Abstract:
Charged particles may undergo two different separations within a single device, without manual intervention to effect the transfer of the particles between separations. In some embodiments, the device may be a Micro-Electro-Mechanical System.

Description:
BACKGROUND 
     This invention relates generally to the analysis of charged particles and particularly to the analysis of proteins and peptides. 
     Techniques such as electrophoresis and chromatography may be used to separate charged molecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. Generally, electrophoresis is used to separate charged molecules on the basis of their movement in an electric field. Chromatography on the other hand, is used to separate molecules based on their distribution between a stationary phase and a mobile phase. 
     Polyacrylamide gel electrophoresis (PAGE) is a standard tool in the study of proteins. Generally, with PAGE, proteins and peptides are exposed to a denaturing detergent such as sodium dodecylsulfate (SDS). SDS binds proteins and peptides. As a result, the proteins/peptides unfold and take on a net negative charge. The negative charge of a given SDS treated protein/peptide is roughly proportional to its mass. An electric field is then applied which causes the negatively charged molecules to migrate through a molecular sieve created by the acrylamide gel. Smaller proteins or peptides migrate through the sieve relatively quickly whereas the largest proteins or peptides are the last to migrate, if at all. Those molecules having a mass between the two extremes will migrate in the gel according to their molecular weight. In this way, proteins that differ in mass by as little as 2% may be distinguished. 
     Polyacrylamide gel electrophoresis may be used in conjunction with other electrophoretic techniques for additional separation and characterization of proteins. For example, native proteins may be separated electrophoretically on the basis of net intrinsic charge. That is, the intrinsic charge of a protein changes with the pH of the surrounding solution. Thus, for a given protein there is a pH at which it has no net charge. At that pH, the peptide will not migrate in an electric field. Thus, when proteins in a mixture are electrophoresed in a pH gradient, each protein will migrate in the electric field until it reaches the pH at which its net charge is zero. This method of protein separation is known as isoelectric focusing (IEF). 
     Isoelectric focusing and SDS-PAGE are commonly used in sequence to separate a protein or peptide mixture first in one dimension by IEF and then in a second dimension by PAGE. Isoelectric focusing followed by SDS-PAGE is commonly referred to as 2D-PAGE. Disadvantageously, 2D-PAGE requires the use of bulky equipment. Further, the chemicals required to run 2D-PAGE separations can be expensive and potentially hazardous. Additionally, running 2D-gels can be time consuming and usually requires a skilled technician to obtain satisfactory results. Even then, results may be variable and difficult to reproduce. 
     Other separation techniques, such as Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOFMS) are available to separate polar compounds including proteins. However, MALDI-TOFMS requires a substantial investment in expensive equipment and labor. 
     Thus, there is a continuing need for improved devices and techniques to separate and characterize charged molecules including nucleic acids and peptides. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is an enlarged perspective view of a device according to some embodiments of the present invention where one or more layers have been stripped away to show various features; 
         FIG. 2  is an enlarged cross sectional view of the unmodified device of  FIG. 1  taken generally along line  2 - 2 ; 
         FIG. 3  is a second enlarged cross sectional view of the unmodified device of  FIG. 1  taken generally along line  3 - 3 ; 
         FIG. 4  is a third enlarged cross sectional view of the unmodified device of  FIG. 1  taken generally along line  4 - 4 ; 
         FIG. 5  is a block flow diagram for the separation of charged particles in two ways according to some embodiments of the present invention; 
         FIG. 6  is a top plan view of an alternate embodiment of the device of the present invention where one or more layers have been stripped away to show various features including electrodes, which are depicted by absolute charge; and 
         FIG. 7  is a top plan view of the same device as  FIG. 6  where other electrodes are depicted by absolute charge. 
     
    
    
     DETAILED DESCRIPTION 
     Referring to  FIG. 1 , a device  10  may be utilized to separate charged molecules such as proteins, peptides and nucleic acids in two different directions or dimensions. Generally, according to some embodiments of the present invention, charged molecules may be sorted and focused in a first direction by field gradient focusing. Thereafter, the molecules may be separated in a second direction by electrophoresis. Thus, according to some embodiments of the present invention, the two separation techniques may be combined such that there is little or no loss or scrambling of the charged molecules after the first separation. 
