Abstract:
An electrophoresis apparatus is generally disclosed for sequentially analyzing a single sample or multiple samples having one or more analytes in high or low concentrations. The apparatus comprises a relatively large-bore transport capillary which intersects with a plurality of small-bore separation capillaries. Analyte concentrators, having antibody-specific (or related affinity) chemistries, are stationed at the respective inter-sections of the transport capillary and separation capillaries to bind one or more analytes of interest. The apparatus allows the performance of two or more dimensions for the optimal separation of analytes.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention relates generally to the analysis of chemical and biological materials and, more particularly, to an improved electrophoresis apparatus which simultaneously performs multiple analyses on a plurality of analytes.  
       BACKGROUND OF THE INVENTION  
       [0002]     Electrophoresis is a known technique for separating and characterizing constituent chemical and/or biological molecules, or analytes, present in simple and complex matrices undergoing analysis. Candidate sample compounds include drugs, proteins, nucleic acids, peptides, metabolites, biopolymers and other substances which exist in simple and complex forms.  
         [0003]     Conventional systems are based on interchangeable cartridges which house a thin capillary tube equipped with an optical viewing window that cooperates with a detector. Sample solutions and other necessary fluids are placed in vials (cups) positioned beneath inlet and outlet ends of the capillary tube by means of a rotatable table.  
         [0004]     When high voltage is applied to a capillary filled with an appropriate solution and/or matrix, molecular components migrate through the tube at different rates and physically separate. The direction of migration is biased toward an electrode with a charge opposite to that of the molecules under investigation. As the molecules pass the viewing window, they are monitored by a UV or other detector which transmits an absorbance or appropriate signal to a recorder. The absorbance or appropriate values are plotted as peaks which supply analytical information in the form of electropherograms.  
         [0005]     Electrophoresis separation relies on the different migration of charged particles in an electric field. Migration speed is primarily influenced by the charge on a particle which, in turn, is determined by the pH of the buffer medium. Electric field strength and molecular size and shape of the analyte also influence migration behavior.  
         [0006]     Electrophoresis is a family of related techniques that perform high efficiency separations of large and small molecules. As one embodiment of this science, capillary electrophoresis is effective for obtaining rapid and high separations in excess of one-hundred-thousand plates/meter. Because it is a non-destructive technique, capillary electrophoresis preserves scarce physical samples and reduces consumption of reagents. A fused silica (quartz) capillary, with an inner bore diameter ranging from about 5 microns to about 200 microns and a length ranging from about 10 centimeters to about 100 centimeters, is filled with an electrically conductive fluid, or background electrolyte, which is most often a buffer. Since the column volume is only about 0.5 to about 30 microliters, the sample introduction volume is usually measured in nanoliters, picoliters and femtoliters (ideally 2% of the total volume of the column). As a consequence, the mass sensitivity of the technique is quite high.  
         [0007]     Improved instrumentation and buffer-specific chemistries now yield accurate peak migrations and precise area counts for separated analytes. But, capillary electrophoresis is still limited by concentration sensitivity.  
         [0008]     To overcome this deficiency, a series of solid-phase micro-extraction devices have been developed for selective and non-selective molecular consolidation. These devices, which are used on-line with a capillary tube, are commonly known as analyte concentrators containing affinity probes to bind target compounds. Typical embodiments are described in U.S. Pat. No. 5,202,010 which is incorporated by reference in this disclosure. Other relevant teachings are provided by U.S. Pat. No. 5,741,639 which discloses the use of molecular recognition elements; and U.S. Pat. No. 5,800,692 which discloses the use of a pre-separation membrane for concentrating a sample.  
         [0009]     Even with the advent of analyte concentrators, there is still a need to improve the sensitivity levels for samples that exist in sub-nanomolar quantities. This deficit is particularly acute in the clinical environment where early detection of a single molecule may be essential for the identification of a life-threatening disease.  
