WO2001090719A1 - Formation de gradient fluide de precision - Google Patents

Formation de gradient fluide de precision Download PDF

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Publication number
WO2001090719A1
WO2001090719A1 PCT/US2001/016120 US0116120W WO0190719A1 WO 2001090719 A1 WO2001090719 A1 WO 2001090719A1 US 0116120 W US0116120 W US 0116120W WO 0190719 A1 WO0190719 A1 WO 0190719A1
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WIPO (PCT)
Prior art keywords
gradient
gradients
density
particles
components
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PCT/US2001/016120
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English (en)
Inventor
Norman Anderson
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Large Scale Proteomics Corporation
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Application filed by Large Scale Proteomics Corporation filed Critical Large Scale Proteomics Corporation
Priority to AU2001261756A priority Critical patent/AU2001261756A1/en
Publication of WO2001090719A1 publication Critical patent/WO2001090719A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
    • G01N1/18—Devices for withdrawing samples in the liquid or fluent state with provision for splitting samples into portions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/04—Investigating sedimentation of particle suspensions
    • G01N15/042—Investigating sedimentation of particle suspensions by centrifuging and investigating centrifugates

Definitions

  • the present invention relates the formation of both linear and non-linear liquid gradients from liquids having very small density differences, and to the formation of gradients having different reagents in different levels of the gradient.
  • the invention further relates to the formation of sets of gradients in parallel. These are particularly useful for fractionation of cellular or subcellular particles from biological samples.
  • Liquids having gradients of temperature, concentration, density and color have been previously prepared. Liquid density gradients have been used for many years, for a variety of purposes, in a number of different industries. The inventor has numerous publications and patents regarding certain aspects of gradient formation and use including: Anderson, N.G. Mechanical device for producing density gradients in liquids. Rev. Sci. Instr. 26: 891-892, 1955; Anderson, N.G., Bond, H.E., and Canning, R.E. Analytical techniques for cell fractions. I. Simplified gradient elution programming. Analyt. Biochem. 3: 472-478, 1962; Anderson, N.G., and Rutenberg, E. Analytical techniques for cell fractions. A simple gradient-forming apparatus. Anal.
  • Density gradients are used to make two basic types of separations.
  • the first separates particles based on sedimentation rate (rate-zonal centrifugation), in which particles are separated based on the size and density and to a lesser extent their shape. The particles will sediment farther if centrifuged for a longer period of time.
  • the second method separates particles based on isopycnic banding density, in which particles reach their equilibrium density level, and do not sediment further with continued centrifugation.
  • One object of the present invention is to produce segments of a liquid density step gradient which differ predictably in their properties, and in the identity and concentration of reagents present in individual segments.
  • Another object of the present invention is to make the sedimentation through a gradient an analytical process for analytical particle recovery from the liquid gradient and analytical measurement of particles in the gradient.
  • a further object of the present invention is a means for producing individual segments of the gradient.
  • the present invention achieves these objectives by using a large number of gradient components where fine differences are achieved by mixing one gradient component with another to prepare an intermediate gradient component. Additional intermediate gradient components may also be similarly made. Even if the exact concentration is uncertain, the range must be correct. This technique assures that inversions and other irregularities do not occur. This technique is also readily automatable and can prepare multiple gradients in the same solution. Of particular benefit is the inclusion of a reagent with specialized properties within a particular region of the gradient to enhance separation and recovery of the sample particles. Preferred uses are for separation and quantification of biological particles in a reproducible manner.
  • Figure 1 shows a mixing pattern for linear gradient construction and how finely graded intermediate level gradient components are prepared.
  • Figure 2 shows a gradient component set and a mixing pattern for non-linear gradient construction.
  • Figure 3 is a side view of an automated gradient pipetter.
  • Figure 4 is a top view of an automated gradient pipetter.
  • Figure 5 shows diagrammatically an enclosed, refrigerated and microprocessor controlled pipetting system.
  • Figure 6A is a Schlieren pattern of a step gradient before diffusion.
  • Figure 6B is a gradient with a colored compound being added to alternating layers of a step gradient.
  • Figure 6C is the gradient of Figure 4B after it has diffused to form a linear gradient.
  • Figure 6D is an optical scan of the colored compound in the gradient of Figure 6C.