     The device  10  may be constructed according to known macro and micro scale fabrication techniques. For example, in embodiments where the device  10  is to be fabricated on the microscale, such as with Micro-Electro-Mechanical System (MEMS), complementary metal oxide silicon (CMOS) or other known semiconductor processing techniques may be utilized to form various features in and on a substrate  12 . With MEMS, electronic and micromechanical components may reside on a common substrate. Thus, according to some embodiments of the present invention, the device  10  may have circuits and MEMS components formed thereon. Further, according to some embodiments of the present invention, MEMS components may include but are not limited to microfluidic channels, reservoirs, electrodes, detectors and/or pumps. 
     The substrate  12  may be any material, object or portion thereof capable of supporting the device  10 . For example, in some embodiments of the present invention, the substrate  12  may be a semiconductor material such as silicon with or without additional layers of materials deposited thereon. Alternately, the substrate  12  may be any other material suitable for forming microfluidic channels therein such as glass, quartz, silica, polycarbonate or poly(dimethylsiloxane) (PDMS). In some embodiments, biocompatible materials such as parylene may be utilized to coat channels or other surfaces thereby minimizing absorption of charged molecules. If parylene is not utilized in a particular embodiment, the substrate  12  may be otherwise treated to minimize reaction between the substrate  12  and the particles to be sorted. 
     Referring to  FIGS. 1 ,  3  and  4 , a first channel  14  may be formed in the substrate  12  for example by etching according to known techniques. The channel  14  may be of any desired length, width, depth and shape. According to some embodiments of the present invention, the channel  14  may be elongate having two ends  16  and  18  extending toward opposing ends of the substrate  12 , although the invention is not so limited. Further, in embodiments where the channel  14  is a microfluidic channel, its width, depth and perhaps length may range from a few micrometers to a millimeter or more in dimension. As shown in  FIGS. 1 and 3 , the channel  14  is generally rectangular in shape. However, the channel  14  may be any suitable shape such as a “V” or “U” shape, although the invention is not so limited. 
     Referring to  FIGS. 1-4 , one or more sidearm or collecting channels  22  may also be formed in the substrate  12 . As with channel  14 , sidearm channels  22  may be etched according to known techniques. Alternately, in embodiments where the substrate  12  is PDMS known techniques such as soft lithography may be used to form channel  14  and sidearm channels  22  in the substrate  12 . 
     Sidearm channels  22  may be coupled to and extend from the length of the channel  14  such that they have one end  24  that opens to channel  14  and a closed end  26  remote from channel  14 . In this way, the channels  22  are in communication with channel  14 . According to some embodiments of the present invention, the channels  22  are generally perpendicular to the channel  14  and parallel to each other, although the invention is not limited in this respect. Further, the sidearm channels  22  may be evenly spaced from each other along the length of channel  14 . However, even spacing between sidearm channels  22  is not a requirement and the channels  22  may be so spaced to fit the needs of a particular application or fabrication parameters. 
     As shown in  FIGS. 1 and 2 , there are three sidearm channels  22 . However, the number of channels  22  in any particular embodiment may depend upon the desired degree of particle focusing. For example, in an application where a higher degree of resolution is required, the device  10  will have more collecting channels  22  than in an application where less resolution is necessary. Thus, the invention is not limited as to the number of collecting channels  22 . Moreover, the channels  22  may be engineered for optimal particle focusing. Accordingly, the collecting channels  22  may be any suitable length, width, depth, shape and distance from each other. As with channel  14 , collecting channels  22  may be micrometers to millimeters in any dimension and rectangular, “V” or “U” shaped as examples. 
     According to some embodiments of the present invention, sidearm channels  22  may be at least partially filled with a sieving media  28 . The sieving media may be disposed in channels  22  during device  10  fabrication. Alternately, sieving media  28  may be disposed in channels  22  at any time post device  10  fabrication. The sieving media  28  may be any media capable of forming a sieve including polyacrylamide, porous silicon, interferometrically-pattern substrates, sintered tantalum, block copolymers or photoresist, although the scope of the invention is not limited in this respect. The choice of sieving media  28  may depend upon the application for which the device  10  is to be used and/or fabrication parameters. 
