A. Field of the Invention
The present invention relates to an apparatus and method for making a multiple density layers or gradients of fluid in a vessel in a highly reproducible manner using a float that floats on the surface of the fluid within the vessel.
B. Description of the Related Art
There are various fields where it is desirable to have density layers or gradients of fluid within a vessel for such purposes as the separation of matter, determining density, etc. Such density layers include, for example, a solution retained in a vessel where the fluid is divided into a plurality of layers, each layer having differing concentrations of a soluble material or solute. For example, a bottom or first layer of fluid may have a concentration of a solute that is X moles per liter; a second layer immediately above the first layer may have a concentration of 0.8X moles per liter; a third layer above the second layer may have a concentration of 0.6X moles per liter; and a fourth layer having a concentration of 0.4X moles per liter.
Liquids having gradients of temperature, concentration, density and color have been previously prepared. Liquid density gradients have been used for many years, for a wide 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. Anal. Biochem. 3: 472-478, 1962; Anderson, N. G., and Rutenberg, E. Analytical techniques for cell fractions. A simple gradient-forming apparatus. Anal. Biochem. 21: 259-265, 1967; Candler, E. L., Nunley, C. E., and Anderson, N. G. Analytical techniques for cell fractions. VI. Multiple gradient-distributing rotor (B-XXI). Anal. Biochem. 21: 253-258, 1967.
A variety of other methods for making density gradients have been developed, and Bock, R. M. and Ling, N.-S., Anal. Chem. 26, 1543, 1954, and Morris, C. J. O. R, and Morris, P., Separation Methods in Biochemistry, Pitman Publishing, 2nd ed. (1976) have reviewed many of these. Only one of these methods allowed gradients to be made from multiple solutions, each having a different combination of reagents (Anderson, et al, xe2x80x9cAnalytical Techniques for Cell Fractions. I. Simplified Gradient Elution Programmingxe2x80x9d, Analytical Biochemistry 3: 472-478, 1962) More recent innovations include the use of pumps and pistons, which are differentially controlled by microprocessors, e.g., the Angelique gradient maker (Large Scale Proteomics Corp. Rockville, Md.). Gradients may also be generated during high speed centrifugation by sedimenting a gradient solute such as cesium chloride or an iodinated x-ray contrast medium such as iodixanol. Gradients may be initially prepared as step gradients and linearized by diffusion, by gentle mixing, or by freezing and thawing. A list of references covering existing methods follows.
Density gradients are used to make two basic types of separations. The first separates particles on the basis of sedimentation rate (rate-zonal centrifugation), in which case particles are separated on the basis of the size and density (and to a lesser extent shape) and particles will sediment farther if centrifuged for a longer period of time. The second separates particles on the basis of isopycnic banding density, in which case particles reach their equilibrium density level, and do not sediment farther with continued centrifugation.
Four types of gradients are in general use with either of these basic methods. The first includes step gradients, made by layering a series of solutions of decreasing density (if the solutions are introduced one above the other), and of increasing density (if the solutions are introduced sequentially to the bottom of the tube). The second type comprises linear continuous gradients usually made by a mechanical gradient maker. These are usually introduced slowly through small tubing to the bottom of the centrifuge tube. Linear gradients for either rate zonal or isopycnic zonal centrifugation are useful for resolving very heterogeneous mixtures of particles.
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 gradient may be designed to separate particles on the basis of both sedimentation rate and isopycnic banding density in the same gradient, in which case some particles reach their isopycnic level at some point in the gradient, while others are still sedimenting. Generally 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 generated in a high centrifugal field by sedimentation of the major gradient solute, and is usually used for isopycnic banding.
Many reasons exist for desiring to control gradient shape. Gradient capacity (i.e., the mass of particles which can exist in a zone without causing a density inversion) 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 is 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, where the highest gradient capacity is required.
An additional reason for desiring to control gradient shape is that when a population of particles is present that differ little in sedimentation rate, these can best be separated by sedimentation through a longer shallower section of the gradient. Such shallow sections are usually near the center of a gradient.
In the majority of density gradient separations, 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. For example, traces of divalent cations are required to control nuclear swelling, whereas such ions are generally deleterious to other subcellular particles. Low concentrations of nonionic detergents remove cytoplasmic contamination from nuclei, but are deleterious to the endoplasmic reticulum. Hence there has been no one procedure or gradient that has been optimized for the systematic separation of the majority of all subcellular particles. There is a need for reproducible means for including in gradients zones containing salts, detergents, enzymes and other reactive substances that would increase the number of different subcellular particles separated in one gradient.
