Abstract:
A molecule separator device for isolating molecules having at least two separable properties and within a solution. The device includes a housing, and at least two molecule collection media disposed within the housing, whereby each such medium captures molecules exhibiting a respective property. In one embodiment, a first membrane captures only molecules with an ionic and/or hydrophobic and/or affinity attraction property while a second membrane captures only such molecules that additionally fall within a particular molecular weight range. A preferred housing is cylindrical for acceptance within a centrifuge, and is constructed of a plurality of releasably-connected compartments. The collection media is sequentially situated and centrifugation of the housing drives the solution through the media. Because of separation and subsequent collection in one device of molecules bearing multiple properties, the present invention permits rapid and efficient isolation of molecules and micro-particulate having a plurality of identification characteristics.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims the benefits of U.S. Provisional Patent Application Ser. No. 60/193,118 filed Mar. 30, 2000, and of U.S. Provisional Patent Application Ser. No. 60/198,529 filed Apr. 20, 2000. 
     
    
     
       STATEMENT RE: FEDERALLY SPONSORED RESEARCH/DEVELOPMENT  
         [0002]    Not Applicable  
           [0003]    BACKGROUND OF THE INVENTION  
           [0004]    This invention relates in general to the separation and capture of molecule types from a solution mixture thereof, and in particular to apparatus and methodology wherein molecules with two or more defined properties such as ionic, hydrophobic, or affinity attractions and molecular weight ranges are captured and retained first for one such property and thereafter for the additional property, with such respective collections accomplished sequentially in a single molecule separator device.  
           [0005]    One of the most important tasks performed during research and other laboratory procedures is the separation of certain components from a mixture of components such that chemical or other analysis can proceed. A usual manner of accomplishing such separations is the employment of filtration devices whereby filtrate is collected by a filter medium as a solution containing the filtrate product passes through the filter medium. The most common of filter media are filter membranes and matrices thereof whose interstices prohibit, and thus capture, particulate whose physical size is too large to pass through as part of the solute.  
           [0006]    While such filter membranes and related matrices (e.g. cloth) work well where particulate to be collected is defined only according to size and the interstices of the filter medium are adequately sized for filtrate retention, the separation of smaller particulate, as exemplified at the molecular level, requires much greater sophistication in order to accomplish separation and collection. Additionally, molecular separation many times involves the need to collect molecules that must possess at least two properties such as ionic, hydrophobic, or affinity attractions plus a limited molecular weight range. To accomplish separation and collection of such micro-particulate, multiple filtration devices must be employed where each device has a one-membrane-type filter for collecting filtrate having one defined characteristic from a solution. Once molecules are collected that possess the first desired property, the filtrate must be transferred to a second filtration device having a second one-membrane-type filter that addresses the second property and collects molecular filtrate meeting the second standard.  
           [0007]    As is thus apparent, where, for example, molecules having at least two defining characteristics are to be isolated from a solution, a user must inefficiently perform filter procedures at least two separate times using at least two separate filtration devices. In view of this now-required inefficient approach, it is a primary object of the present invention to provide a molecule separator device where molecules having a plurality of properties can be separated and collected with one separator device.  
           [0008]    Another object of the present invention to provide a molecule separator device where such molecule separation is accomplished sequentially within a single housing.  
           [0009]    Yet another object of the present invention to provide a molecule separator device where respective dedicated membrane media provide filtrate collection.  
           [0010]    Still another object of the present invention is to provide methodology for separating and capturing molecules having a plurality of properties utilizing a single separator device.  
           [0011]    These and other objects of the present invention will become apparent throughout the description thereof which now follows.  
         BRIEF SUMMARY OF THE INVENTION  
         [0012]    The present invention is a molecule separator device for separating and isolating molecules having at least two separable properties and present in a solution comprising the molecules. The separator device includes a housing for accepting pressured passage there through of the solution, and at least two molecule collection media disposed within the housing, wherein each such medium captures molecules exhibiting a respective property respectively capturable by the media. In a preferred embodiment, a first molecule-collection chromatography membrane captures and retains only molecules with an ionic, hydrophobic, or affinity attraction property while a second molecule-collection ultrafiltration membrane captures and retains additional such molecules that additionally fall within a particular molecular weight range. Conversely, these exemplary membranes can be in reverse order such that the first molecular collection membrane is an ultrafiltration membrane while the second membrane possesses the ionic, hydrophobic, or affinity attraction property. A preferred housing is generally cylindrical for operational acceptance within a generally cylindrical fixed-angle or swinging-bucket chamber of a centrifuge head, and is constructed of a plurality of liquid-tight, releasably-connected compartments in communication with each other. The collection media is situated in a sequential relationship among the compartments while centrifugation of the housing drives the solution through the media. Removing and replacing appropriate compartments during the molecule collection process permits separate and replaceable reservoir, wash, and collection sites to yield filtrate product as so chosen for further analysis, processing, or use, or for discard where a separation goal is the provision of clean solute. Because of separation and subsequent collection of molecules bearing two or more properties, the present invention permits rapid and efficient isolation of molecules and/or micro-particulate having multiple identification characteristics. 