  • Figure 7A shows diagrammatically non-sedimenting reagents in zones in a density gradient and particles in a sample zone about to be sedimented through said zones.
  • Figure 7B shows diagrammatically the same gradient after sample particles have passed through the gradient, leaving reagent extractable substances in the reagent zones.
  • Figure 8 is a plot of density vs. fraction number for a non-linear gradient.
  • Figure 9 is a flow chart of the operation of the apparatus in the present invention.
  • Figure 10 provides another flow chart of activities in preparing a gradient.
  • gradient includes a non-homogenous fluid composition where one portion contains a quantitative difference in a particular property from another portion.
  • the differences may be in concentration, density, color, temperature, osmotic pressure, absorbency, particle amount or size, electrical and magnetic properties such as resistance, etc.
  • density and concentration gradients are generally of most interest. The gradient need not be completely stable if it is controllable for a period of time.
  • a “linear gradient” is a gradual, even and constant change in a gradient property from one end of the gradient to an opposite end.
  • a “step gradient” is an abrupt change in the gradient property between two fluid portions in the gradient. It is typically made by layering one liquid over a different liquid.
  • a “non-linear gradient” is a gradient with an uneven distribution of or non-constant change in the gradient over the length of the gradient. While step gradients are non- linear, others such as exponential or irregular are also considered non-linear gradients.
  • a “gradient component” is a homogenous fluid having a particular property that is desired to be located in a particular region of a gradient. For example, to create a gradient, one requires at least two gradient components or solutions unless the gradient is self-forming (e.g. cesium chloride gradients).
  • the gradient components being used to create a gradient may contain different amounts of a compound, composition, particle etc, (same features), or may contain chemically or physically different features (compounds, compositions, particles etc.). A large number of different gradient components are known per se.
  • a “reagent capable of reacting” includes any composition that engages in an interaction such as binding, catalysis, chemical reaction, precipitation etc.
  • An “intermediate level” gradient component is one which is known to have a property intermediate between two other gradient component materials because the intermediate level gradient component is formed by mixing one gradient component material with another gradient component material.
  • the term “intermediate level” is not meant to mean exactly intermediate. Instead the term is used to indicate a level more similar to one parent gradient component than another.
  • a “secondary intermediate level” gradient component results from mixing an intermediate level gradient component with another gradient component material which itself may be an intermediate level gradient component.
  • “Tertiary”, “quaternary” etc. intermediate level components are also so formed in a like manner. In each situation, the resulting gradient material will be intermediate between the two gradient component materials used to form it.
  • a “sedimentation container” is a vessel that a sample is allowed to or is forced to pass through.
  • Centrifuge tubes are typical sedimentation containers for small particles. For larger particles, the force of gravity is sufficient to sediment such particles. Separation is enhanced by a density gradient in the sedimentation container.
  • isolated when referring to a particle or macromolecule, means that it is essentially free of other components originally found in the sample.
  • purified refers to a state where the relative concentration of a particle or macromolecule is significantly higher than in the starting composition before it is purified. Purity and homogeneity are typically determined using standard analytical techniques.
  • a purified or isolated product will comprise more than 80% of all similar species present in the preparation.
  • the product is purified to greater than 90%) of all species present. More preferably, the product is purified to greater than 95% and most preferably, the product is purified to essential homogeneity, or wherein other species are not significantly detected by conventional techniques.
  • binding includes any physical attachment or close association, which may be temporary or permanent as in a chemical bond. Generally, an interaction of hydrogen bonding, hydrophobic forces, van der Waals forces etc. facilitates physical attachment between the ligand molecule of interest and the receptor.
  • the "binding" interaction may be brief as in the situation where binding causes a chemical reaction to occur. This is typical when the binding component is an enzyme and the analyte is a substrate for the enzyme. Reactions resulting from contact between the binding component and the analyte are within the definition of binding for the purposes of the present invention. Binding is preferably specific.
  • the binding may be reversible, particularly under different conditions.
  • bound to or associated with refers to a tight coupling of the two components mentioned.
  • the nature of the binding may be chemical coupling through a linker moiety, physical binding or packaging such as in a macromolecular complex.
  • all of the components of a cell are “associated with” or “bound to” the cell.
  • the present invention achieves its objectives by using a large number of gradient components where fine differences are achieved by mixing one gradient component with another to prepare an intermediate gradient component. Additional intermediate gradient components may also be similarly made. This technique is also readily automatable and can prepare multiple gradients using the same set of basic solutions.