Density gradient separations are important in proteomics research. High resolution two-dimensional electrophoresis (2DE) is widely used to produce global maps of the proteins in extracts prepared by solubilizing whole cells or tissues. By careful control of the procedures employed, use of staining procedures which are quantitative, and computerized image analysis and data reduction, quantitative differences in the abundance of individual proteins of xc2x115% has been achieved (Anderson, N. Leigh, Nance, Sharron L., Tollaksen, Sandra L., Giere, Frederic A., and Anderson, Norman G., Quantitative reproducibility of measurements from Coomassie Blue-stained two-dimensional gels: Analysis of mouse liver protein patterns and a comparison of BALB/c and C57 strains. Electrophoresis 6: 592-599, 1985; Anderson, N. Leigh, Hofmann, Jean-Paul, Gemmell, Anne, and Taylor, John, Global approaches to quantitative analysis of gene-expression patterns observed by use of two-dimensional gel electrophoresis. Clin. Chem. 30: 2031-2036, 1984). There is a need for precision subcellular fractionation that will allow changes in abundance of minor proteins to be accurately detected and measured in data which sums the abundance of all proteins found in all of the fractions of one sample.
This technology allows changes in gene expression, as reflected in protein abundance, to be studied under a wide range of conditions, and has led to the development of databases of protein abundance changes in response to a wide variety of drugs, toxic agents, disease states. In such studies large sets of data must be acquired and intercompared. Hence all stages in one pharmaceutical study, for example, must be standardized for inter comparability.
2DE maps of whole cells or tissues typically contain a thousand or more protein spots in sufficient abundance to allow each protein to be analyzed by mass spectrometry and identified and characterized. However, it is known that a very much larger number of proteins are actually present in tissue samples analyzed than are actually observed. The number present varies with cell or tissue type, and is believed to be up to ten or twenty times the number detected.
Different subcellular particles and the soluble fraction of the cell (the cytosol) contain many location-specific proteins which constitute only trace fractions of the total cell protein mass. Hence the total number of proteins resolved from one cell type or tissue could be greatly increased if the 2DE analysis were done on cell fractions rather than on whole cell or tissue extracts as has previously been demonstrated (Anderson, N. L., Giere, F. A., et al, Affects of toxic agents at the protein level: Quantitative measurements of 213 mouse liver proteins following xenobiotic treatment. Fundamental and Appl. Tox. 8: 39-50, 1987). If a drug effect study is to be done on cell fractions, however, the fractionation procedures must be quantitative, in the sense that the same organelles, or even mixtures of organelles are used in all analyses to be intercompared. There exists, therefore, an emerging need for high resolution density gradient separations using precision gradients in proteomics research. Making precision gradients reproducibly and in parallel has proven to be difficult, particularly when the gradients are shallow.
The protein composition of tissues such as liver varies diurnally, hence all the tissues from one group of animals 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. Further, gradient fraction recovery must also 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 be isopycnically banded, as is done in two-dimensional or s-xcfx81 fractionation, then these subsequent steps must also be carried out in parallel. This, in turn, requires that the gradients be made in parallel.
Precision gradients are difficult to make in practice, and it is further difficult to confirm that a set of gradients are all identical without destroying them for analysis. Existing swinging bucket rotors generally allow six gradients to be centrifuged simultaneously. Larger numbers may be centrifuged if the lower resolution of vertical or near vertical tube rotors is accepted. Therefore if existing density gradient formers are to be used, a set of six or more of them operating in parallel will be required.
With any gradient maker, small amounts of turbulence or non-laminar flow typically cause solutions of differing concentrations to at least partially mix, thereby reducing the effectiveness and usefulness of the density layers. There is therefore a need for a method for decelerating fluids flowing into a tube, and for moving them slowly into position to form distinct bands.
One of many uses of density layers and gradients is in the fields of cell separation, sub-cellular fractionation and analysis, and density gradient methods are used in molecular biology and in polymer chemistry. Little attention has been paid to forming sets of precision-made gradients that are highly reproducible for cell separation. There is therefore a requirement for precision gradients adapted to cell separation.
One high resolution system is disclosed in xe2x80x9cDevelopment of Zonal Centrifugesxe2x80x9d, by N. G. Anderson, National Cancer Inst. Monograph 21, 1966) and employs zonal centrifuge rotors. The rotors are of high capacity, and process one sample at a time. However, the rotor volumes are too high for many applications. Angle head or vertical rotor tubes may also be employed (Sheeler, P., Centrifugation in Biology and Medicine, Wiley Interscience, N.Y., 1981, 269pp) using either step or continuous gradients. However these do not provide the resolution obtained with swinging bucket rotors.