       
    
    
     BRIEF SUMMARY OF THE DRAWINGS  
       [0013]    An illustrative and presently preferred embodiment of the invention is shown in the accompanying drawings in which:  
         [0014]    [0014]FIG. 1 is a perspective view of a first embodiment of a molecule separator device for capture or collection of molecules and/or micro-particulate;  
         [0015]    [0015]FIG. 2 is a perspective view of a separated compartment structure for the separator device of FIG. 1;  
         [0016]    [0016]FIGS. 3 a - 3   e  illustrate use of the embodiment of FIG. 1;  
         [0017]    [0017]FIG. 4 is a side perspective view of a second embodiment of a molecule separator device; and  
         [0018]    [0018]FIG. 5 a - 5   g  illustrate use of the embodiment of FIG. 4. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0019]    Referring first to FIGS. 1 and 2, a molecule separator device  10  is shown. The device  10  includes a housing  12  constructed of two releasably connected, liquid-tight, separable compartments  14 ,  16  attached to each other by conventional friction fit between adjacent compartments. Within the housing  12  are two sequentially disposed membranes  24 ,  26  for collecting filtrates. In particular, the first membrane  24  is a chromatography membrane operating as a cationic or anionic ion-exchange membrane, hydrophobic membrane, affinity membrane, or a combination thereof for attracting molecules exhibiting ionic and/or hydrophobic and/or affinity attractions. The first membrane  24  can have a porosity non-limitedly exemplified in the range of 0.1 to 10 microns and is fabricated of any appropriate microporous material including nylon, polycarbonate, polyethersulfone, glass fiber, polypropylene, polysulfone, cellulose acetate, regenerated cellulose, and mixed esters of cellulose or other polymeric material as would be recognized by a skilled artisan. The second membrane  26  preferably is anisotropic (asymmetrical) and can be fabricated of the same materials as the first while providing ultrafiltration in speaking toward molecular weight characteristics for capturing molecule filtrate. Thus, a chosen molecular weight range can be exemplified in values from about 5×10 2  to about 3×10 6  Daltons.  
         [0020]    As shown in FIG. 1, the upper compartment  14  of the housing  12  has an upper reservoir chamber  28  immediately above the first membrane  24  and a lower reservoir chamber  30  immediately below the first membrane  24 . The lower compartment  16  includes an upper chamber  32  immediately above the second membrane  26  and a fluid collection chamber  34  immediately beneath the second membrane  26 . FIG. 2 shows an independent compartment  36  attachable to the upper compartment  14  during certain washing procedures as described later. The housing  12  can be constructed of a semi-rigid material such as polypropylene or of any other plastic or polymeric material as would be evident to a skilled artisan. Likewise, housing size can be as required to provide volumetric accommodations as required for a particular task. A screw-type closure cap  38  with an aperture  40  there through closes the housing  12 . As is apparent, the housing  12  resembles the configuration of a standard centrifuge tube, thus permitting placement of the separator device  10  within a standard fixed-angle or swinging-bucket chamber (not shown) of a centrifuge head (not shown). While centrifugation is the preferred manner of pressurized force, the aperture  40  in the screw cap  38  is provided to accept a pressure nozzle such as the outlet of a hypodermic syringe (not shown) whose pressure can be applied to force the solution through the separator device  10 .  
         [0021]    A description of an exemplary operation of the separator device  10  is accompanied by the illustrations of FIGS. 3 a - 3   e . First, the upper compartment  14  and an independent compartment  36  are attached as shown in FIG. 3 a . A subject solution is placed within the upper reservoir chamber  28  of upper the compartment  14 , the cap  38  is secured in place as shown in FIG. 3 b , and the resulting unit is centrifuged (fixed angle or swinging bucket) or pressurized for as long as necessary (many times about 0.5 minute) to accomplish liquid movement through the unit. As expected, the force moves the liquid quickly through the first membrane  24  as target molecules are collected. Since this first membrane  24  has a relatively large pore size, virtually any sized molecules or micro-particulate can pass through unimpededly, and only target molecules or micro particulate with ionic, hydrophobic, or affinity attractions will be retained. Alternatively, dependent upon the properties of the passing solution, target molecules or micro-particulate may pass through the membrane while contaminant is retained. The cap  38  is removed, an appropriate buffer solution is added to the upper compartment  14  which is re-capped, and a second period of centrifugation or pressurization is completed to assure removal of any contaminants from the target molecules, while the molecules or micro-particulate remain bound to the first membrane  24 . Elution of target molecules is accomplished as the independent compartment  36  with solute therein is removed and replaced with the lower compartment  16  as shown in FIG. 3 c . The upper reservoir chamber  28  is then filled with an appropriate elution buffer to remove the target molecules from the first membrane  24  and the separator device  10  is centrifuged for several minutes as the target molecules now pass through the first membrane  24  are captured because of size by the second membrane  26 . The upper compartment  14  (FIG. 3 d ) is removed and, thereafter, the upper reservoir chamber  15  is filled with a final washing buffer and centrifuged for several minutes for product desalting and placing the target molecules in a desired buffer such as physiological saline. Finally, an independent compartment  36  (FIG. 3 e ) is placed onto the compartment  16 , and the resulting unit is inverted and centrifuged or pressurized for final product collection as the target molecules are forced from the second membrane  26  and into the independent compartment  36 .  