         [0010]     Known capillary electrophoresis instruments are also limited by low-throughput, i.e., the number of samples that can be analyzed in a specified period of time. U.S. Pat. No. 5,045,172, which is incorporated by reference, describes an automated, capillary-based system with increased analytical speed. The &#39;172 patent represents a significant improvement over the prior art. But, throughput is still relatively low because the instrument uses only one capillary which performs single sample analyses in approximately 30 minutes.  
         [0011]     U.S. Pat. No. 5,413,686 recognizes the need for a multi-functional analyzer using an array of capillary tubes. Like other disclosures of similar import, the &#39;686 patent focuses on samples having relatively high concentrations. There is no appreciation of the loadability and sensitivity necessary for analyzing diluted samples, or samples present at low concentrations in a variety of liquids or fluids.  
         [0012]     Based on these deficiencies, there exists an art-recognized need for an electrophoresis instrument having higher loadability, better detectability of constituent analytes, faster throughput and multi-functional capability for analyzing a plurality of components in a single sample and/or a plurality of samples with high and low concentrations using a variety of chromophores, detectors and/or pre-concentration devices.  
       OBJECTS OF THE INVENTION  
       [0013]     Accordingly, it is a general object of the present invention to provide an improved electrophoresis apparatus having at least one transport capillary, at least one separation capillary and an analyte concentrator positioned therebetween;  
         [0014]     It is another object of the present invention to provide an electrophoresis apparatus having greater operating efficiency, detectability and throughput.  
         [0015]     An additional object of the present invention is to provide a user-friendly, sample preparation step which is designed to eliminate unwanted analytes that occupy binding sites and contaminate the inner walls of capillaries or channels.  
         [0016]     A further object of the present invention is to provide an electrophoresis apparatus that can analyze multiple samples having a single constituent, or multiple constituents of a single sample.  
         [0017]     It is a further object of the present invention to provide an electrophoresis apparatus which uses more than one analyte concentrator to sequentially bind more than one analyte in a single complex matrix, or in multiple matrices of simple or complex configuration.  
         [0018]     It is yet another object of the present invention to provide an electrophoresis apparatus having enhanced loadability and sensitivity which is capable of analyzing samples present in a wide range of concentrations, including those found at low concentrations in diluted liquids or fluids with simple or complex matrices.  
         [0019]     It is a further object of the present invention to provide an electrophoresis apparatus that delivers high-throughput for screening and analyzing a wide variety of samples in multiple application areas, utilizing a single or multiple dimension separation principle or mode.  
         [0020]     Another object of the present invention is to provide an electrophoresis apparatus which uses more than one separation method to sequentially permit binding to, and elution from, an analyte concentrator to effect the separation of one or more analytes.  
         [0021]     It is another object of the present invention to provide an automated, miniaturized desk-top electrophoresis apparatus for bioanalysis and other applications.  
         [0022]     Additional objects of the present invention will be apparent to those skilled in the relevant art.  
       SUMMARY OF THE INVENTION  
       [0023]     In one aspect of the invention, a sample including a number of analytes of interest is passed through a relatively large-bore transport capillary orthogonal to a plurality of smaller-bore separation capillaries. An analyte concentrator is positioned at each intersection of the transport capillary and separation capillaries.  
         [0024]     After the sample has been passed through each of the analyte concentrators, and after the analytes of importance are captured by each concentrator matrix, a selected buffer is applied to each analyte concentrator to free the system of salts and other non-relevant components. For example, a typical buffered solution is sodium tetraborate having a pH in the range of 7.0 to 9.0. The bound analytes are then eluted from each concentrator matrix in a sequentially time-controlled fashion using an aliquot or plug of an optimal eluting solution. The process continues until each of the analytes has been removed from the concentrator matrices and passed through the detector by high resolution electrophoresis migration. To increase the sensitivity of the analytes, an additional analyte concentrator containing a chromophoric reagent may be placed in one or more of the separation capillaries to react with the analyte present in that capillary. Alternatively, the eluting solution may contain a chromophoric reagent allowing decoupling and derivatization to occur simultaneously. The derivatized analytes can then be isolated in the separation capillary.  