  • the present invention involves a system for producing liquid density gradients by using a series of liquids of increasing density prepared by mixing a starting set of liquids of differing density. This is performed using an automatic microprocessor controlled system for pipetting and mixing the liquids according to a set or adjustable computer program, and a device for introducing aliquots of the series of liquids into centrifuge tubes to produce a gradient. It is particularly desirable that uniformly sharp interfaces can be made between the individual liquids in the gradients whether they are density gradients or a different type of gradient.
  • intermediate gradient component liquid When preparing an exact intermediate gradient component liquid, it is preferred to use equal volumes or equal weights of each parent liquid.
  • the choice of liquids to mix and formation of intermediate gradient liquids may be set according to a prescribed pattern, conveniently determined by a computer controlled by a set of instructions to use the prescribed pattern.
  • By carefully controlling the volume or weight of each parent liquid one may directly prepare the final gradient component to be added to the sedimentation vessel. In such a situation, one need not make intermediate liquids which are used only to make other intermediate liquids.
  • One advantage of using multiple-step gradients is that they will reproducibly convert from step to continuous gradients by diffusion over time. This occurs more rapidly and reproducibly if the steps are small. Additionally, the smaller the steps, the tighter the control, a particular advantage with non-linear gradients. With non-linear gradients, one may have a first interface between steps, which is very small, and a second interface between steps, which is very large. One can optimize diffusion time for one interface but it will not be optimal for the other interface and may even result in destabilization of the density gradient. By using many steps, these problems are minimized. With non-linear gradients, it may be preferred to have uneven sized steps to compensate for uneven differences in the step gradient components and uneven amounts of liquid for various steps. Many reasons exist for controlling gradient shape.
  • Gradient capacity i.e., the mass of particles which can exist in a zone without causing a density inversion
  • Gradient capacity is a function of gradient slope, and a steep gradient can support a greater mass of particles per unit gradient length than can shallow gradients.
  • the greatest particle mass concentration in a gradient separation usually occurs immediately beneath the sample zone shortly after centrifugation or other sedimentation has started. As different particles separate in the length of the gradient, the possibility of an overloaded zone diminishes. For this reason, it is desirable to have a short steep gradient section immediately under the sample zone, followed by a shallower gradient section. It is particularly important to avoid inversions, as they may be unstable, trap particles at the wrong location and render the gradient needlessly overly complex.
  • complex gradients By using more gradient steps and different gradients superimposed on each other complex gradients may be produced, e.g. a density gradient and a salt gradient superimposed on or an osmotic band included in the density gradient.
  • a density gradient and a salt gradient superimposed on or an osmotic band included in the density gradient may be produced, e.g. a density gradient and a salt gradient superimposed on or an osmotic band included in the density gradient.
  • Complex gradients may have a concentration of density material may be very different from concentration of non-density material (e.g. a solvent), which may be very- different from a macromolecule material (e.g. an enzyme) as long as each material is not a sedimentable particle that would be moved substantially by the sedimentation process.
  • concentration of density material e.g. a solvent
  • macromolecule material e.g. an enzyme
  • Each gradient component may be in a linear or non-linear format independently.
  • a step gradient formed with cobalamine as an indicator is shown in Figure 6B immediately after it has been formed. It is optional to use a readily detectable component with approximately the same molecular weight as the density gradient material so that oth will codiffuse together. Other readily detectable components may also be used and detected by a variety of different techniques. When an optical scan (or other appropriate scan depending on the detection method) of the tube yields a continuous sine wave ( Figure 6D), the gradient is even enough to be used. Other readily detectable components may also be used and detected by a variety of different techniques.
  • the second type comprises continuous linear gradients that may be made by a mechanical gradient maker.
  • the gradient may be introduced slowly through small tubing to the bottom of the centrifuge tube.
  • liquid gradient components may be pumped through the small tubing or the small tubing may have a funnel attached thereto for easy addition of liquids.
  • the third type of gradient is non-linear, and may be designed to separate particles having a very wide range of sizes or densities.
  • Non-linear gradients may be designed to separate particles on the basis of either sedimentation rate or isopycnic banding density, or both types of separations may occur in the same gradient, in which case some particles reach their isopycnic level at some point in the gradient, while others are still sedimenting.
