially described above, wherein the pH barriers 25 are chosen out of a relatively broad range, e.g. pH 3, pH 5, pH 7, and pH 9.
  • the components are introduced to further pH multiplexers 22, 23, and 24, wherein pH definition is more narrow, and thereby a much more precise fractionation is possible, e.g. a fractionation of components, which differ in their isolelectric points by at least 0.05 pH units.
  • the electrophoretic volume or chamber is filled with solutions of varying viscosity.
  • the volume or chamber is attached to a centrifugal device capable of generating gravitational force along the one axis.
  • analyte molecules are separated by isoelectric pH multiplex properties along one axis, and by density along another axis.
  • the density separation is used as a further concentration method if proteins are loaded between two density cushions in a step gradient.
  • the inventive devices obtain a fine division of incoming fluid into collection regions with very narrow pH definition.
  • the losses increase with depth.
  • any and all low/high pass pH filters are ideally loss- less, i.e. they implement a step function in pH.
  • Creation of a true pH step gradient at the delta region, the amplitude of the buffering capacity, and the voltage applied over the delta region are critical in this respect.
  • Using a device on the scale of a microchip according to the invention large voltages can be applied, being advantageous to minimise losses.
  • Critical can be maintenance of solubility and control of field strength with respect to temperature, which determines the time available for molecules to electrophoretically leave the delta region.
  • inventive solution to this problem involves duplicate analysis of samples by complementary pH multiplexes, such that for instance one multiplex separates analytes between pH regions 3-7, and 7-11, while a second complementary multiplex separates identical analytes between pH 5-9.
  • Another preferred application of the disclosed multiplex pH system is its use in combination with a coupled parallel HPLC/CE separation.
  • the benefits of each technique are non-overlapping, thus an optimal reduction of complexity of the analytes is achieved.
  • the pH barriers chosen in the array determine the optimum IPG gel into which HPLC/CE sorted analytes are delivered.
  • Another preferred embodiment of the subject invention is an innovative and optimised device for reduction of sample complexity which thereby should facilitate analysis of the full dynamic range of analytes. If e.g. in the device according to Fig. 3 the last level of the tree is controlled by an appropriate high sensitivity detec- tor, the device is able to discriminate which fractions are high and low abundance. If the total flow through of a given channel of the above described pH multiplex is higher than a computer established threshold, an appropriate pulse is sent, and the outlet of this channel is closed.

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Abstract

L'invention concerne un dispositif pour séparer les composants d'une pluralité de mélanges, constitués en particulier de molécules biologiques, par focalisation isoélectrique. Le volume, dans lequel la séparation électrophorétique a lieu, est bordé de surfaces qui sont au moins partiellement revêtues de molécules tampon fixes, permettant ainsi de définir des barrières de pH distinctes. Ce dispositif est en outre caractérisé par de faibles distances entre des surfaces opposées, lesdites surfaces étant constituées par des pièces de silice ou de plastique.
PCT/EP2000/002312 2000-03-15 2000-03-15 Focalisation isoelectrique pour micropreparation Ceased WO2001068225A1 (fr)

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PCT/EP2000/002312 WO2001068225A1 (fr) 2000-03-15 2000-03-15 Focalisation isoelectrique pour micropreparation
AU38120/00A AU3812000A (en) 2000-03-15 2000-03-15 Micropreparative isoelectric focussing

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003019172A3 (fr) * 2001-08-31 2003-12-18 Diagnoswiss Sa Appareil et procede de separation d'un analyte
WO2005026715A1 (fr) * 2003-09-12 2005-03-24 Proteosys Ag Focalisation isoelectrique serielle de gradients de ph immobilises
WO2005089910A1 (fr) * 2004-03-17 2005-09-29 Ciphergen Biosystems, Inc. Filtre a plusieurs compartiments et procede de filtrage au moyen de ce filtre
WO2007147862A1 (fr) * 2006-06-20 2007-12-27 Becton, Dickinson & Company Procédé et dispositif pour la séparation et l'appauvrissement de certaines protéines et particules à l'aide d'une électrophorèse
WO2009144621A1 (fr) * 2008-05-27 2009-12-03 Koninklijke Philips Electronics N. V. Biopuce pour le fractionnement et la détection d'analytes
WO2009147554A1 (fr) * 2008-05-27 2009-12-10 Koninklijke Philips Electronics N. V. Biopuce de focalisation isoélectrique
WO2010023589A1 (fr) * 2008-08-28 2010-03-04 Koninklijke Philips Electronics N.V. Procédé de préparation d’un gradient de ph dans une biopuce à focalisation isoélectrique
US8945914B1 (en) 2010-07-08 2015-02-03 Sandia Corporation Devices, systems, and methods for conducting sandwich assays using sedimentation
US8962346B2 (en) 2010-07-08 2015-02-24 Sandia Corporation Devices, systems, and methods for conducting assays with improved sensitivity using sedimentation
US8988881B2 (en) 2007-12-18 2015-03-24 Sandia Corporation Heat exchanger device and method for heat removal or transfer
US9005417B1 (en) * 2008-10-01 2015-04-14 Sandia Corporation Devices, systems, and methods for microscale isoelectric fractionation
US9244065B1 (en) 2012-03-16 2016-01-26 Sandia Corporation Systems, devices, and methods for agglutination assays using sedimentation
US9261100B2 (en) 2010-08-13 2016-02-16 Sandia Corporation Axial flow heat exchanger devices and methods for heat transfer using axial flow devices
US9795961B1 (en) 2010-07-08 2017-10-24 National Technology & Engineering Solutions Of Sandia, Llc Devices, systems, and methods for detecting nucleic acids using sedimentation