         [0025]     To separate and analyze multiple samples with the electrophoresis apparatus of the invention, individual separation capillaries are provided, each of which contains an analyte concentrator that enriches the analytes present in diluted solutions of low concentration. Multiple elutions are carried out in a manner similar to that performed when analyzing a single sample. Effective results can also be achieved using solutions that contain an appropriate eluting chemical and a chromophoric reagent to simultaneously elute the targeted analyte and enhance sensitivity. As with a single-sample analyzer, an extra analyte concentrator may be placed in one or more of the separation capillaries to allow on-line derivatization of analytes to achieve even further enhancement of concentration sensitivity. In addition, an extra analyte concentrator may be placed in one or more of the separation capillaries to permit biochemical reactions, such as the on-line cleavage of proteins to generate peptides.  
         [0026]     An analyte concentrator may also be used to quantify enzymatic products generated by the action of one or more pharmacological agents during a specific enzyme reaction. Furthermore, the use of an analyte concentrator coupled to a different mode of electrophoresis can be used to differentiate structurally related substances present in biological fluids or tissue specimens. For example, the identification and characterization of natural proteins from artificially-made proteins or other chemicals in serum.  
         [0027]     All reactions described above can be performed in an apparatus containing a format that includes either capillaries or channels. In addition, the migration of analytes can be accomplished by an electrical or mechanical pump.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0028]      FIG. 1  is a perspective view of the electrophoresis apparatus of the present invention;  
         [0029]      FIG. 2  is an enlarged, elevated view of a plurality of analyte concentrators stationed at the respective intersections of a large bore transport capillary and an equal plurality of small bore separation capillaries;  
         [0030]      FIG. 3  is an elevated view of a second embodiment of the present invention, showing a plurality of analyte concentrators stationed at the respective intersections of an alternative transport channel and an equal plurality of separation channels;  
         [0031]      FIG. 3A  is an enlarged view of the described intersection containing the analyte concentrator microstructure;  
         [0032]      FIG. 4  is an enlarged, elevated view of an analyte concentrator stationed at the intersection of a transport capillary and a separation capillary;  
         [0033]      FIG. 5  is an elevated view of an analyte concentrator in the form of a cross-shaped capillary;  
         [0034]      FIG. 6  is an elevated view of the electrophoresis apparatus of the present invention, showing an analyte concentrator disposed along the length of a separation capillary;  
         [0035]      FIG. 7  is a perspective view of a third embodiment of the present invention, showing a plurality of separation capillaries connected to a single outlet capillary for sequential detection; and  
         [0036]      FIG. 8  is a perspective view of a fourth embodiment of the present invention, showing a plurality of separation capillaries adapted to analyze multiple samples according to the techniques described in this specification. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0037]      FIG. 1  illustrates electrophoresis apparatus  10  of the present invention. In its elementary mode (e.g.,  FIG. 8 ), apparatus  10  performs single sample studies on chemical or biological matrices having constituents or analytes of interest. But, according to the operating principles shown and described, apparatus  10  can perform multiple analyses by detecting and measuring the presence of a plurality of analytes (for example, three). Suitable and representative analytes may include narcotics, glucose, cholesterol or pharmaceutical drugs that may be present in urine or whole blood, as well as small and large molecular weight substances having simple and complex structures.  