     During subsequent processing, channels  14  and  22  may be covered by a second layer  20  to form closed channels  14  and  22 . Alternately, in other embodiments, the layer  20  (and any additional layers) covers at least a portion of the length of channel  22 . In this way openings (not shown) may be formed at one or both ends  24  and  26  of channels  22  such that the user of the device  10  may gain access to the channels  22 . 
     Generally, any material that is suitable for the substrate  12  may form the second layer  20 . However, substrate  12  and layer  20  are not required to be the same material in any given embodiment. For example, in some embodiments, the substrate  12  may be a semiconductor material whereas the layer  20  is a dielectric or vice versa. As such, the invention is not to be limited by the materials chosen to form the substrate  12  and layer  20 , or the manner in which they are combined. 
     Referring to  FIGS. 1-3 , reservoirs  30  and  32  may be formed at least through layer  20  into substrate  12  to couple with ends  16  and  18  respectively of channel  14 . In this way, the length of the channel  14  at each end  16  and  18  is extended by the diameter or length of the reservoirs  30  and  32 . In embodiments that include additional layers, at least a portion of reservoirs  30  and  32  will be formed through the additional layers such that the reservoirs  30  and  32  will not be completely covered, thereby allowing the user of the device  10  to access the reservoirs. As shown in  FIG. 1 , reservoirs  30  and  32  are generally circular. However, the reservoirs  30  and  32  may be any shape and depth that is suitable for the particular application in which the device  10  is to be used and/or allowed by processing parameters. 
     Referring back to  FIGS. 1-4 , the device  10  may undergo additional processing to form various electrodes in association with reservoir  30 , channel  14  and/or channels  22 . The electrodes should not substantially obstruct the sort or separation of charged particles. A first electrode  34  may be disposed within reservoir  30 . According to some embodiments, the electrode  34  may be a ground or reference electrode adapted to receive either negative or positive voltage when the device  10  is in use. For example, when negatively charged particles are to be classified, electrode  34  will be negatively charged. Alternately, where positively charged particles are to be separated according to some embodiments of the present invention, electrode  34  will be positively charged. 
     As shown in  FIGS. 1-4 , electrodes  36  are proximate to the channel ends  24  such that they extend into the channel  22 . Thus, each electrode  36   a ,  36   b  and  36   c  is separated from electrode  34  by a different distance. Moreover, according to some embodiments of the present invention, electrodes  36   a ,  36   b  and  36   c  receive a voltage such that the potential difference between electrode  34  and electrodes  36   a ,  36   b  and  36   c  differs. As such, an electric field strength gradient with respect to reference electrode  34  may be applied to a solution to cause charged particles in the solution to migrate in channel  14 . 
     In some embodiments, the applied electric field strength gradient may be positive (or negative depending upon the particles to be sorted) and linear, increasing from reservoir  30  toward reservoir  32 . However, other electric field strength gradients may be produced as well. For example, the electric field gradient may be linear for a period of time and non-linear at a different point in time. Further, the device  10  may be physically adapted to generate non-linear gradients, for example by varying the number and/or distance between electrode  34  and electrodes  36  in a nonlinear fashion. Thus, device  10  may be adapted to produce a wide variety of electric field gradients for the separation of charged particles in an electric field. 
     Although electrodes  34  and  36  are shown in the figures as being disposed in reservoir  30  and the ends  24  of channels  22  respectively, the positioning (and number) of the electrodes  34  and  36  may be varied according to design preferences and/or experimental needs. For example, electrodes  36  may be disposed in channel  14 , proximate the ends  24  of the channels  22 . Alternately, electrodes  36  may be external to the channels  14  and  22 , yet proximate thereto. Thus, the gradient electrodes  34  and  36  may be positioned on device  10  in any manner that is capable of applying a voltage or electric field gradient to a solution to cause charged particles in the solution to move through channel  14  in the direction of the electric field. 