  • Such combined separations involve larger and denser particles which band near the bottom of the gradient, while other smaller, and usually lighter particles are still sedimenting in the upper portion of the gradient.
  • the fourth type of gradient is one generated in a high centrifugal field by sedimentation of the major gradient solute.
  • the gradient is formed by the action of the centrifugal field.
  • Particles, which differ little in sedimentation rate, are preferably separated by sedimenting them through a longer shallower section of the gradient, which may be located near the center of a gradient.
  • particles with widely differing sedimentation rates may be separated through a steeper section of the gradient.
  • a steep gradient section may be included near the bottom. Particles may be retained either because they sediment more slowly through this concentrated, and often viscous section, or because they band isopycnicly.
  • the sedimentation vessel has a preferably smaller cross section toward the bottom section of the sedimentation vessel.
  • the present invention is exemplified by the fractionation of subcellular particles from mammalian cells and different types of mammalian cells.
  • different cell types from other higher organisms are separable using the same basic techniques such as fractionating cells from plants, fungi, etc.
  • the techniques are applicable to all cells even including single celled organisms, yeast, bacteria, etc.
  • the present invention is suitable for separation and isolation of such agents for research and diagnostic purposes.
  • viruses have different densities and different sedimentation coefficients. This permits easy separation and isolation of viruses by density gradient techniques.
  • the fractionation system of the present invention may also be used for many other materials for diverse purposes including the manufacture and separation of various particles for abrasives, catalysts, pigments, fiuidized bed material and regeneration thereof (particularly for chemical manufacture), wastewater treatment, clays, vaccine, protein, DNA and other bioactive delivery systems, magnetic particles, food products, cosmetics etc.
  • the gradients and their chemical composition are designed to optimize the separation of one or a few particles types. This accounts for the very large number of different gradient recipes that have been published for subcellular fractionation. Those used for the isolation of mitochondria, for example, are usually quite different from those used to isolate nuclei.
  • the protein composition of tissues such as liver, varies diurnally, hence all the tissues from one group of organisms are prepared at the same time of day, and, to be comparable, must be fractionated in parallel, on the same time schedule, and, if gradients are to be used, in identical gradients. Plants in particular respond to light and their metabolism as determined by protein abundance also differs with time of day. Further, gradient fraction recovery should be done from all gradients in parallel, under identical conditions. If the initial separations are done partly or entirely on a sedimentation rate basis, and if the recovered fractions are to then each to be isopycnicly banded, as is done in two-dimensional or s-p fractionation, then these subsequence steps must also be carried out in parallel. There is therefore a need for systems for making a set of gradients simultaneously and repeatably.
  • n is the number of solutions at one level, the next will include 2n-l solutions.
  • Any number of mixing levels can be used.
  • An advantage of this method is that one can be certain that mixing a solution, such as 6 with an equal or unequal volume of solution 7, will produce solution 8 which will have a density between that of 6 and 7, and not outside the range of 6 to 7.
  • a mixture will be produced that will lie between, and if equal volumes are used, almost exactly between the two solutions mixed. This method is of great advantage when very shallow gradients are to be formed.
  • Two variables may be introduced in the dilution pattern of Figure 1 to make simple non-linear gradients.
  • the first is to alter the ratios between components at one or more mixing steps to introduce either plateaus or sharp density increments (or decrements).
  • the second is to vary the amount of each final component actually pipetted into the centrifuge tube.
  • the scheduled reagents may pipetted directly and rapidly into the centrifuge tubes, using either floats (WO 01/12507) or other means to prevent mixing between layers.
  • One otherwise unused tube may contain for the sample, which is added after the gradient has diffused the scheduled amount to smooth the gradient.
  • Integral to the system is a device for changing pipette tips at intervals not only to provide fresh uncontaminated tips, but also to change their capacity.
  • Equivolume mixtures are made according to the pattern indicated, and all blank circles represent positions not used. Used positions are either half black or completely black, and only vessels indicated as completely black are used as sources of liquid for pipetting into the centrifuge tubes. Thus the pipetting pattern can be complex and is best done under microprocessor control. Other types of gradients may be likewise formed. Non equivolume mixtures may be used to prepare the liquids used in preparing the gradients.
  • temperature control the entire system preferably by refrigeration ( ⁇ 5°C).