         [0038]     As shown in  FIG. 1 , apparatus  10  includes platform  12  having side wall  14 . Sample cup  15  is mounted laterally on side wall  14 . A large-bore (150-300 mm in length×500-2000 μm I.D.), non-selective introduction capillary  16  and large-volume (1-3 ml) analyte concentrator  17  connect sample cup  15  to a first input of valve  18  which is coupled, by capillary  20 , to waste container  22  positioned on side wall  14  adjacent to sample cup  15 . In a typical configuration, analyte concentrator  17  comprises a matrix-like assembly of the type shown in U.S. Pat. No. 5,202,010. The collective mass of the matrix is provided by large quantities of microstructures such as beads, platelets, chips, fibers, filament or the like. Individual substrates can be made from glass, plastic, ceramic or metallic compositions, and mixtures thereof. Coated or otherwise deposited onto the microstructures are immobilized analyte-specific antibodies or other affinity chemistries which are suitable for characterizing and separating particular analytes of interest. Representative antibodies include those which act against peptide hormones such as insulin, human growth hormone and erythropoietin. These antibodies are readily available from commercial vendors such as Sigma-Aldrich Co., St. Louis, Mo. and Peninsula Laboratories, Belmont, Calif.  
         [0039]     The present invention contemplates a user-friendly, sample preparation step which is designed to eliminate unwanted analytes that occupy binding sites and contaminate the inner walls of capillaries or channels. This procedure will now be described with specific reference to apparatus  10  of  FIG. 2 .  
         [0040]     A first output of valve  18  is placed in the closed position and a quantity of solution from sample cup  15  is introduced into analyte concentrator  17 . Depending on its pre-selected matrix, analyte concentrator  17  traps, in a non-specific manner, many (up to 100 or more) different analytes, including the analytes under investigation. Sample cup  15  is then replaced by a buffer container (not shown). This replacement step may be accomplished by a rotatable table mechanism of the type described in U.S. Pat. No. 5,045,172. Thereafter, a quantity of buffer is injected through analyte concentrator  17  to remove excess amounts of sample and unwanted sample components. Because valve  18  remains closed during this operation, excess and unwanted samples are passed into waste container  22 .  
         [0041]     The remainder of apparatus  10  can now be considered. A second output of valve  18  communicates with transport capillary  24  which, as shown by  FIG. 2 , intersects a plurality, here shown as three, of narrow-bore (20-75 μm) separation capillaries  28 ,  30  and  32 . Analyte concentrators  34 ,  36  and  38  are sequentially stationed at the intersections of transport capillary  24  and separation capillaries  28 ,  30  and  32  to trap or bind different analytes of interest.  
         [0042]     A first end (the left as viewed in  FIG. 1 ) of separation capillary  28  is initially placed in buffer solution cup  40 . In like manner, a first end of separation capillary  30  is placed in buffer solution cup  42 ; and a first end of separation capillary  32  is placed in buffer solution cup  44 . A major portion of separation capillaries  28 ,  30  and  32  extend in parallel over the upper surface of platform  12  through detection zone  45  where the analytes respectively present in each of the separation capillaries are identified by an otherwise conventional detector  46 . Separation capillaries  28 ,  30  and  32 , which terminate at ground connection  48 , may be secured to the upper surface of platform  12  by holders  49 . Platform  12  can also take the form of an interchangeable cartridge with pre-positioned capillaries and analyte concentrators properly secured and aligned with respect to the optical system. In yet another embodiment, best shown in  FIG. 3 , transport channel  24 A and separation channels  28 A,  30 A and  32 A, having uniform and concave shapes, can be engraved, etched or otherwise formed into a glass or plastic microchip using known lithography or other manufacturing techniques. Analyte concentrators  34 A,  36 A and  38 A are disposed at the respective intersections of transport channel  24 A and separation channels  28 A,  30 A and  32 A as previously described.  
