Abstract:
Analysis of complex media (e.g.—blood and seawater) is difficult because the media are composed of particles of different sizes and chemical profiles. Disclosed is a method for the detection of a constituent in a medium that enhances the molecular selectivity of a detector by separating the detector from the medium by a membrane of specified permeability. Proxy reporters are employed to enhance particle specificity. The novel combination of the invention has application to chemical detection in a broad range of fields.

Description:
CROSS-REFERENCE TO RELATED DISCLOSURES 
     This application claims priority to a provisional application filed Aug. 11, 2006, bearing application No. 60/822,143. 
    
    
     FIELD OF INVENTION 
     This invention relates to analytical detection of molecules within a sample, and more specifically, to a novel analysis system that quantifies the presence of a molecule by proxy. 
     BACKGROUND OF THE INVENTION 
     A complex medium is a system that has a large number of distinct chemical components. Particles in a complex medium vary widely in size and chemical profile. These media do not readily lend themselves to chemical analysis of specific constituent of group of constituents because the components of a complex medium are frequently difficult to separate. Examples of complex media include blood, seawater and crude petroleum. 
     There is a need for an efficient method of assaying a sample of a complex medium to determine the presence of a particular constituent. Efficient assaying of complex media will enhance chemical detection across a broad range of fields. For example, the medical practitioner will assess the presence of toxins in blood or sera with greater accuracy and specificity. The environmental practitioner will assess the presence of pollutants in a body of water with greater speed and resolution. 
     The present invention is a method that adds a tag to a constituent particle, separates the tag from the particle, passes the tag through a membrane, and then analyzes the collection of tags. The invention is distinct from the prior art as it passes tags through a membrane designed to accommodate the measurement of particles of disparate sizes and chemical profiles. The invention offers greater selectivity than the prior art because it allows the researcher to more narrowly focus molecular recognition by manipulating the membrane permeability and mass selectivity 
     A tag is an identifying marker that can be attached to and removed from a molecule without permanently altering the molecule. Tags are used to help identify a target molecule because it may be easier to detect and manipulate the tags than to detect and manipulate the target particle itself. 
     A membrane is a barrier that separates two phases and delineates the molecules that can pass between the two phases. The use of a membrane may conserve resources that the researcher would have expended using conventional methods of separation, e.g., distillation. Membrane structures vary according to the nature of the reactants and the properties of the molecules that are selected to pass between phases. Membranes may be homogenous or heterogenous, symmetric or asymmetric and solid or liquid. The researcher can charge the membrane, render it bipolar or neutral. Method of transport varies as well. The researcher can select a membrane that uses a charge, temperature or pressure gradient. 
     A detector is any apparatus capable of registering and/or quantifying the presence of a target. 
     SUMMARY OF INVENTION 
     An embodiment of the invention includes the steps of preselecting proxy reporters that bind to target molecules, introducing the reporters to a fluid sample, the reporters provided in a sufficient concentration to saturate all available target molecules, purifying the sample to remove excess reporters, unbinding the remaining reporters in the sample from their respective molecules, providing a membrane selectively permeable to the unbound reporters, migrating the unbound reporters across the membrane leaving the fluid sample substantially unaltered, and quantifying the unbound reporters that migrated across the membrane whereby the quantification correlates to the presence of target molecules in the sample. 
     The quantification may be performed by an analytical detector including, but not limited to optical detectors, electrochemical detectors and mass spectrometers. The unbinding step may be effectuated by any suitable method known in the art including photocleaving, chemical reaction, antibody recognition, molecular imprinted polymer reaction, oligonucleotide linkage and metal affinity reaction. 