  • refrigeration ⁇ 5°C
  • temperature affects the volume measurement of liquids being handled. This is particularly important for forming non-linear gradients and reagent containing zones in the gradient. It is even important in linear gradient formation as it effects the diffusion time needed. Even when using non-sedimenting reagents, as is preferred, the reagents will slowly diffuse at a rate that is affected by the temperature.
  • FIG. 1 While the design shown in the Figures 1, 2 and 4 is adapted for equivolume measurements, they may be adapted to pipette unequal volumes to directly prepare the final gradient component liquids. In such a design, only two pipette tips are needed for preparation of the gradient components, and for each parent liquid.
  • the system may employ two arms, each with its own pipette and computer control to prevent the arms from interfering with each other. Arms using radial moving motors are preferred to X and Y coordinate movement on rails if two automated pipetting devices are to be used. Optionally, different lines and pumps to move parent liquids to the tubes may be used.
  • Distinct zones may be formed which remain almost stationary relative to sedimenting particles, providing the distinct zone constituents have sedimentation coefficients that are negligible relative to the sedimenting particles.
  • a zone or region having a salt composition, osmotic pressure, or enzyme activity may be beneficial for the separation of certain subcellular components of a sample.
  • FIG. 7A shows a centrifuge tube 80 containing a gradient 81 having reagent zones 83 and 84, composed of reagents having negligible sedimentation coefficients under the centrifugation conditions employed, and particle-containing sample zone 82.
  • the particles 85 After centrifugation, as shown in Figure 7B, the particles 85 have traversed the reagent zones, and have left behind extracted proteins in zones 86 and 87.
  • Gradients may also be prepared to contain increments of one component, which stabilizes the gradient, and a second, which sequentially extracts material from sedimenting particles.
  • gradients may be prepared using sucrose and sodium chloride which sequentially extract nuclei or other subcellular particles.
  • sucrose and sodium chloride which sequentially extract nuclei or other subcellular particles.
  • a variety of different combinations may be prepared using the gradient producing and pipetting system described in which sedimenting particles undergo changes in pH, ionic composition, ionic composition or organic solvent concentration.
  • Density gradients may thus include not only gradients of dissolved solutes, but gradients of solvents including D 2 O, dimethyl sulfoxide, or organic solvents including chlorinated and brominated alcohols.
  • one or more reagents are added to one or more solutions in the set of gradient components so that reagent zones are made in the final gradient.
  • the reagent will not change the density, volume or other undesired chemical properties of the gradient component. This is important to not affect the shape of the overall gradient. This can best be done by replacing a chemical in the gradient component with the reagent in proportions to not affect the other features of the final gradient.
  • Reagents to be added in very small amounts such as enzymes, detergents, solvents or salts may also be placed in otherwise unused positions on plate 42 of Figures 3 and 4, and added to gradient solutions as programmed.
  • the present invention is readily adapted to the addition of one or more reagents to one or more components of the gradient.
  • Detergents may be added as a single band to solubilize membrane bound particles or to prevent non-particle components from being membrane bound. By recovery of the band, proteins disassociated from particles may be isolated and purified. Particles without these bound components may be recovered from a different region of the gradient.
  • solvents, salts, chaotropic or disaggregating agents e.g. high concentrations of urea).
  • the pipetting machine is microprocessor-controlled, and measures and mixes all gradient components from an initial series of operator-made solutions.
  • the intermediate mixtures may be further mixed by the pipetter by several cycles of solution withdrawal and expulsion controlled by the first phase of the pipetting program.
  • a pipette device can comprise a base on which the remaining parts are positioned, a plurality of movable or rotatable arms, supports and joints for supporting and positioning a plurality of pipettes and a receiver to hold the vessels in which the gradient is made. Movement of the arms can be along horizontal or vertical axes or planes. The movements can be controlled by a computer or microprocessor.
  • the arms, supports and joints can be affixed directly to the base or may be attached to the base by an extendable member.
  • a robot capable of moving a pipette or part of a pipette device accurately and with controllable working in three coordinates is used.
  • various simulation programs and other scheduling systems may be used to have the robot generate a large number of different gradients automatically.
  • the pipette may have removable pipette tips that are readily replaceable within the system.
  • the liquids used to form the gradient may be arrayed is separate vessels, such as tubes in a rack, or in different regions of an integral multiple container sheet, such as a multi-well plate.