     Further, according to some embodiments of the present invention, there is a one to one correspondence between the number of sidearm channels  22  and electrodes  36 . The scope of the invention however, is not limited in this respect and there may be any number of gradient producing electrodes  36 . In embodiments where at least some of the gradient producing electrodes  36  are proximate to or disposed in sidearm channels  22 , particles having similar mobility characteristics will focus and collect therein. 
     Still referring to  FIGS. 1-4 , one electrode  40  in an electrode pair  38  and  40  may be disposed at or near the closed end  26  of sidearm channel  22 . The other electrode  38  in the pair may be disposed in channel  14  opposite electrode  40 . In other embodiments, electrodes  38  may be disposed at or near the open end  24  of sidearm channels  22 , or they may be absent altogether. Further, in embodiments of the present invention where electrodes  36  are disposed at or near the open end  24  of collecting channels  22 , the electrodes  36  may be utilized to form a pair with electrodes  40 . Thus, embodiments of the present invention are not limited to the number and location of electrode pairs  40  and  38  or  36  so long as when an electric field is applied to a solution, charged particles in the solution are caused to migrate in the collecting channels  22 . 
     The formation of electrodes  34 ,  36 ,  38  and  40  and their corresponding leads may be achieved by various fabrication techniques as is known in the art. For example, in some embodiments, contact holes (not shown) may be etched in the layer  20  and/or substrate  12 . Thereafter, a conductive material such as gold, copper, aluminum, or titanium/platinum may fill the holes and be deposited on the substrate  12  or layer  20 . If the substrate  12  and/or layer  20  is a conductive or semiconductive material, an insulating layer may be deposited prior to the metal layer. Patterning and etching may then be carried out to form the traces of electrodes  34 ,  36 ,  38  and  40 . Reservoirs  30  and  32  and other openings such as at one or both ends  24  and  26  of the collecting channels  22  may be etched at the same time as the traces in some embodiments. This is but one example of how electrodes may be formed on device  10 . The invention should not be construed as being limited by this or any other fabrication technique. Further, the process described herein is representative and should also not be considered as limiting. That is, the various features of device  10  may be formed in any way that will achieve the desired result both on the micro and macro scale. 
     As shown in the figures, the leads to the electrodes all extend in the same direction so that they are exposed on one side of the device  10 . Other arrangements may be considered without affecting the scope of the invention. For example, leads may extend in various directions to be exposed on one or more sides of the device  10 . Further, the electrodes shown in the figures all communicate to the top surface of layer  20 . However, electrodes may, in some embodiments, be formed to communicate with the top or bottom surface of substrate  12 . Thus, the manner in which the electrodes  34 ,  36 ,  38  and  40  are formed and receive voltage are not limiting and may be directed by design choice and/or process parameters. 
     In embodiments of the present invention where electrodes  34 ,  36 ,  38  and  40  leads are formed on layer  20 , a layer  42  may be deposited on the device  10  according to known techniques to insulate the electrodes/leads. As such, in some embodiments reservoirs  30  and  32  and other openings may be subsequently formed according to known techniques such as by patterned etching. 
     The electrodes  34 ,  36 ,  38  and  40  may receive voltage from any suitable power supply. The power supply may be external or internal. Thus, the scope of the present invention is not to be limited by the manner in which voltage is supplied to the electrodes. 
     Referring to  FIG. 5 , prior to device  10  use, a sample may be prepared for loading into reservoir  30  as shown in block  50 . Generally, the sample may be suspended in a liquid such as a buffer at a given pH. However, the invention is not so limited and the sample may be prepared in any manner that will achieve the desired particle separation. Where the device  10  is used in biological applications, the sample may be a pre-purified mixture of charged particles such as nucleic acids or proteins, although the invention is not so limited. As described herein for exemplary purposes only, the mixture of particles to be sorted using device  10  are peptides and proteins. However, the device  10  may be used to sort any charged particles, biological, pre-purified or not. Further, a mixture of uncharged molecules that are individually associated with a charged carrier may be separated using device  10 . Thus, the type of particles to be separated and characterized using device  10  are not limited. 