         [0043]     When the sample preparation step is complete, valve  18  is opened to the main system and a buffer (e.g., sodium tetraborate) is passed through introduction capillary  16  and analyte concentrator  17 . At this time, the analytes of interest are released from analyte concentrator  17  using an eluting solution, along with other analyte constituents present in the sample. The analytes of interest and all the other analytes captured and released by concentrator  17  are passed through transport capillary  24  to analyte concentrators  34 ,  36  and  38  which, as described below with reference to  FIG. 3 , contain a large quantity of microstructures that are capable of binding different analytes of interest; that is, each of the analyte concentrators  34 ,  36  and  38  select and isolate a different one of the analytes under investigation. Excess amounts of sample then pass through the other end of transport capillary  24  to waste container  27 . Transport capillary  24  is subsequently washed with running buffer until unwanted substances are removed.  
         [0044]     Separation capillaries  28 ,  30  and  32  are filled hydrodynamically (pressure or vacuum) with an appropriate electrophoresis separation buffer which occupies the entire volume of the capillary or channel. Immobilized analytes on a solid support are stable for long periods of time. As a result, large numbers of analytes can be sequentially separated over time, thereby providing high throughput for the apparatus of the present invention. Separation capillary  28  is first activated by introducing a plug of an appropriate eluting buffer from cup  40  by hydrodynamic (pressure or vacuum) or electrokinetic methods to desorb or elute analytes bound to analyte concentrator  34 . The eluting buffer is immediately followed by a freshly prepared electrophoresis separation buffer present in replacement cup  40 . Then, the power supply connected to cup  40  is activated to begin the process of analyte separation.  
         [0045]     As shown in Table 1, with insulin taken as representative, a typical analysis involves the targeted analyte of interest, its corresponding antibody, an appropriate buffer and eluting solution.  
                           TABLE 1                       Antigen   Antibody   Sep. Buffer+   Eluting Solution*                   Insulin   Anti-insulin   Sodium   Magnesium Chloride           antibody   tetraborate   or Ethylene Glycol               (pH 8.5)                 +Concentrations of electrophoresis separation buffer may range from 50 mM to 200 mM.            *Elution of other antigens or haptens may require a different eluting method. Effective eluting buffers include a 2 M solution of Magnesium Chloride and a 25% solution of Ethylene Glycol.             
 
         [0046]     When the initial separation is complete, the next cycle, using separation capillary  30  and analyte concentrator  36 , is performed in a similar manner, i.e., the analyte is eluted from concentrator  36  and then separated by eletrophoresis migration in separation capillary  30 . During these operations, the power supply is connected to one analyte concentrator-separation capillary system at a time.  
         [0047]     Separated analytes are then passed sequentially to detection zone  45  where each analyte is recognized and measured by detector  46  using, for example, known UV or fluorescence techniques. In one embodiment of the present invention, a single, bi-directional detector is indexed laterally above platform  12  to detect analytes of interest in separation capillaries  28 ,  30  and  32  or separation channels  28 A,  30 A and  32 A. Other sub-assemblies could include a single, fixed detector and movable platform  12  which operates to position separation capillaries  28 ,  30  and  32  or separation channels  28 A,  30 A and  32 A beneath the detector. Multiple detectors and movable platforms configured for X, Y and Z indexing are also contemplated.  
         [0048]      FIG. 4  illustrates the location of analyte concentrator  34  stationed at the intersection of transport capillary  24  and separation capillary  28 . As shown in  FIG. 4 , and in U.S. Pat. No. 5,203,010, porous end plates or frits  50 , which permit fluid flow, are provided in transport capillary  24  and separation capillary  28  to act as barriers for holding microstructures  54  in analyte concentrator  34 .  
         [0049]     Alternatively, as shown in  FIG. 5 , analyte concentrator  55  can be fabricated by using two constricted areas with no frits at all. Analyte concentrator  55 , in the form of a cross-shaped capillary, has inner diameter  61  and  63  pre-formed in relation to inner diameter  57  of transport capillary  24  and inner diameter  59  of separation capillary  28 .  
         [0050]     Analyte concentrator capillary  55  contains a plurality of previously described microstructures  54  which are larger than inner diameters  57  and  59 . They are typically coated with non-specific chemistries such as C-18 or highly specific antibodies or antigens having an affinity for one of the analytes under investigation. Several other well-known chemistries can also be used.  