  • the robot is informed or senses the locations of all of the vessels or containers of gradient forming liquid and the location of the resulting vessel being filled by mixing or layering of two or more liquids.
  • a number of simple computer programs can determine the patterns and schedules to be formed such as that shown in Figure 2.
  • a separate container may be present or reserved for reagents for forming reagent-containing zone as gradient component(s), the final gradient and the sample to be added to and passed through the gradient.
  • the layering of the gradient components to form the gradient may be done by a number of methods, including slow and careful layering one on top of another, but is preferably performed with the apparatus and techniques of WO 01/12507 which comprise floats 45 as shown in Figures 3 and 4 which decelerate fluids introduced above them and cause said fluids to flow slowly and evenly past the float, and to form sharp interfaces between successively introduced layers.
  • WO 01/12507 which comprise floats 45 as shown in Figures 3 and 4 which decelerate fluids introduced above them and cause said fluids to flow slowly and evenly past the float, and to form sharp interfaces between successively introduced layers.
  • disposable vessels, sheets, pipette tips etc. are preferred.
  • Plastic and glass materials are ideally suited for these purposes.
  • the pipetting machine is microprocessor-controlled, and measures and mixes all gradient components from an initial series of operator-made solutions.
  • the intermediate mixtures are further mixed by the pipetter by several cycles of solution withdrawal and expulsion controlled by the first phase of the pipetting program.
  • the shape of the gradient is controlled by (a) the number, composition, and distribution pattern of the initial solutions used, (c) the mixing and source pattern employed, (d) the volumes of the aliquots used to make intermediate solutions, and (d) by the volumes actually pipetted out of each source vessel into the centrifuge tubes.
  • FIGs 3 and 4 show diagrammatically a pipetting machine 30 in accordance with the present invention.
  • the pipetting machine 30 includes a base 41 that supports a support plate 43 and vertical supports 40.
  • the plate 42 is formed with apertures that support a plurality of tubes.
  • the vertical supports 40 support a horizontal first track 39 that in turn supports a guide 55 that is adapted to selectively move back and forth along the first track 39 that in turn supports a guide 55 that is adapted to selectively move back and forth along the first track 39.
  • Horizontal movement of the guide 55 along the first track 39 is effected by a motor (not shown).
  • a second track 37 extends from the guide 55 in a direction generally perpendicular to the first track 39.
  • a vertical track 36 is supported on the second track 37 such that the vertical track 36 is selectively moveable along the horizontal length of the second track 37.
  • Horizontal movement of the vertical track 36 along the second track 37 is effected by a stepping motor 38.
  • a motorized syringe or pipette 33 is supported on the vertical track 36 for selective up and down movement along the vertical length of the vertical track 36. Up and down movement of the motorized pipette 33 is effected by a stepper motor 35.
  • the motorized pipette 33 includes a probe 32 able to pick up and release a disposable plastic tip 31.
  • the motor controlling movement of the guide 55, the motor 38 and motor 35 are all controlled by a computer 95, shown in Figure 5.
  • the three dimensional movement allows the system to pipette solutions between tubes in plate 42, and other tubes arrayed behind them on the plate or rack 42, all containing gradient component solutions, and to transfer aliquots of the set of gradient producing solutions into the centrifuge tubes 44, into which the gradient components are loaded.
  • the motorized pipette 33 includes suction control (not shown) such that liquid from any of the tubes may be aspirated into and dispensed from the pipette back to any of the tubes.
  • FIG 4 a top view of the pipetting machine 30 is shown with pipette holder 50 positioned over one gradient solution tube.
  • the solution tubes actually used are shown in black, with interconnecting lines indicating the source of mixture.
  • the entire apparatus 90 is preferably enclosed in an insulated transparent temperature-controlled cabinet 91 to keep all reagents at a constant temperature, preferably at about 5 C using chilled air passed through connection 94 from chiller 93. Maintaining isothermal conditions are important to avoid thermal currents mixing the gradient. Operation of the apparatus is controlled by computer 95 and control programs are observed on CRT 96.
  • the computer 95 may be a specialized computer programmed to control the pipetting machine 30 or may be a general-purpose computer such as a standard personal computer with software adapted to control the pipetting machine 30.
  • the computer 95 includes an input device (not shown), such as a keyboard and/or mouse that enables a user to enter information that enables the pipetting machine 30 to produce any desired density gradient within the tubes 44.
