Abstract:
A method and system are disclosed for manipulating the flow of gases into and out of a microfluidic circuit to regulate pressure within the circuit or to provide for the delivery of gases to or removal of gases from the circuit. Pressure within the microfluidic circuit may be increased or decreased to modify physical or chemical properties of fluid within the circuit, or to modify reaction kinetics. Gaseous reactants may be added to the circuit, and reaction products or excess reactant gases may be removed the circuit according to the invention. Warm or cool air or other gas may be flowed over liquid reactants within the circuit to perform a warming or cooling function. Various biochemical reactions or processes, including for example polymerase chain reaction (PCR) and ligand-receptor binding, may be performed with the use of the inventive method and system.

Description:
This application claims the benefit of provisional application 60/204,306 filed May 15, 2000. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the field of microfluidic circuitry for biochemical processes or reaction. It relates more specifically to the regulation of pressure and movement of gases into and out of microfluidic circuits. 
     BACKGROUND OF THE INVENTION 
     The tools of microfabrication developed primarily by the integrated circuit and microsystems industries may be used to fabricate micrometer-sized structures that are designed to manipulate small volumes of fluids. This area of development is known as microfluidics. Microfluidic structures have been fabricated to integrate and miniaturize many of the fluid handling and analysis steps involved in traditional biological and chemical analysis. The present and forthcoming integrated and miniaturized systems are expected to offer several advantages over traditional methods. These advantages include greater quality control, faster analysis times, higher throughput of sample processing, and lower costs primarily related to smaller sample volumes and smaller amounts of chemicals and reagents required to analyze these smaller volumes. 
     Many different methods of fluid manipulation and sample analysis have and will be created. The numerous varying chemical and physical properties associated with the multitude of possible sample types make it difficult, if not impossible, for one sample manipulation or analysis technique to be used for all microfluidic applications. For example, there are many methods of pumping fluids through a microfluidic processing circuit, some of which are useful for ionic solutions, some of which are more appropriate for larger flow rates, and some of which utilize no moving parts. These include electrokinetics, electro-hydrodynamics, and pressure driven flow. Pressure driven flow can be further broken down into pneumatics, hydraulics, capillarity and centrifugal flow. 
     In most utilizations of pressure driven flow the microfluidic circuit is open to the atmosphere at one or more points downstream of the moving fluid so that air displaced by the moving fluid is allowed to escape the circuit. This prevents unwanted buildup of pressure that may oppose the desired fluid movement. The fluid may be prevented from escaping the circuit through the air displacement ducts by use of capillary valves, porous hydrophobic membranes, or similar methods, where air may escape but the fluid is contained. 
     The passive, or mostly passive, behavior of the air displacement ducts and possible corresponding capillary valves, are generally sufficient for their intended purpose. However, benefit may be gained by altering the pressure inside the microfluidic circuit, which cannot be readily carried out with passive air displacement ducts. 
     As the physical and chemical properties of a fluid, or the reaction rates of a reaction in which the fluid is involved, may vary with pressure, in some applications it would be beneficial for the fluid within a fluid circuit to experience a pressure higher or lower than the normal ambient pressure to which it is exposed via the air ducts. 
     Some reactions involve the production of gaseous phases that it is desirable to remove from the reaction chamber. This may be needed to prevent buildup of pressure within the system, to keep the gases from possibly poisoning further reactions, or to allow for analysis of the gases that are generated. In addition, some reactions may benefit from the delivery of gaseous materials, such as for the delivery of gaseous reagents, or for heating or cooling the system. Therefore, it would be desirable to provide a method for removing gaseous components from or introducing gaseous components to the microfluidic system. 
     SUMMARY OF THE INVENTION 
     The present invention discloses a method and system for manipulating the flow of gaseous materials into and out of a microfluidic circuit via air displacement ducts common in many pressure driven microfluidic systems. Air displacement ducts are used in combination with valves, pumps, and other pressure regulation devices to provide for the delivery or removal of gaseous reaction constituents or products, and to allow for the control of ambient pressure, and hence reaction pressure, within a microfluidic system. The air displacement ducts are not in direct communication with the atmosphere, but rather are connected to active pressure regulation devices such as valves and pumps that allow the air flow to be controlled through each duct individually, if desired. These active pressure regulation devices may be integrated within the microfluidic substrate, or they may be permanent or semi permanent members of an external system to which the microfluidic system is interfaced. 
     One object of the invention is to allow manipulation of the pressure within a microfluidic circuit in an effort to change the reaction process of a sample when compared to normal atmospheric conditions. 
