Abstract:
A three dimensional microfluidic device is formed by placing a membrane between two fluid containing features. The membrane is positioned to cover the area where features intersect. In one embodiment the membrane is porous. Electric pulses are applied such that molecules in the fluid with faster membrane transit times go through the membrane, while longer transit time molecules withdraw back from the membrane between pulses.

Description:
RELATED APPLICATIONS  
       [0001]     This application is a divisional of U.S. patent application Ser. No. 10/372,016, filed Feb. 21, 2003, which claims priority to U.S. Provisional Patent Application Ser. No. 60/359,118, filed Feb. 21, 2002, which is incorporated herein by references. 
     
    
     GOVERNMENT SUPPORT  
       [0002]     This invention was made with government support awarded by National Institute of Health (Princeton) # R01-HG01516. The Government has certain rights in this invention. 
     
    
     FIELD OF THE INVENTION  
       [0003]     The present invention relates to microfluidic devices, and in particular to a microfluidic device having a porous membrane.  
       BACKGROUND OF THE INVENTION  
       [0004]     Microfluidic devices have many applications in chemical and biological assays, such as drug screening, nucleic acid separation and protein separation. Some filter cartridges use a porous membrane having many holes etched through a substrate for high performance liquid chromatography (HPLC), DNA separation and protein separation. The throughput for such cartridges is relatively low, and the cost per assay is high.  
       SUMMARY OF THE INVENTION  
       [0005]     A three dimensional microfluidic device is formed by placing a membrane between two fluid containing features. The membrane is positioned to cover the area where features intersect. In one embodiment the membrane is porous. Electric pulses are applied such that molecules in the fluid with faster membrane transit times go through the membrane, while longer transit time molecules withdraw back from the membrane between pulses.  
         [0006]     In one embodiment, a three dimensional microfluidic device is formed by placing a membrane between two micropatterned chips. The patterning in one embodiment comprises intersecting channels, wherein the membrane is positioned to cover the area where the channels intersect. In one embodiment, channels are formed in polycarbonate chips. A porous membrane is placed between the chips. The chips are positioned such that the channels intersect at approximately a right angle. The chips are then bonded. In one embodiment, the chips are formed of plastic, and are thermally bonded under pressure.  
         [0007]     In a further embodiment, reservoirs are formed on the chips at each end of each channel. The channels are created in the chip by use of an embossing master, such as a patterned silicon wafer. The reservoirs are formed by drilling. A hydraulic press is used to emboss both chips, and is also used to thermally bond the chips and membrane under pressure. In a further embodiment, the surfaces of the channels are oxidized, changing the surfaces from hydrophobic to hydrophilic.  
         [0008]     A method of molecule separation is performed using the microfluidic device. In one embodiment, DNA is placed in one reservoir, and moved to the membrane by a low voltage. A short electric pulse is applied to drive the DNA through the porous membrane. After the pulse, short DNA molecules have moved completely through the porous membrane, while longer DNA molecules have only partially moved into the holes of the porous membrane. After the pulse, when voltage is zero, longer DNA molecules recoil out of the holes of the porous membrane. After multiple iterations of electric pulses, short DNA molecules have moved completely through the porous membrane, while longer DNA molecules have not moved through the porous membrane, resulting in separation of the DNA molecules by length. The electric pulses are varied to provide separation of different length molecules. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0009]      FIG. 1  is an exploded three dimensional perspective view of a microfluidic device formed in accordance with the present invention.  
         [0010]      FIG. 2  is block diagram showing top view illustrating features of the microfluidic device of  FIG. 1 .  
         [0011]      FIG. 3  is a block diagram showing use of the microfluidic device of  FIG. 1  in the separation of molecules.  
         [0012]      FIGS. 4A, 4B ,  4 C,  4 D,  4 E and  4 F are a series of block diagrams showing the formation of the microfluidic device of  FIG. 1 .  
         [0013]      FIG. 5  is a SEM image of a polycarbonate membrane for the microfluidic device of  FIG. 1 .  
         [0014]      FIG. 6  is a cross sectional representation of a molecule moving through a single pore of a membrane.  
         [0015]      FIG. 7  is a graphical representation of the intensity of tagged DNA molecules that have passed through the porous membrane.  
         [0016]      FIG. 8  is an exploded view of a three dimensional multilevel microfluidic device. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0017]     In the following description, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural, logical and electrical changes may be made without departing from the scope of the present invention. The following description is, therefore, not to be taken in a limited sense, and the scope of the present invention is defined by the appended claims.  