     Channels  14  and  22  and the reservoirs  30  and  32  may be filled with a fluid, as indicated in block  50 . The fluid may be the same fluid that the sample is dissolved in, although the invention is not so limited. Accordingly, any number of fluids may used to fill the channels  14  and  22  and the reservoirs  30  and  32 . 
     Before, during or after sample loading in reservoir  30 , an electric field gradient may be applied to the solution to cause charged proteins/peptides in the sample to migrate in channel  14  as outlined in block  52 . For example, the voltage to electrodes  34  and  36   a ,  36   b  and  36   c  may be adjusted until the desired gradient is established. In this example, a positive field strength gradient is generated such that the potential difference between electrodes  34  and  36   a  is the least and the potential difference between electrodes  34  and  36   c  is the greatest; the potential difference between electrode  34  and  36   b  is there between to create a linearly increasing positive field strength gradient in channel  14 . As a result, negatively charged proteins and peptides will leave well  30  and migrate through channel  14  toward reservoir  32 . In contrast, positively charged and uncharged proteins/peptides will tend to remain in the reservoir  30 . However, if positively charged particles are to be separated, the polarity of electrodes  34  and  36  may be reversed to generate a negative electric field gradient thereby causing positively charged particles to migrate in the electric field. 
     According to some embodiments of the present invention, the potential difference between the first electrode  34  and any one of the electrodes  36  may range from about 0.1 volts (V) to about 300 V. For example, in one embodiment, the potential at electrodes  36   a ,  36   b  and  36   c  may be 25 V, 50 V and 100 V respectively. However, embodiments of the invention are not limited to voltages between 0.1 V and 300 V. That is, some embodiments may utilize voltages outside of the stated range, which may depend upon the size of the device  10  and/or the channel  14 . 
     Likewise, before, during or after sample loading in reservoir  30 , a convective fluid flow may be established in channel  14  as indicated in block  54 . For example, fluid may be moved from fluid source reservoir  32  toward reservoir  30  through the channel  14 . Generally, when charged particles electrophoresed in a voltage gradient are opposed by a convective fluid flow they will sort based on their mobility. This technique of particle sorting or separating is typically known as field gradient focusing. Thus, through the use of field gradient focusing, and under a given set of conditions, molecules having similar mobility characteristics will stop migrating or focus at a unique position in channel  14  where the forces due to the electric field gradient and convective fluid flow balance or are cancelled out. As a result, one or more bands or groups of similarly focused particles will be distributed along the length of channel  14 . 
     For example, proteins having similar charge that migrate about the same distance in channel  14  in opposition to the calculated convective fluid flow may focus at or near one of the electrodes  36   a ,  36   b  or  36   c . The proteins that focus near each electrode  36   a ,  36   b  and  36   c  will collect in the respective sidearm channel  22 . Thus, according to this example, there will be at least three groups of similarly focused proteins, one group collecting in each channel  22   a ,  22   b  and  22   c . Increasing the number of collecting channels  22  and electrodes  36  along the length of channel  14  increases the number of focusing and accumulation points, hence the resolution of the system. 
     The force of convective fluid flow is calculated to enhance focusing of charged molecules at or near the sidearm channels  22 . A conventional external pump may establish the convective flow of fluid. Alternately, in some embodiments, the convective flow of fluid may be established by a MEMS pump such as an electroosmotic pump or piezoelectric micropump. However, embodiments of the present invention should not be limited by the means for establishing convective fluid flow whether it is by pump, gravitational pull or other means. 
     Referring to  FIG. 1 , the gradient producing electrodes  36  are disposed in or proximate to the open ends  24  of channels  22 . When in this configuration, similarly focused proteins/peptides may be actively induced to collect in the open end  24  of the collecting channel  22  that is proximate to the focusing point of the charged particle. Alternately, in embodiments where the electrodes  36  are disposed in channel  14  near the open ends  24  of the channels  22 , similarly focused proteins may diffuse into the adjacent collecting channel  22  to accumulate. Nonetheless, once accumulated in a collecting channel  22 , similarly focused molecules may be prevented from diffusing through the length of the channel  22  by the sieving media  28  disposed within the channel  22 . Further, molecules accumulated in a sidearm channel  22  may try to return to the first channel  14 . However, the same forces that originally caused the molecule to enter the channel  22  cause it to reenter or remain in the same sidearm channel  22 . Because the channels  22  are physically separated the charged molecules do not move laterally between the channels  22 . 