     Another object of the invention is to allow for the delivery or removal of gaseous reactants or products from the microfluidic system, or simply to deliver hot or cold air to heat or cool a microfluidic sample. In most applications the air remaining in the fluid circuit is at approximately the same pressure as the ambient atmosphere, due to the presence of the air displacement ducts. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 is a perspective view of a first embodiment of the invention having two air displacement channels; 
     FIG. 2 is a cross-sectional view of the embodiment of the invention shown in FIG. 1; 
     FIG. 3 is a perspective view of a second embodiment of the invention having two air displacement channels leading to outlets in the substrate; 
     FIG. 4 is an embodiment of the invention having two air displacement channels leading to a single outlet; 
     FIG. 5 is a cross-sectional view of the embodiment of the invention shown in FIG. 4; 
     FIG. 6 is a perspective view of another embodiment of the invention including a microfluidic circuit having one inlet leading to a well with one outlet channel, and two cross channels joined thereto. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Pressure driven microfluidic circuits, such as those connected with external pumping systems, generally require a system outlet that allows air or other gaseous fluids to be displaced out of the system as the fluid circuit fills with liquid fluid. This system outlet may be designed to allow the exit of liquid fluid as well as air (or other gaseous fluid), or it may be designed to only allow displaced air to escape, and not liquid. In addition, some embodiments may allow air to enter or exit depending on the movement of liquid within the system, and some embodiments provide for multiple air displacement ducts, at different sites along the fluid circuit, to allow for more complex fluid manipulation. 
     An example of a fluid circuit that permits air to escape as liquid fluid enters the circuit is shown in FIGS. 1 and 2. It should be noted that the term “air” is used because it is typically the case that air is displaced from within the fluid circuit, but that it is contemplated that other gases or gaseous mixtures may also travel through the air displacement ducts of the invention. Liquid fluid is pumped into microfluidic circuit  1  by pump  2 , via tubing  3  to inlet  4  of microchannel  5 . Microchannel  5  leads to chamber  6 . Microchannel  7  exits chamber  6  and branches into microchannels  8  and  9 , leading to chambers  10  and  11 , respectively. Air displacement ducts  12  and  13  are connected to chambers  10  and  11 , and thus indirectly to microchannels  8  and  9 . In FIG. 1, microfluidic circuit  1  is formed in surface  20  of substrate  21  and is covered by a lid  22  sealingly placed on top of substrate  21  to contain the fluid within the microfluidic circuit  1 . The ends of air displacement ducts  12  and  13  extend past the edge of lid  22  to communicate with the external atmosphere. This allows for the circuit to be in communication with the outside, so that air displaced by the incoming liquid fluid can pass out of the circuit. FIG. 2 is a cross-sectional view of the device in FIG. 1, with inlet  4  shown at the left. 
     If the air displacement ducts allow direct communication between the inside of the microfluid circuit and the external ambient atmosphere, as shown in FIGS. 1 and 2, any reaction or process involving the fluid within the microfluidic circuit will take place at ambient atmospheric conditions, as far as pressure is concerned. The air displacement ducts typically are not capable of ensuring equi-temperature or equi-humidity with the ambient atmosphere. Also, the equi-pressure condition can only be ensured if the fluid in the system is static. Dynamic fluid movement may alter the pressure conditions within the circuit due to a possible pressure drop across the air displacement duct. 
     Many air displacement ducts are designed to allow only the movement of air or other gaseous materials, and not liquid fluid. This can be done by including within the air displacement duct a capillary stop barrier, a porous hydrophobic membrane, or another structure that allows air to flow but impedes liquid movement. In previous work of the inventors, small channels in hydrophobic materials were used as air escape ducts. The air escape ducts were on the order of 10-15 μm in diameter, whereas the normal fluid channels were 10-20 times larger. This allowed for air to escape readily, but required relatively large pressures to force the fluid through the small air ducts due to their hydrophobic nature. However, it should be stated that any channel downstream of fluid in a microfluidic circuit could be considered an air duct, whether air passes through it exclusively, or whether first air and then fluid as the fluid advances through the circuit. 
     Many important bio-chemical processes take place at elevated temperatures (greater than 20° C.). These include incubation steps for cell proliferation, some ligand-receptor binding events, and polymerase chain reaction (PCR). Some reactions are performed at elevated temperatures to accelerate the rate of the reaction or process, or because the elevated temperature is required for the reaction to take place at all. At some elevated temperatures, particularly ones approaching the boiling point of the fluid involved, steam generation may increase to a level that effects the concentration of the reactants in the sample, or causes downstream channels to be saturated with vapor. This may be detrimental to the efficiency of the present reaction or of downstream processes, or it may be the mechanism of the intended reaction, such as promoting evaporation to increase the concentration of a species of interest. 
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                  Vapor pressure of water at various temperatures. 
               