     An alternative embodiment of the invention includes the steps of preselecting a first proxy reporter that binds to a first target molecule, preselecting a second proxy reporter that binds to a second target molecule, introducing the first and second proxy reporters to a fluid sample, the reporters provided in a sufficient concentrations to saturate all available target molecules, purifying the sample to remove excess reporters, unbinding the remaining reporters in the sample from their respective molecules, providing a membrane selectively permeable to the unbound reporters whereby the first proxy reporter migrates through the membrane at a first rate and the second proxy reporter migrates through the membrane at a second rate, migrating the unbound reporters across the membrane leaving the fluid sample substantially unaltered, and quantifying the unbound reporters that migrated across the membrane on a temporal basis whereby the quantification correlates unbound reporters migrating through the membrane at the first rate with the presence of the first target molecule in the sample and the unbound reporters migrating through the membrane at the second rate with the presence of the second target molecule in the sample. The first and second proxy reporters may be variable mass units, the first proxy reporter having a mass value quantitatively distinct from a mass value of the second proxy reporter. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which: 
         FIG. 1  is a diagrammatic view of a fluid sample containing three types of molecules. 
         FIG. 2  is a diagrammatic view of proxy reporters introduced to the fluid sample and binding to their target molecule type. 
         FIG. 3  is a diagrammatic view of a fluid sample purified to remove excess reporters. 
         FIG. 4  is a diagrammatic view of a fluid sample exposed to light to photocleave the reporters from the target molecules. 
         FIG. 5  is a diagrammatic view of a fluid sample in a first phase separated by a selectively permeable membrane from a second phase. 
         FIG. 6  is a diagrammatic view of a fluid sample showing the migration of unbound reporters across the membrane from the first phase to the second phase. 
         FIG. 7  is a diagrammatic view of a fluid sample wherein two types of proxy reporters bind to their respective target molecules and excess proxy reporters remain in the sample unbound. 
         FIG. 8  is a diagrammatic view of a fluid sample exposed to light to photocleave the bound proxy reporters from their respective target molecules. 
         FIG. 9  is a diagrammatic view of a fluid sample showing a first type of proxy reporters have migrated across the selectively permeable membrane at a first time interval. 
         FIG. 10  is a diagrammatic view of a fluid sample showing a second type of proxy reporters have migrated across the selectively permeable membrane at a second time interval. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The invention discloses a method for analyzing and detecting specified molecules using separable tags where the detector is separated from the sample by a barrier membrane that allows said tags to pass but prevents said sample molecules from passing. The invention discloses the attachment of a removable tag, probe, marker or other identifier to a molecular, chemical or cellular target. In a preferred embodiment, this tag is a variable mass unit tag. A variable mass unit tag is a tag that can be shaped to specification because the variable mass unit can be manipulated to alter the molecular weight of the tag. For example, a complex medium is composed of particles between 500 Daltons and 900 Daltons. The membrane chosen is permeable only to particles with less mass than 400 Daltons. A tag of about 350 Daltons will penetrate the membrane. The membrane is impermeable to the remaining molecules, including those of the target constituent. In an alternative embodiment, the tag is a proxy-reporter probe. In an alternative embodiment, the tag is a membrane transporter. In an alternative embodiment, the tag is a tag of low molecular weight. These embodiments are merely exemplary and are not intended to limit the scope of the claimed invention. 
     The invention discloses the attachment of a removable tag, probe, marker or other identifier to a molecular, chemical or cellular target. In a preferred embodiment, this tag is photocleavable or photolabile. The Masscode™ genotyping system developed by Quiagen is an exemplary system of attaching tags. In an alternative embodiment, the tag is attached and separated from the target molecule through a chemical reaction. In an alternative embodiment, the tag attaches to and separates from the target molecule using an antibody recognition mechanism. In an alternative embodiment, the tag attaches to and separates from the target molecule using a molecular imprinted polymer. In an alternative embodiment, the tag attaches to the target molecule using an oligonucleotide linkage. In an alternative embodiment, the tag attaches to and separates from the target molecule through metal affinity. 
     The invention discloses the use of a membrane to separate the detector from the complex medium. An appropriate membrane is selected based on parameters known to one skilled in the art. Membrane permeability is selected that is compatible with both the tags that are intended to pass through the membrane and the particles from the medium that are not intended to pass through the membrane. Material of the membrane is selected to be compatible with the medium under examination. 