         [0018]     A microfluidic device formed in accordance with the present invention is shown in an exploded view at  100  in  FIG. 1 . A first plastic layer or chip  110  has a channel  120  formed therein. A second plastic chip  130  also has a channel  140  formed therein. The chips are formed of a polymeric optical grade plastic, such as ZEONOR® in one embodiment. Polyethylene, polypropylene, other plastics and other materials such as semiconductor materials are used in further embodiments. Channels are just one example of micropatterning to produce microfeatures that is achievable. Many different microfeatures may be produced, including but not limited to sensors, reservoirs, and any other structure that may produced.  
         [0019]     The two chips are positioned relative to each other such that the channels  120  and  140  are positioned at approximately right angles to each other in one embodiment, and a membrane  150  is positioned between the two chips where the channels intersect. The intersection creates a substantially square aperture covered by the membrane separating the two channels, top and bottom, from each other. In one embodiment, the membrane is porous. The membrane  150  is large enough to entirely cover and extend partially beyond the intersection of the channels  120  and  140  in one embodiment such that substances mainly travel through the membrane to move from one channel to the other channel. The chips and membrane are bonded to form a three dimensional microfluidic device. In one embodiment, the membrane  150  is substantially flat, with essentially no wrinkles.  
         [0020]     In further embodiments, the channels or other micropatterning are not perpendicular, and the membrane is formed in a suitable shape to cover the intersection of the patterning as desired. The membrane is formed in size corresponding to the size of the chips in a further embodiment, and more than one set of channels are formed in the chips. In still further embodiments, the channels are not formed in straight lines, and may also intersect in more than one point.  
         [0021]     In one embodiment, the porous membrane is a Nuclepore® (Trademark of Whatman PLC) polycarbonate porous membrane that has many small holes etched through a substrate. The hole size is about 15 nm-5 um and the thickness of the membrane is about 6 um-20 um. The hole size is comparable to the size of some DNA and protein, and is suitable for use as an entropic trap for filtration of DNA and protein. The service temperature of this membrane is high, therefore, it is easily integrated into microfluidic systems by the use of thermal bonding between polymers.  
         [0022]     In further embodiments, membranes have pores in only some portions, and allow for cross flow filtration. Many other uses are available, such as for running cleaning solutions between two membranes, adding nutrients to solutions, introduction of reagents from one channel to cells in a channel opposite the membrane, diffusive transport access and many others. In addition, this method of juxtaposing two fluid channels may facilitate the implementation of fuel cell methods with lower cost of contruction, greater efficiency, or other benefits. The use of a membrane sandwiched between micropatterned chips provides a basic construction tool for fabrication of micro devices.  
         [0023]      FIG. 2  provides a top view of a further three dimensional microfluidic device at  200 . Intersection channels  210  and  220  are formed, with channel  210  being a bottom channel and channel  220  forming a top channel. Channel  210  has a reservoir  225  and  230  formed at each end of the channel. Similarly, channel  220  has a reservoir  235  and  240  formed on each end of the channel. A membrane  250  is disposed between the channels at their intersection. In one embodiment, the channel width was approximately 40 um and depth approximately 20 um. In essence, the range of sizes of channels and other micropatterning is very great depending on the intended use. The reservoirs were substantially larger in order to hold substances to be separated, such as DNA and protein. It should be noted that many other sizes of channels are utilizable depending on the size of membrane achievable and the desired throughput of the device.  
         [0024]      FIG. 3  provides a top view of the device of  FIG. 2  with a voltage source  310  applying a 100 volt electric potential across reservoir  235  and reservoir  230 , with reservoir  230  grounded. In one embodiment, T2 and T7 DNA were placed in reservoir  230 . Since T2 and T7 DNA molecules are negatively charged, they flow from reservoir  230  to  235 . They pass through the membrane with essentially no leaking. The voltage is varied in different embodiments to obtain different rates of flow and filtration as desired. In yet further embodiments, other means of causing flow are provided, such as heat pumps, differential pressures, gravity, capillary action, and osmosis to name a few.  
         [0025]      FIGS. 4A, 4B ,  4 C,  4 D,  4 E, and  4 F depict a process of forming a three-dimensional microfluidic chip in accordance with the present invention. In  FIG. 4A , a silicon wafer  412  is covered in a photoresist  414  for patterning. Silicon wafer  412  is a three inch silicon wafer that is used as an embossing master. A Shiply 1813 photoresist (Microchem, Newton, Mass.) is spin coated at 3000 RPM for 90 seconds on the silicon wafer in one embodiment. The thickness of the photoresist is approximately 1.3 um. Other photoresists and silicon wafers are also options.  