             
          
           
               
                 deg C 
                 MmHg 
                 psi 
               
               
                   
               
             
          
           
               
                 5 
                 6.5 
                 0.13 
               
               
                 10 
                 9.2 
                 0.18 
               
               
                 15 
                 12.8 
                 0.25 
               
               
                 20 
                 17.5 
                 0.34 
               
               
                 25 
                 23.8 
                 0.46 
               
               
                 30 
                 31.8 
                 0.62 
               
               
                 35 
                 41.2 
                 0.80 
               
               
                 40 
                 55.3 
                 1.07 
               
               
                 45 
                 71.9 
                 1.39 
               
               
                 50 
                 92.5 
                 1.79 
               
               
                 55 
                 118 
                 2.28 
               
               
                 60 
                 149.4 
                 2.89 
               
               
                 65 
                 187.5 
                 3.63 
               
               
                 70 
                 233.7 
                 4.52 
               
               
                 75 
                 289.1 
                 5.59 
               
               
                 80 
                 355.1 
                 6.87 
               
               
                 85 
                 433.6 
                 8.39 
               
               
                 90 
                 525.8 
                 10.17 
               
               
                 95 
                 633.9 
                 12.26 
               
               
                 100 
                 760 
                 14.70 
               
               
                 105 
                 906 
                 17.52 
               
               
                 110 
                 1075 
                 20.79 
               
               
                 115 
                 1268 
                 24.53 
               
               
                 120 
                 1489 
                 28.80 
               
               
                 125 
                 1741 
                 33.67 
               
               
                 130 
                 2026 
                 39.19 
               
               
                 135 
                 2347 
                 45.40 
               
               
                 140 
                 2711 
                 52.44 
               
               
                 145 
                 3117 
                 60.29 
               
               
                 150 
                 3570 
                 69.05 
               
               
                   
               
             
          
         
       
     