     Molecules may be focused and then collected in sidearm channels  22  by either batch or continuous mode according to some embodiments of the present invention. During batch mode, the entire sample is loaded in reservoir  30  for separation and collection in the sidearm channels  22 . In contrast, in continuous mode, one or more samples may be continuously loaded into reservoir  30  for separation and collection in the channels  22  over a period of time. Nevertheless, in both modes the longer the first separation is allowed to run, the greater the recovery of molecules. In other words, more molecules will tend to accumulate in the sidearm channels  22  over a longer period of time. 
     After a desired length of time, field gradient focusing may be terminated such that the focused particles that have accumulated at or near the open end  24  of sidearm channels  22  may undergo further separation in the channels  22 . For example, referring to  FIG. 5 , proteins may be denatured by a detergent such as SDS and/or a reducing agent as indicated in block  56 . SDS may be infused into the sidearm or collecting channels  22 , for example by hydrodynamic pressure or gel electrophoresis, although the scope of the invention is not limited in this respect. SDS binds proteins and peptides to give the molecules a net negative charge, which is roughly proportional to mass. 
     Conventional electrophoresis by SDS-PAGE utilizes a polyacrylamine gel as a molecular sieve. Similarly, according to some embodiments of the present invention, one or more sidearm channels  22  may be filled, partly or entirely, with a sieving media  28  during device  10  fabrication. In this way, charged particles may be caused to migrate through the molecular sieve thereby sorting the particles in a second direction or dimension as indicated in block  58 . For example, when a potential is applied across electrodes  38  and  40 , the negatively charged proteins/peptides will be drawn toward the positive electrode. However, the sieve impedes the progress of the charged particles. Generally, proteins and peptides having the least molecular weight migrate the fastest through the sieve toward closed ends  26  of the channels  22 . Thereafter, proteins/peptides migrate in the channels  22  towards the closed end  26  according to their molecular weight, with the sieve impeding the larger proteins to a greater extent than smaller proteins/peptides. Thus, the proteins and peptides first sorted in the electric field gradient may be further separated in channels  22 . 
     After a given amount of time, the electric field between electrodes  38  and  40  may be removed to stop the second separation. The separated particles may be detected by any known means. For example, aliquots of eluant may be removed from channels  22  at timed intervals for further analysis. Alternately, in some embodiments the charged particles may be stained, or if radioactive, a film may be exposed. Largely, the user of the device  10  decides what technique should be used for particle detection. Thus, the scope of the present invention should not be limited in this respect. 
     Referring to  FIG. 6 , a device  110  may be utilized to simultaneously sort positively and negatively charged particles by field gradient focusing in a first dimension. Referring to  FIG. 7 , the same device  110  may thereafter be utilized to electrophoretically sort the charged particles that have focused and accumulated in the sidearm channels  22  in a second dimension, in substantially the same way as described above with respect to device  10 . In fact, the device  110  is similar to device  10  in many respects. For example, the device  110  has two halves,  112  and  114 , which in some embodiments are generally mirror images. The two halves  112  and  114  are generally mirror images in that both halves include the same sample-receiving reservoir  30  for descriptive purposes. Otherwise, the two halves  112  and  114  may be mirror images in that each half includes substantially the same structures configured in substantially the same way, allowing for some variations. Nonetheless, the two halves  112  and  114  of device  110  are not required to be substantially alike and may take on a variety of configurations, all within the scope of the present invention. 
     The first half  112 , may be configured such that it is nearly identical to any embodiment described with respect to device  10 . As shown in  FIGS. 6 and 7 , a distinction between the first half  112  of device  110  and device  10  is the presence of reservoirs  44  disposed at the distal ends  26  of the collecting channels  22 . In this way, the reservoirs  44  are in communication with the channels  22 . Because the length of the reservoirs  44  increases the length of channels  22 , the positive electrode  40  of the electrode pair  38  and  40  is disposed in reservoir  44  proximate the closest edge of the layer  20 , although the invention is not so limited. As shown in  FIGS. 6 and 7 , the electrodes  34 ,  36 ,  38 ,  40 ,  46  and  48  are schematically represented by either a (+) or (−) charge. 