  • a user inputs information specifying the density required in each layer of a density gradient, such as the density gradient shown graphically in Figure 8, and described in greater detail below.
  • FIG. 9 is a flowchart showing one example of operational steps conducted by the computer 95 to control the pipetting machine 30.
  • the computer 95 initializes with a start-up procedure. After start-up, a user is prompted at step S2 to enter parameters regarding one layer of the desired density gradient.
  • the computer 95 determines whether all data with respect to the desired gradient layers have been inputted. If more information regarding a layer needs to be inputted, then step S2 is repeated. If all information regarding the layers of the density gradient have been inputted, then operation proceeds to step S4 where the computer 95 is directed to calculate all necessary intermediate solutions necessary to produce the final density gradient.
  • step S5 the computer 95 manipulates the pipetting machine 30 to deliver appropriate liquids to one of the intermediate tubes to create an intermediate solution.
  • step S6 a decision is made determining whether or not all intermediate solutions or mixtures have been produces. If more mixtures need to be produced, step S5 is repeated. If all intermediate solutions have been produced, then step S7 is performed. At step S7, each intermediate solutions is loaded one by one, into the appropriate tube 44 to produce the density gradient.
  • step S8 a decision is made determining whether of not another identical density gradient is to be produced. If so, steps S5, S6 and S7 are repeated as necessary. Once all required tubes 44 are supplied with the requested density gradients, operation moves to step S9 where the computer 95 stops and the program ends or returns to a standby state.
  • the computer may have a graphical interface wherein the density gradient may be inputted by manipulation of a curve on a graph similar to that shown in Figure 8.
  • the computer may be pre-programmed with an index having information relating to many possible variations of intermediate solutions to be formed in the production of various density gradients.
  • the computer 95 may alternatively be pre-programmed with a plurality of density gradient profiles similar to the information displayed in Figure 8 such that a user chooses one of the plurality of density gradient profiles for automatic production of one or more density gradients delivered to the tubes 44.
  • fractionation system of the present invention may also be used for many other materials for diverse purposes including the manufacture and separation of various particles for abrasives, catalysts, pigments, fluidized bed material and regeneration thereof (particularly for chemical manufacture), wastewater treatment, clays, vaccines, proteins, DNAs and other bioactive delivery systems, magnetic particles, food products, cosmetics etc.
  • the various components of an invention of interest can be ordered at the benchtop using a rack that holds multiple containers.
  • the rack can be any horizontally oriented structure having a plurality of means for positioning individual gradient components in an upright position.
  • the means for restraining and positioning the components could be spaces to receive vessels in the structure or receptacles therein.
  • the gradient components can be arranged in particular configurations, for example based on the expected location thereof in the gradient.
  • a component forming an intermediate layer may be positioned in the rack between components that will comprise a layer above and a layer below the intermediate layer.
  • Other configurations can be contemplated and arranged to facilitate access to the components and gradient formation.
  • EXAMPLE 1 GRADIENT SET-UP
  • a method and system for preparing solutions for a making a 17 step gradient is illustrated diagrammatically in Figure 1.
  • the tubes may be either separate vessels as shown, or may be molded into one or more multiwelled plate(s). Rows 1-5 of tubes or containers are arranged so that the first row contains two tubes, the second 3, the third 5, the fourth 9, and the fifth 17, i.e., each row contains one less than double number in the preceding row.
  • the present invention may continue the process with as many rows as desired for all or part of each row to make as fine of a gradient and in whatever proportions desired. From each row liquid is pipetted from adjacent tubes to the one between in the succeeding rows to produce a mixture, and is pipetted into the one directly below without addition except as described below.
  • linear gradients suitable for fractionating rat liver homogenates may be prepared.
  • the homogenate may be fractionated into soluble phase, endoplasmic reticulum, and mitochondria by centrifuging for twenty minutes at 4°C, at a rotor speed of 20,000 rpm.
  • a non-linear complex gradient shown in Figure 8, was prepared using the method and system illustrated in Figure 2.
  • This gradient resolves the cytosol, endoplasmic reticulum, mitochondria and nuclei, using an iodinated gradient material such as Iodixanol®.
  • This system uses 5 rows 11-15 of vessels or tubes having decreasing volumes in descending rows.
