Patent Publication Number: US-6660493-B2

Title: Hydrodynamic enhanced dielectrophoretic particle trapping

Description:
The United States Government has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of Lawrence Livermore National Laboratory. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to particle trapping, particularly to trapping of DNA and cells/spores using dielectrophoretic forces, and more particularly to hydrodynamic enhanced dielectrophoretic particle trapping by introducing a side stream of fluid into the main stream of fluid containing particles for forcing the particles closer to electrodes producing the dielectrophoretic forces. 
     Trapping of DNA and cells/spores using dielectrophoretic (DEP) forces is being considered for performing sample preparation protocols for polymerized chain reaction (PCR) based assays for counter biological warfare applications, as well as for a clinical tool to determine genetic information and other medical applications. A key element of the sample preparation process is to enable controlled concentration and/or movement of DNA, for example, prior to detection. DEP forces are strongest near the electrodes which create manipulating fields. The region of effective force is less than 100 μm from the electrodes. Small channels manufactured to bring the fluid containing the particles close to the electrodes have been considered, but this enhances the probability of clogging the small channels, since biological materials are very sticky and plug channels easily. 
     The present invention solves the problem by introducing a side stream into the main stream to force or squeeze the fluid containing particles close to the electrodes such that the particles would be affected by the DEP forces, but would allow for a relatively open or larger channel to prevent clogging. The invention utilizes a series of electrodes located along a length of an electrophoretic channel. Since DEP forces induce a dipole in the sample particles, these particles can be trapped in non-uniform fields located along the channel, and which are produced by the electrodes. Thus, the present invention provides for hydrodynamic enhanced dielectrophoretic particle trapping. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide enhanced particle trapping using dielectrophoretic forces. 
     A further object of the invention is to provide hydrodynamic enhanced dielectrophoretic particle trapping. 
     Another object of the invention is to provide enhanced dielectrophoretic particle trapping by forcing the particle containing fluid close to electrodes which produce the dielectrophoretic forces. 
     Another object of the invention is to provide hydrodynamic enhanced dielectrophoretic particle trappings by introducing a side stream into the main particle containing stream to squeeze the main stream close to electrodes which produce dielectrophoretic forces such that the particles are affected by the dielectrophoretic forces thereby enhancing particle trapping. 
     Other objects and advantages of the present invention will become apparent from the following description and accompanying drawings. Basically, the present invention provides for trapping of particles using dielectrophoretic (DEP) forces. More specifically the invention involves a method and apparatus for hydrodynamic enhanced DEP particle trapping. This is accomplished by the use of side stream flows to direct main stream flows. Since DEP forces are effective only very close to the electrodes (less than 100 μm), it is important to direct the cells and DNA close to the electrodes. This is accomplished by the invention by using side stream flows. Use of side stream flows in lieu of making smaller channels reduces the chance of blockage of the flow channels, which is very common in biosystems. The apparatus of the invention includes a series of electrodes, which may be photolithographically patterned along the side of a sample flow or fluidic channel, with an AC field placed between pairs of electrodes. The AC field induces a dipole in the DNA or cell or spore which at certain frequencies, traps the particles along the edges of the electrodes. The sample or incoming flow stream containing the cells and DNA is forced close to the electrodes using a side stream flow, which improves the efficiency of DEP trapping. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The accompanying drawings, which are incorporated into and form a part of the disclosure, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. 
     FIG. 1 is a top diagrammatic view of an embodiment of a sample preparation/assay system utilizing hydrodynamic enhance dielectrophoretic particle trapping in accordance with the present invention. 
     FIG. 2 is a side view of a portion of the FIG. 1 system. 
     FIG. 3 is a top view of a fluidic channel in which is located to DEP electrodes and a hydrodynamic (side stream) for carrying out the invention. 
     FIG. 4 is a partial side view of the FIG. 3 device illustrating the main (sample) flow stream and the side (hydrodynamic) flow stream. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is directed to trapping of DNA and cells/spores using dielectrophoretic (DEP) forces to perform sample preparation protocols for PCR based assays, for applications such as counter biological warfare, determining genetic information, etc. A key element for PCR sample preparation is the use of DEP forces to concentrate the DNA prior to detection. DEP forces are strongest near the electrodes. By introducing a side stream into the main stream containing the particles, the main stream is squeezed such that the particles are forced toward the electrodes and are thus more affected by the DEP forces. This invention enables the use of relatively open channels thereby preventing clogging which results from the use of small channels. 