     The first half  112  of device  110  may include a second electrode pair  46  and  48 . The electrode pair  46  and  48  carries a low voltage for the detection of charged particles as they emerge from the sieving media during the second electrophoretic separation. For example, as a molecule of a given molecular weight emerges from the sieving media and moves toward electrode  40 , it may be detected by a slight change in conductivity as it passes through the electric field generated by electrodes  46  and  48 . Thereafter, the molecule may be further analyzed as desired by the user of the device  110 . 
     As shown in  FIG. 7 , the electrode pairs  46  and  48  are disposed on the same side of channel  22 , proximate to the distal end  26 . In other embodiments, the electrode pairs  46  and  48  may be disposed on the other side of the channels  22  or in the reservoirs  44 . Further, as with electrodes,  34 ,  36  and  40 , the electrode pairs  46  and  48  may be connected to an edge of the device  110  via leads that communicate with the upper surface of layer  20 . Alternately, in some embodiments, the electrode pair  46  and  48  may communicate with the under surface of device  110 . Thus, the exact configuration and location of the electrode pair  46  and  48  is not limited. They may be placed anywhere that will allow an electric potential to be generated for the detection of particles as they migrate toward reservoirs  44  and that does not substantially block particle migration. Although the reservoirs  44  and detection electrodes  46  and  48  are not shown with respect to device  10 , it should be readily appreciated that device  10  could be easily adapted to include these features. 
     Referring to  FIGS. 6 and 7 , the second half  114  of device  110  may generally be the mirror image of the first half  112 . In other embodiments, the two halves  112  and  114  are not mirror images yet retain the same structural components. For example, the channels  22  of the first half  112  may extend toward one side of the device  110  whereas the channels  22  of the second half  114  may extend toward the opposing side of device  110 . Further, channels  22   a ,  22   b  and  22   c  may be spaced apart from reservoir  30  in a manner that is different from the channels  22   d ,  22   e  and  22   f . Additionally, the spacing between channels  22   a ,  22   b  and  22   c  may differ from the spacing between channels  22   d ,  22   e  and  22   f . Thus, the two halves  112  and  114  may be include the same types of structural attributes yet be configured in a number of ways to achieve the desired sorting. 
     Halves  112  and  114  may differ in the polarity of the electric field or voltage gradient generated for the implementation of field gradient focusing. Generally, the device  110  may have one or more ground electrodes  34  disposed in reservoir  30  in a manner that will not obstruct particle separation. A first voltage gradient between electrode  34   a  (or  34   b ) and electrodes  36   a ,  36   b  and  36   c  may cause a first particle type (in solution) having a first absolute charge to migrate in channel  14   a  in opposition to convective fluid flow. As such, particles of the first type will focus at various points along the length of channel  14   a  and accumulate in collecting channels  22   a ,  22   b  and  22   c  as described with respect to device  10 . 
     In some embodiments of the present invention, a second field strength gradient may be generated in channel  14   b . Note though that the two gradients are in the same direction or dimension with respect to the second electric field applied between electrodes  38  and  40 . The two voltage gradients may be generated at generally the same time or sequentially although embodiments are not so limited. The second gradient may be between electrode  34   b  (or  34   a ) and electrodes  37   a ,  37   b  and  37   c . This gradient is adapted to cause a second particle type having a second absolute charge to migrate in the second gradient in opposition to convective fluid flow. As such, particles of the second type will focus at various points along the length of channel  14   b  and accumulate in collecting channels  22   d ,  22   e  and  22   f  according to their mobility characteristics. 