         [0026]     In  FIG. 4B , an HTG contact aligner is used for standard photolithographic processing to pattern ridges in the photoresist by use of a mask  422 . In  FIG. 4C , the silicon is etched using SF 6  in one embodiment. The ridge  432  of photoresist is not etched. The etching is performed in a Plasmatherm ICP770 to a depth of 20 um. After the etching process, the photoresist is removed with acetone and plasma etching to form the embossing master as shown in  FIG. 4D  having a ridge of silicon  442  for forming channels in plastic chips.  
         [0027]     A first plastic chip  452  is cleaned such as by acetone for two minutes in an ultrasonic bath, and cut into a desired size, such as 2.0 cm×2.0 cm. The chip is then placed in contact with the silicon master with heat (approximately 130 degrees C.) and pressure from both top and bottom for embossing of the chip for about 7 minutes as shown in  FIG. 4E . A second chip is processed in the same manner. The times, pressures and temperatures may be varied as desired.  
         [0028]     Two of the chips are then equipped with four holes for reservoirs. The holes are approximately 2 mm in one embodiment and are formed by use of a conventional drill with low RPM to prevent melting of the plastic. The holes are formed in any manner suitable, such as photolithographic processing at the same time as the channels.  
         [0029]     In  FIG. 4F , two chips and a membrane are positioned relative to each other as shown in  FIG. 1 , and heated to approximately 85 degrees C. under pressure for approximately 10 to 15 minutes using a thermal press machine. The same machine is used in one embodiment for both embossing of the chips and bonding of the chips to form the microfluidic chip.  
         [0030]     In one embodiment, a H 2 SO 4 /CrO 3  solution is injected into the microfluidic channels to oxidize the surface of the plastic. The oxidation changes the surface of the plastic from hydrophobic to hydrophilic.  
         [0031]     A SEM image of a membrane is shown in  FIG. 5 , illustrating the pores. The pores comprise holes that are approximately 0.05 to 10 um in width, and approximately 6.0 to 11.0 um thick. The material is biologically inert.  
         [0032]      FIG. 6  is a representation of hole  610  in a porous membrane  620 . Membrane  620  contains thousands of such pores in one embodiment. Hole  610  is approximately 100 nm wide, and is shown with a molecule  630  partially inserted into the hole. This is caused by application of an electric field.  
         [0033]     A method of molecule separation is performed using the microfluidic device by application of electric fields across the membrane as shown in  FIG. 3 . In one embodiment, DNA is placed in one reservoir, and moved to the membrane by a low voltage. A short electric pulse is applied to drive the DNA through the porous membrane. After the pulse, short DNA molecules have moved completely through the porous membrane, while longer DNA molecules have only partially moved into the holes of the porous membrane. After the pulse, when voltage is zero, longer DNA molecules recoil out of the holes of the porous membrane by a process referred to as entropic recoil. After multiple iterations of electric pulses, short DNA molecules have moved completely through the porous membrane, while longer DNA molecules have not moved through the porous membrane, resulting in entropic recoil separation of the DNA molecules by length. The electric pulses are varied to provide separation of different length molecules.  
         [0034]      FIG. 7  is a graphical representation of the intensity of tagged DNA molecules that have passed through the porous membrane. As the voltage across the membrane was increased, the relative intensity of the molecules increases.  
         [0035]      FIG. 8  is an exploded view of a three dimensional multilevel microfluidic device. Three layers, top layer  810 , middle layer  815  and bottom layer  820  are separated by two porous membranes  825  and  830 . Each adjacent layer has a structure, such as a microfluidic channel. Top layer  810  has a channel  835 . Middle layer  815  has a structure such as a channel on each side,  840  and  845  respectively for fluid transport. Bottom layer  820  also has a channel  850 . Channels of adjacent layers may partially overlap, and may or may not be separated from each other by one of the membranes. Middle layer  815  also has a via  855  formed through it, connecting channels  840  and  845 . The via  855  provides for fluid flow between multiple levels. While particular structures, and positions of the structures are described in this example device, other arrangements are also within the scope of the invention, such as four layers, and different shaped membranes. Many different variations may be utilized.  
       CONCLUSION  
       [0036]     The present invention involves the use of a membrane positioned between two micro patterned surfaces. Many different types of membranes are used in various embodiments. While the membrane is described as substantially flat, it may also be contoured as desired, such as accordion shaped in portions to increase effective surface areas. The micro patterned surfaces are also formed of multiple different types of materials using many different processes. The membrane is coupled to the patterned surfaces in one of many different manners. Thermal bonding coupled with pressure is just one method of adhering the membranes and micro patterned surfaces.