There has been no reliable method for reproducibly locating and recovering organelle zones purely on the basis of the physical parameters of sedimentation rate and isopycnic banding density. Mathematical analyses, based on analysis not only of the biological particles separated, but of the gradients themselves have been required. These have been tedious and idiosyncratic to the rotors and conditions employed. The basic problem in preparing density gradients in tubes is that the liquid volume elements of either step (layers), or continuous gradients must be introduced into tubes very slowly or mixing will occur. This problem is only partially overcome by introducing the gradient into a set of tubes in an angle-head rotor during rotation.
Methods for producing one or a few gradients in parallel have been developed, but fraction recovery is generally done one at a time. The gradients are rarely identical, and it is difficult to introduce the sample layer on top of the gradient without mixing. Hence there is no published data on the quantitative high-resolution protein analysis of cell fractions of animals subject to various experimental treatments. If multiple, parallel identical gradients are to be prepared using gradient engines (for instance, see xe2x80x9cMechanical device for producing density gradients in liquidsxe2x80x9d by N. G. Anderson,. Rev. Sci. Instruments 26: 891-892, 1955) one must have one machine for each tube being filled. Centrifugal gradient distributing heads have been built (see xe2x80x9cA Method For Rapid Fractionation of Particulate Systems by Gradient Differential Centrifugationxe2x80x9d by J. F. Albright, and N. G. Anderson, Exptl. Cell Research 15: 271-281, 1958), however the gradients actually produced tend to be uneven, and a refrigerated centrifuge is required. There is, therefore, a continuing need for simple gradient makers that produce identical gradients in parallel in sufficient number to satisfy current requirements. There is a further need for a simple, disposable and easily sterilizable system for making reproducibly sharp step gradients. An additional need exists for a system or device that can produce very narrow-step density gradients in which diffusion can rapidly and reproducibly even out the steps. A further need exists for a system or device which allows individual gradient steps to be rapidly pipetted into centrifuge tubes, either manually or robotically, and in which the introduced fluid does not disturb the underlying gradient. A still further need exists for a gradient making device in which the composition of the successive layers, while forming a stable density series, differ in composition relative to salts, enzymes, detergents or other reactive materials.
One object of the present invention is to provide a rapid, simple and reproducible method and apparatus for forming a multiplicity of liquid density gradients in vessels.
Another object of the present invention is to provide a rapid, simple and reproducible method and apparatus for forming a multiplicity of liquid density gradients in vessels for rate-zonal separations, for isopycnic banding separations, or a combination of the two.
Yet another object of the present invention is to provide an apparatus and method for reproducibly producing a plurality of liquid density gradients in a plurality of corresponding vessels, each vessel having a specific predetermined liquid density gradient.
An additional object of the present invention is to provide means for making liquid density gradients in which aliquots of a liquid density series are rapidly pipetted into the centrifuge tubes without regard to potential stirring or mixing.
A further object of the invention is to decelerate the aliquots ejected from pipettes or automatic pipetters, and to cause them to flow evenly into position without disturbing the underlying fluids.
A further object of the present invention is to provide means for making the linear or complex gradients by making them initially as step gradients having very small density differences per step.
A further object of the present invention is to produce step gradients in which the steps are so small that diffusion rapidly evens out the gradient.
A still further object of the present invention is to make the gradient making components disposable and easily sterilizable.
It is a further object of the present invention to make possible construction of sets of identical gradients in a short period of time.
It is an additional object of the present invention to make possible addition of the sample layer on top of the gradient at any time after the gradient is formed.
In accordance with one aspect of the present invention, there is a method for producing liquid density gradients in a vessel using a float within the vessel includes the steps of:
inserting the float in the vessel;
introducing a first liquid into the vessel;
introducing a second liquid into the vessel such that the second liquid contacts at least one surface of the float upon entering the vessel, contact between surfaces of the float and the second liquid allowing the second liquid to form a layer above the first liquid thereby forming separate layers of liquid; and
repeating the second introducing step with successive introducing steps with a third, fourth and so on liquid.
The float used in the above method slows the velocity of fluid such that flow of liquid is laminar thereby limiting mixing of the two liquids.
In accordance with another aspect of the present invention, an apparatus for producing liquid density gradients includes a vessel and a float positionable in the vessel. The float is formed with at least one surface that is shaped to inhibit acceleration of fluid introduced into the vessel thereby restricting turbulent flow of the fluid.
An outer peripheral surface of the float and the inner surface of the vessel are sized such that in response to fluid being introduced into the vessel above the float, the fluid undergoes capillary action moving downward beneath the float in the vessel.