     For example, a negative voltage gradient may be generated with respect to the ground  34   b  and electrodes  37   a ,  37   b  and  37   c . As shown schematically in  FIG. 6 , there is a relative increase in the negative potential and distance between the electrodes  37   a ,  37   b  and  37   c  with respect to ground  34 . Thus, when in use, positively charged proteins will migrate in the negative gradient generated in channel  14   b . A convective flow of fluid opposes the negative electric field gradient in the same manner as described with respect to device  10 . Thus, bands of proteins having similar positive mobility characteristics will focus at or about electrodes  37   a ,  37   b  and  37   c . Proteins that are similarly focused collect in an adjacent sidearm channel  22  as previously described. Accordingly, device  110  has been adapted to sort both positively and negatively charged proteins or other particles in a first dimension or direction at the same time using field gradient focusing. 
     The convective fluid flow in device  110  may be similar to that of device  10 . For example, fluid circulates from fluid source reservoirs  32   a  and  32   b  to the central reservoir  30 . Further, one or more pumps, as is known in the art, may establish and maintain the fluid flow. A pump may be a MEMS pump fabricated on the substrate  12 . Alternately, the pump may be an external pump, which may also be a MEMS pump in some embodiments. 
     In contrast to device  10 , the force or rate of fluid flow in each branch  14   a  and  14   b  of the channel  14  does not have to be the same. The flow rate in branch  14   a  may be greater or less than the flow rate of fluid in branch  14   b . In this way, each half  112  and  114  of the device  110  may be adapted to separate or sort the differently charged particles in a manner that is best suited to enhance focusing along the length of the channel  14   a  or  14   b  and proximate to the sidearm channels  22 . Flow rate in the two branches  14   a  and  14   b  may be established by utilizing two different pumps and/or providing branches  14   a    14   b  with different cross sectional areas as examples. 
     Referring to  FIG. 7 , after the negatively charged and positively charged particles have been sorted and focused in channel branch  14   a  and  14   b  respectively, the particles may undergo a second separation in the sidearm channels  22  in the same manner as described with respect to device  10 . Further, as with device  10 , the channels  22  of device  110  are filled with sieving media  28 . Thus, the focused and accumulated particles will remain at or near the openings  24  until subsequent separation. 
     Prior to subsequent separation, positively and negatively charged proteins/peptides may be treated with SDS and/or a reducing agent as described with respect to device  10 . Consequently, all proteins will carry a net negative charge that is roughly proportional to their mass. Thereafter, the groups of proteins in each channel  22  are electrophoresed through the molecular sieve  28  as described with respect to device  10 . Thus, as shown in  FIGS. 6 and 7 , up to six groups or bands of proteins may undergo a second sort via electrophoresis; each group electrophoretically separated according to molecular weight in one of the channels  22   a ,  22   b ,  22   c ,  22   d ,  22   e  or  22   f.    
     During or after electrophoretic separation, protein bands may be detected by any desired means. With respect to the device  110 , bands of proteins may be detected in each channel  22  as they pass through the conductivity detector (electrodes  46  and  48 ) disposed in the distal end  26  of the channels  22 . Thereafter, protein bands may be collected for further study. As such, in some embodiments, reservoirs  44  may be accessible to the device  110  user. 
     The device  110  may be fabricated in generally the same way as device  10 , accounting for additional features such as channels, electrodes and a third reservoir. Generally, channel  14 , sidearm channels  22  and reservoirs  30 ,  32  and  44  are formed in substrate  12 . A second layer  20  may cover channel  14  and channels  22 . In contrast, reservoirs  30 ,  32  and  34  may be formed through layer  20  and any other additional layers. Electrodes  34 ,  36 ,  37 ,  38  and  40  (and optionally  46  and  48 ), and associated leads may be formed during additional processing of device  110 . The electrodes may be disposed in any manner that will achieve the desired electric field so long as particle separation is not obstructed. Further, if disposed within the reservoir  30  or  44 , channel  14  or sidearm channel  22 , the depth to which the electrode extends may be one of choice and/or of processing parameters. In embodiments where the electrodes/leads are formed on the surface of layer  20 , a top layer  42  (not shown) may cover the leads. 
     While the present invention has been described with respect to a limited number of embodiments, those skilled in the art will appreciate numerous modifications and variations therefrom. It is intended that the appended claims cover all such modifications and variations as fall within the true spirit and scope of this present invention.