         [0022]    [0022]FIGS. 4 and 5 a - 5   g  show a second preferred embodiment and use of a molecule or micro-particulate separator device  50 . In particular, the separator device  50  includes a housing  52  constructed of two releasably connected, liquid-tight, separable compartments  54 ,  56 , each having one separable reservoir  53 ,  57 , with compartments  54 ,  56  and reservoirs  53 ,  57  held to each adjacent structure by conventional friction fit. Within the housing  52  are two sequentially disposed membranes  63 ,  65  for collecting two different filtrates. In particular, the first membrane  63  is anisotropic (asymmetrical) and can be fabricated of any appropriate polymeric material with ultrafiltration pore size including nylon, polycarbonate, polyethersulfone, glass fiber, polypropylene, polysulfone, cellulose acetate, regenerated cellulose, and mixed esters of cellulose or polymeric materials as would be recognized by a skilled artisan while providing ultrafiltration in speaking toward molecular weight characteristics for capturing molecule filtrate. Thus, a chosen molecular weight range can be exemplified in values from about 5×10 2  to about 3×10 6  Daltons. The second membrane  65  is a chromatography membrane operating as a cationic or anionic ion-exchange membrane, hydrophobic membrane, affinity membrane, or a combination thereof for attracting molecules exhibiting ionic and/or hydrophobic and/or affinity attractions. The second membrane  65  can have a porosity non-limitedly exemplified in the range of 0.1 to 10 microns and is also fabricated of nylon, polycarbonate, polyethersulfone, polysulfone, cellulose acetate, glass fiber, polypropylene, regenerated cellulose, and mixed esters of cellulose or other polymeric materials.  
         [0023]    As shown in FIG. 4, the upper compartment  54  of the housing  52  has an upper reservoir chamber  58  immediately above the first membrane  63  and a lower reservoir chamber  60  immediately below the first membrane  63 . The lower compartment  56  includes an upper chamber  62  immediately above the second membrane  65  and a fluid collection chamber  64  immediately beneath the second membrane  65 . The housing  52  can be constructed of a semi-rigid material such as polypropylene or of any other polymeric material as would be evident to a skilled artisan. Likewise, housing size can be as required to provide volumetric accommodations as required for a particular task. As is apparent, the housing  52  resembles the configuration of a standard centrifuge tube, thus permitting placement of the separator device  50  within a standard fixed-angle or swinging-bucket chamber (not shown) of a centrifuge head (not shown).  
         [0024]    A description of an exemplary operation of the separator device  50  is accompanied by the illustrations of FIGS. 5 a - 5   g . First, a subject solution is placed within the upper chamber  62  of the lower compartment  56  (FIG. 5 a ), the upper and lower compartments  54 ,  56  are attached as shown in FIG. 5 b , and the resulting unit is centrifuged (fixed angle or swinging bucket) for as long as necessary (many times about 0.5 minute) to accomplish liquid movement through the membrane. As expected, the centrifugal force moves the liquid quickly through the second membrane  65  as target molecules are collected. Since this second membrane  65  has a relatively large pore size, virtually any sized molecule or micro-particulate can pass through unimpededly, and only target molecules with ionic or hydrophobic or affinity attractions will be retained. Alternatively, dependent upon the properties of the passing solution, target molecules or micro-particulate may pass through the membrane while contaminant is retained. Next, an appropriate buffer solution is added to the upper chamber  62  of the lower compartment  56 , and a second centrifugation is completed to assure removal of any contaminants from the target molecules while the molecules remain bound to the second membrane  65 . The reservoir  57  is then removed and emptied, and filled with an elution buffer. Upon reassembly, the separator device  50  is inverted (FIG. 5 e ) and inserted into the centrifuge for centrifugation to remove the target molecules or micro-particulate from the second membrane  65  and capture them because of size at the first membrane  63 . Thereafter, while remaining in the now-upside down position, the lower reservoir chamber  60  is filled with an appropriate buffer to wash the target molecules free of high salt of the elution buffer while retaining the molecules at the first membrane  54 . Finally, the reservoir  53  is emptied (FIG. 5 f ), the reservoir  57  is removed and replaced with a new reservoir  57   a  (FIG. 5 g ), and the resulting unit is inverted and centrifuged for final product collection as the target molecules are forced into the reservoir  57   a . Alternatively, of course, the device  50  may be inverted at the beginning of the process such that the ultrafiltration membrane is the first contact membrane.  
         [0025]    As is apparent, the molecule separator devices above described provide rapid two-stage separations within a single, convenient, and molecular-property specific apparatus. Additionally, as recognized by the skilled artisan, there are numerous possible combinations of chromatography membranes and ultrafiltration membranes for producing unique purification results. Therefore, while an illustrative and presently preferred embodiment of the invention has been described in detail herein, it is to be understood that the inventive concepts may be otherwise variously embodied and employed and that the appended claims are intended to be construed to include such variations except insofar as limited by prior art.