         [0051]     In the embodiment illustrated by  FIG. 5 , microstructures  54  are retained or confined in the interior of analyte concentrator  55  by making inner diameter  57  of transport capillary  24  smaller than inner diameter  61  of analyte concentrator  55 . In like manner, inner diameter  59  of separation capillary  28  is smaller than inner diameter  63  of analyte concentrator  55 . For example, inner diameters  57  and  59  may be one-quarter to one-half the size of inner diameters  61  and  63 .  
         [0052]     To increase detection sensitivity for a particular analyte, a chromophore may be added to the eluting buffer to elute and tag the bound analyte for the purpose of enhancing the ultraviolet absorptivity, fluorescence, phosphorescence, chemiluminescence or bioluminescence of the analyte as it passes through detector  46 .  
         [0053]     In an alternative technique to increase detection sensitivity, additional analyte concentrator  60  may be placed in one of separation capillaries  28 ,  30  and  32 , as shown in  FIG. 6 . Analyte concentrator  60  has a plurality of microstructures  54  coated with a chromophoric agent or antibody that binds to a portion of a chromophoric agent which increases ultraviolet absorptivity, fluorescence or phosphorescence when bound to a minute quantity of a specific analyte. Frits  62  are located at the input and output of analyte concentrator  60 , and narrow capillary  64 , which intersects with separation capillary  28 , carries a buffer to periodically cleanse microstructures  54  in analyte concentrator  60  after each analysis.  
         [0054]     An analyte tagged with a chromophoric agent is more readily identified by the apparatus of the present invention, thereby increasing the sensitivity of analyte detection by as much as 100 times or more. Many different chromophoric agents emit light when they bind a specific functional group to form a product molecule in an electronically excited state.  
         [0055]     The alternative embodiment illustrated by  FIG. 7  is similar to that shown in  FIG. 1 . But, the  FIG. 7  embodiment is different because the output ends of separation capillaries  28 ,  30  and  32  are connected to each other at the interface with a single outlet capillary  66  which cooperates with on-column detector  86  that senses ultraviolet (UV) or fluorescent energy. The exit position of outlet capillary  66  may also be connected (as shown) to off-column detector  88  which comprises an electrochemical, mass spectrometry, circular dichroism detector or nuclear magnetic resonance detector.  
         [0056]     The electrophoresis apparatus of  FIG. 7  employs multiple separation capillaries or channels for sample concentration, but only one outlet capillary for sample detection. This coordinated separation by individual capillaries may be sequentially activated and controlled by well-known electronic circuitry. Like the  FIG. 1  embodiment, preceding analytes are completely separated and detected before the next separation operation is activated.  
         [0057]     The electrophoresis apparatus of  FIG. 8  is similar to that of  FIG. 7 , but it is adapted to work with multiple samples (here, e.g., three) having a simple or complex component. There is no introduction capillary  16  or sample cup  15  as provided by embodiments of  FIG. 1  and  FIG. 7 . Separation capillaries  28 ,  30  and  32  are equipped with single analyte concentrators  34 ,  36  and  38 , respectively. Individual samples are directly and sequentially delivered to separation capillaries  28 ,  30  and  32  and the analytes of interest are captured using suitable chemistries as previously described. The capillaries may be washed with buffer until all unwanted substances are removed. Like the  FIG. 7  embodiment, separation capillaries  28 ,  30  and  32  are activated in series one after the other. When all the analytes are separated in a single capillary, the apparatus begins the next separation cycle. In each of the described embodiments, apparatus  10  provides greater efficiency and higher throughput when compared to prior art devices.  
         [0058]     Various modifications and alterations to the present invention may be appreciated based on a review of this disclosure. These changes and additions are intended to be within the scope and spirit of this invention as defined by the following claims.