  • the initial Iodixanol ® concentrations are for tube 16, 10%; 17, 15%; 18, 17.5%; 19, 20%; 20, 22.5%; 21, 27%; and 22, 50% w/v.
  • the pipetting scheme is indicated by the cross connections between the circles. Only a fraction of the tubes is required to produce a given complex gradient. This plate yields 22 gradient steps. In the instance shown, only two tubes are required in row 15.
  • the 22-step gradient is pipetted out of the vessels shown in solid black using the microprocessor-controlled pipetter.
  • Figure 8 shows the non-linear gradient produced using the plate and diagram of Figure 2 giving physical density (g/mL) as a function of step numbers.
  • the raw data is given in the following Table 1.
  • a non-linear complex gradient shown in Figure 8, was prepared using an alternative method and system from that in Example 2.
  • This system uses two parent solutions of 10% and 60% Iodixanol ® . Twenty-three tubes are used each to represent the exact 22 gradient components and one tube for the 60% cushion. The proportions of each parent solution needed to prepare each solution are calculated by a computer that controls a two-arm pipetting device (one pipette for each parent solution). Once the 22 solutions are prepared, the 22-step gradient is pipetted out of the vessels using the microprocessor-controlled pipetter with a clean pipette tip changed for each. An indistinguishable gradient producing comparable results to that of Example 3 results.

Abstract

L'invention concerne des procédés permettant de reproduire des gradients liquides présentant un haut degré de précision. Différentes régions dans le gradient sont préformées par mélange préalable de liquides utilisables pour d'autres composants du gradient de manière à former un composant de gradient intermédiaire qui est, ensuite, ajouté au réservoir. Ce système est particulièrement adapté à la fabrication dans un même liquide et dans un même réservoir d'une multitude de gradients différents non linéaires et se chevauchant. Les procédés permettant de reproduire des gradients très complexes sont illustrés dans la figure 2, les rangées (11-14) comprenant sept solutions de départ (16-22) qui augmentent en densité en ordre numérique, mais qui peuvent présenter des compositions très différentes.
PCT/US2001/016120 2000-05-19 2001-05-18 Formation de gradient fluide de precision WO2001090719A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114632435A (zh) * 2022-03-21 2022-06-17 江苏冠军科技集团股份有限公司 一种高闪点溶剂的制备装置、方法及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3519400A (en) * 1967-01-25 1970-07-07 Atomic Energy Commission Method of centrifugal separation and recovery of chemical species utilizing a liquid medium
US3863901A (en) * 1972-10-13 1975-02-04 Ceskoslovenska Akademie Ved Apparatus for mixing of liquids at an adjustable ratio
US4290300A (en) * 1978-10-18 1981-09-22 Joseph Carver Sucrose density gradient system
US5496517A (en) * 1989-12-22 1996-03-05 Beckman Instruments, Inc. Laboratory workstation using thermal vaporization control
US5922211A (en) * 1996-03-26 1999-07-13 Nees; Stephan Method for centrifugal particle separation, particularly for use in the biological sector

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3519400A (en) * 1967-01-25 1970-07-07 Atomic Energy Commission Method of centrifugal separation and recovery of chemical species utilizing a liquid medium
US3863901A (en) * 1972-10-13 1975-02-04 Ceskoslovenska Akademie Ved Apparatus for mixing of liquids at an adjustable ratio
US4290300A (en) * 1978-10-18 1981-09-22 Joseph Carver Sucrose density gradient system
US5496517A (en) * 1989-12-22 1996-03-05 Beckman Instruments, Inc. Laboratory workstation using thermal vaporization control
US5922211A (en) * 1996-03-26 1999-07-13 Nees; Stephan Method for centrifugal particle separation, particularly for use in the biological sector

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EBBING D.D.: "General chemistry", HOUGHTON MIFFLIN COM., 1984, pages 74 - 77, XP002947101 *
GUENTHER W.B.: "Density gradient columns for chemical displays", J. CHEM. ED., vol. 63, no. 2, 1986, pages 148 - 150, XP002946900 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114632435A (zh) * 2022-03-21 2022-06-17 江苏冠军科技集团股份有限公司 一种高闪点溶剂的制备装置、方法及其应用
CN114632435B (zh) * 2022-03-21 2023-02-28 江苏冠军科技集团股份有限公司 一种高闪点溶剂的制备装置、方法及其应用

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