     FIGS. 1 and 2 schematically illustrate a PCR sample preparation system which incorporates the hydrodynamic enhanced DEP particle trapping of the present invention, as exemplified in FIGS. 3 and 4 and described in detail hereinafter. FIG. 1 is a top view of the overall system and FIG. 2 is a side view of a portion of the FIG. 1 system. As shown, the system incorporates four (4) sections or functions which include sample fractionation indicated at  10 , sample concentration indicated  11 , DNA concentration indicated at  12 , and DNA motion/reagent mix indicated at  13 . The sample fractionation section  10  includes a flow channel  15  in which electrodes  16 - 17  for DEP are mounted, with channel  15  having inputs or inlets  18  and  19  into which are directed a focusing buffer  20  and a sample  21  (from an aerosol collector, for example) and outlets  22  and  23 , connected to a channel  24  and to waste  25 . 
     Channel  24  extends though section  11 - 13  of the system and includes 3 inlets, a sample inlet  26 , a lysing solution inlet  27 , and a focusing buffer inlet  28 , see FIG. 2, for sample  26 ′, lysing solution  27 ′ and focusing buffer  28 ′ and is provide with a waste outlet  29 , a PCR reagent inlet  30  and outlet  31 , and exit  32 , for waste  29 ′ and reagent  30 ′ and  30 ″. The channel  24  is also provided with electrode sets indicated at  33  for section  11 ,  34  for section  12  and  35  for section  13  and with a single electrode  36 , see FIG. 2, which extends the length of electrode sets  33 ,  34 , and  35 . The electrode sets  33 - 35  and single electrode  36  are electrically connected to an AC power source  37  as in FIG.  3 . The channel  24  terminates via a detector which includes ports  38 . As charged particles, such as DNA,  39  from outlet  22  of channel  15  of sample fractionation section  10  pass along channel  24  the electrodes of electrode sets  33 ,  34 , and  36  are each sequentially activated to control the concentration of the particles via electrical fields produced by the sequentially activated electrodes. As seen in FIGS. 1 and 2 a sample  26 ′ containing particles  39  is introduced into flow channel  24 , wherein the particles (cells and spores) are captured on the electrodes of electrode set  33  by DEP forces. As seen in FIG. 2, a focusing buffer  28  via inlet  28  and a lysing solution  27 ′ are introduced into channel  24 , the lysing solution  27 ′ breaking open the spores to release the DNA and the focusing buffer  28 ′ squeezing sample toward the electrodes  62 . The DNA travels downstream to another set  34  of electrodes where the DNA is captured. The DNA is walked down the channel  24  to a low-flow area, section  13 , via electrode set  35 , where PCR reagents  30  are introduced. The sample is then released for the PCR process and detection. 
     A key factor to the success of the system of FIGS. 1-2 is that flows in small dimensional (&lt;500 μm) channels is laminar. Mixing between streams is limited to diffusion, which is not very effective. Thus, side stream flows can be used to direct other flows. Since DEP forces are effective only very close (&lt;100 μm) to the electrodes, it is important to direct the cells and DNA close to the electrodes. This can be accomplished using side stream flows, as shown at in FIGS. 1-2 at inlet  28  and the focusing buffer flow indicated at  40 , and illustrated in greater detail in FIGS. 3-4 described hereinafter. Use of side stream flows in lieu of making smaller channels reduces the chance of blockage of the flow channels, which is very common in biosystems. 
     FIGS. 3 and 4 illustrate a simplified embodiment of the hydrodynamic enhanced DFP particle trapping of the FIGS. 1-2 system, and corresponding components are given corresponding reference numerals. As clearly seen in FIG. 4, the sample fluid with particles  26 ′ passing via inlet  26  and channel  24  is squeezed close to electrode  36  by the side stream or focusing fluid  28 ′ via inlet  28 , whereby the particles in sample fluid  26 ′ are affected by the DEP forces and trapped along electrode  36  as indicated at  39  in FIG.  2 . 
     It has thus been shown that the present invention enables hydrodynamic enhanced dielectrophoretic particle trapping and enables movement and concentration of particles in a fluidic channel via DEP forces through sequentially activated electrodes, which produce trapping via electric fields. The invention solves the problem of directing the particles close to the electrodes for increase DEP force affect thereon without the use of small channels, thereby reducing potential clogging of the channels while increasing the efficacy of DEP trapping. The invention is particularly applicable for use in counter biological warfare as well as a clinical tool to determine genetic information via PCR processing. 
     While particular embodiments of the invention have been described and illustrated to exemplify and teach the principles of the invention, such are not intended to be limiting. Modifications and changes may become apparent to those skilled in the art and it is intended that the invention be limited only by to scope of the appended claims.