     In an alternative embodiment, more than one target constituent is examined. Parameters known to those skilled in the art are used to determine a membrane configuration and tag mass that will allow for a temporal separation as well as a physical separation among the target constituents. Two molecular tags of different mass will pass through the membrane at different rates. The difference in the mass between the tags corresponds to the difference in the rate of passage across the membrane. The greater the mass, the slower the passage across the membrane. 
     The invention discloses the use of an analytical detector to ascertain details about a sample. In one embodiment, the detector is an optical detector. In another embodiment, the detector is an electrochemical detector. In a preferred embodiment the detector is a mass spectrometer. 
     Turning now to  FIG. 1 , first phase  10  is a fluid sample containing molecules  20 ,  30  and  40 . Molecules  20 - 40  may include non-organic and/or organic matter such as cellular structures or cells themselves. In  FIG. 2 , proxy reporter  50  is introduced  60  into first phase  10 . It can be seen that proxy reporter  50  only binds to molecule  40  and that a sufficient quantity of proxy reporter  50  is provided to the fluid sample to saturate all possible target molecules. Accordingly, the fluid sample would necessarily have at least some excess and unbound proxy reporter  50 . 
     In  FIG. 3 , excess and unbound proxy reporter  50  is removed  70  from fluid sample whereby a one-to-one relationship remains between bound proxy reporter  50  and molecule  40 . In  FIG. 4 , light  80  photocleaves proxy reporter  50  from molecule  40 . The fluid sample still maintains a one-to-one relationship between (now) unbound proxy reporter  50  and molecule  40 . In  FIG. 5 , first phase  10  is separated from second phase  100  by selectively permeable membrane  90 . In the current exemplary embodiment, selectively permeable membrane  90  permits the migration of proxy reporter  50  but does not permit molecules  20 - 40  to pass. Coupled to second phase  100  is analytical detector  110 . 
     In  FIG. 6 , proxy reporters  50  have migrated from first phase  10 , across selectively permeable membrane  90 , to second phase  100 . It should be noted that the fluid sample in first phase  10  is substantially unaltered from its original state. Proxy reporters  50  are quantified by analytical detector  110  which directly correlates to the quantity of molecule  40  in the fluid sample. The ability to quantify the presence and/or concentration of a molecule in a fluid sample without substantially altering the original fluid sample has significant utility for a large number of diagnostic, detection and monitoring applications. 
     In  FIG. 7 , first proxy reporter  50  binds to molecule  40  as previously illustrated in  FIGS. 2-3 . In addition, second proxy reporter  120  binds to molecule  120 . In an embodiment of the invention, first proxy reporter  50  and second proxy reporter  120  have differing masses. Similar to that shown in  FIG. 2 ,  FIG. 7  shows excess proxy reporters that are unbound to their respective target molecules. This insures that the one-to-one correlation between proxy reporter and target molecule constitutes an accurate assumption. 
     In  FIG. 8 , excess and unbound first proxy reporter  50  and second proxy reporter  120  have already been removed from the fluid sample. Light  80  photocleaves first proxy reporter  80  from molecule  40  and second proxy reporter  120  from molecule  20 . 
     In  FIG. 9 , small massed proxy reporter  50  crosses from first phase  10  to second phase  100  across selectively permeable membrane during time interval x. Analytical detector  110  quantifies proxy reporter  50  resident in second phase  100  to provide a one-to-one correlation to molecule  40 . In  FIG. 10 , larger massed proxy reporter  120  crosses from first phase  10  to second phase  100  across selectively permeable membrane during time interval y. The larger mass of proxy reporter  120  moves more slowly through selectively permeable membrane  90  providing a temporal analysis for analytical detector  110  to quantify the proxy reporter  120  resident in second phase  100 . As noted in the previous example above, fluid sample in first phase  10  is returned to its original state. 
     It will be seen that the advantages set forth above, and those made apparent from the foregoing description, are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. 
     It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween. Now that the invention has been described,