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3844925A (en) * 1973-07-02 1974-10-29 Center For Blood Res Molecular fractionation
EP0287513A2 (fr) * 1987-04-11 1988-10-19 Ciba-Geigy Ag Procédé de focalisation isoélectrique et moyens pour la mise en oeuvre de ce procédé
US5376249A (en) * 1992-11-25 1994-12-27 Perseptive Biosystems, Inc. Analysis utilizing isoelectric focusing
US5540826A (en) * 1995-03-15 1996-07-30 Protein Technologies, Inc. Multi-channel separation device
EP1044716A1 (fr) * 1999-03-13 2000-10-18 Michael Dr. Cahill Focalisation isoélectrique micropréparative

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3844925A (en) * 1973-07-02 1974-10-29 Center For Blood Res Molecular fractionation
EP0287513A2 (fr) * 1987-04-11 1988-10-19 Ciba-Geigy Ag Procédé de focalisation isoélectrique et moyens pour la mise en oeuvre de ce procédé
US5376249A (en) * 1992-11-25 1994-12-27 Perseptive Biosystems, Inc. Analysis utilizing isoelectric focusing
US5540826A (en) * 1995-03-15 1996-07-30 Protein Technologies, Inc. Multi-channel separation device
EP1044716A1 (fr) * 1999-03-13 2000-10-18 Michael Dr. Cahill Focalisation isoélectrique micropréparative

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7390389B2 (en) 2001-08-31 2008-06-24 Diagnoswiss S.A. Apparatus and method for separating an analyte
WO2003019172A3 (fr) * 2001-08-31 2003-12-18 Diagnoswiss Sa Appareil et procede de separation d'un analyte
WO2005026715A1 (fr) * 2003-09-12 2005-03-24 Proteosys Ag Focalisation isoelectrique serielle de gradients de ph immobilises
WO2005089910A1 (fr) * 2004-03-17 2005-09-29 Ciphergen Biosystems, Inc. Filtre a plusieurs compartiments et procede de filtrage au moyen de ce filtre
US20100252435A1 (en) * 2006-06-20 2010-10-07 Gerhard Weber Method and device for separation and depletion of certain proteins and particles using electrophoresis
WO2007147862A1 (fr) * 2006-06-20 2007-12-27 Becton, Dickinson & Company Procédé et dispositif pour la séparation et l'appauvrissement de certaines protéines et particules à l'aide d'une électrophorèse
AU2007263005B2 (en) * 2006-06-20 2013-07-25 Becton, Dickinson And Company Method and device for separation and depletion of certain proteins and particles using electrophoresis
CN101512334B (zh) * 2006-06-20 2013-08-07 贝克顿迪肯森公司 用电泳来分离和去除某些蛋白和颗粒的方法与装置
US8988881B2 (en) 2007-12-18 2015-03-24 Sandia Corporation Heat exchanger device and method for heat removal or transfer
WO2009147554A1 (fr) * 2008-05-27 2009-12-10 Koninklijke Philips Electronics N. V. Biopuce de focalisation isoélectrique
WO2009144621A1 (fr) * 2008-05-27 2009-12-03 Koninklijke Philips Electronics N. V. Biopuce pour le fractionnement et la détection d'analytes
WO2010023589A1 (fr) * 2008-08-28 2010-03-04 Koninklijke Philips Electronics N.V. Procédé de préparation d’un gradient de ph dans une biopuce à focalisation isoélectrique
US9005417B1 (en) * 2008-10-01 2015-04-14 Sandia Corporation Devices, systems, and methods for microscale isoelectric fractionation
US8962346B2 (en) 2010-07-08 2015-02-24 Sandia Corporation Devices, systems, and methods for conducting assays with improved sensitivity using sedimentation
US8945914B1 (en) 2010-07-08 2015-02-03 Sandia Corporation Devices, systems, and methods for conducting sandwich assays using sedimentation
US9795961B1 (en) 2010-07-08 2017-10-24 National Technology & Engineering Solutions Of Sandia, Llc Devices, systems, and methods for detecting nucleic acids using sedimentation
US9261100B2 (en) 2010-08-13 2016-02-16 Sandia Corporation Axial flow heat exchanger devices and methods for heat transfer using axial flow devices
US9244065B1 (en) 2012-03-16 2016-01-26 Sandia Corporation Systems, devices, and methods for agglutination assays using sedimentation

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