     Source: CRC Handbook of Chemistry and Physics, 65th Ed., p D-193, CRC Press, Boca Raton, Fla. 1984. 
     In these, and other instances where steam generation, or boiling, is not preferred, the present invention provides for all ducts connected to downstream channels to be pressurized in order to pressurize the complete fluidic system. As the ambient pressure is increased, the fluid pressure is also increased. This causes the vapor pressure of the fluid to equal the ambient pressure at higher temperatures, causing a reduction in steam generation, or raising the boiling point of the fluid. Table 1 shows the relationship between water vapor pressure and temperature. Assuming normal atmospheric pressure (14.7 psi) an increase in microsystem pressure of only 2.8 psi will increase the boiling point of water by approximately 5 degrees. If other factors in the reaction require a temperature near the normal boiling point of water, pressurizing the system only slightly will allow the normal boiling point to be reached, but at a reduction in steam generation. In another case, as can be seen from Table 1, if the internal pressure were reduced by approximately 14 psi the vapor pressure of water drops to near room temperature and water may boil without heating. 
     FIG. 3 shows fluid circuit  25 , which is a modification of fluid circuit  1  of FIGS. 1 and 2, in which air displacement ducts  26  and  27  pass into substrate  21  and exit on the side of substrate  21 . Tubes  31  and  32  can be readily connected to outlets  28  and  29  of air displacement ducts  26  and  27 , respectively. Air displacement ducts  26  and  27  can then be connected to pressure regulation devices  32  and  33  (which may be the same or separate pressure sources), such as a pump or a pressurized gas supply regulated by a valve. Since the volumes of microfluidic systems are usually quite small (total volume may range from 1-1000 μL for one circuit, depending on its complexity), it is not necessary to use a high volume displacement pump to substantially change the pressure of a system; a low-volume pump, such as a syringe pump, is may be used effectively to add or extract gas from the system. 
     FIGS. 4 and 5 are perspective and sectional views of a microfluidic circuit similar to that shown in FIG. 3, but with air displacement ducts  30  and  31  joined together to form a single air displacement duct  32  leading to outlet  33  for connection to a single pressure regulation device. 
     In work done by the inventors, microfluidic circuits were fabricated on the surface of a Teflon™ substrate using excimer laser ablation. The circuits usually contained a minimum of one inlet and one outlet. The inlet and outlet were fabricated by cross drilling small holes from the side and top of the substrate so that they connect and a clear channel exists from the side of the substrate up to the surface of the substrate, where the top hole is connected to the surface channels, as depicted in FIGS. 3-6. The outlet, or final air escape duct, was fabricated in the same manner. A friction fit of tubing into the inlet and outlet was sufficient for delivering pressures of up to 25 psi. It is assumed they would also allow for a reduction in pressure of a few psi. It was found that the air connection did not need to be perfectly leak proof, so long as the positive or negative gas flow was greater than any leakage that occurred. The top of the substrate was covered with a hydrophobic film, such as Teflon AF™ coated vinyl membrane, and pushed onto the substrate with a polycarbonate lid held by thumbscrews. It was not necessary for the lid to be perfectly sealed, since all surfaces were hydrophobic, which reduces the problems associated with micro cracks. 
     EXAMPLE 1 
     Polymerase Chain Reaction (PCR) is a process that consists of a cyclical heating and cooling of a reaction mixture. The high temperature in the cycle approaches the boiling point of water, and can even surpass it at higher elevations. In the case when the boiling point of water needs to be elevated to prevent boiling and evaporation, this can be accomplished by increasing the ambient pressure inside the microfluidic circuit. FIG. 4 shows an embodiment of the invention in which two air displacement ducts  30  and  31  are joined into a single duct  32  prior to the channel being fed into the substrate  21  and out the end. The outlet of the duct would be interfaced with tubing  37 , valve  36 , and a pump  34  to control air pressure in a manner similar to that in which inlet of the fluid circuit is connect to a pump for fluid delivery. FIG. 5 shows a side view of the system of FIG.  4 . 
     Fluid circuit  40  is filled with water up to a series of parallel processing chambers  10  and  11 . Air displacement ducts  30  and  31  are joined into a single air displacement duct  32  and connected to the system outlet  33 . A valve  36  connected to the outlet tubing  37  is opened to allow for air to be displaced out of the system as it fills with liquid fluid. Once chambers  10  and  11  are filled, the system is pressurized by pumping air via a pressure regulation device  34  (which may be, for example, a syringe) connected to outlet tubing  37  into circuit  40  via the outlet  33 . A pressure gauge  35  may be connected to the system to indicate the pressure reached, and to monitor pressure during cycling. Once the desired pressure is reached, valve  36  may be closed and pressure regulation device  34  removed or turned off. Liquid fluid pump  2  may need to be activated to compensate for any backward movement of the fluid caused by the increase in downstream air pressure. The whole substrate can then be heated in the thermal cycling process. 
     EXAMPLE 2 
     FIG. 6 illustrates an alternative embodiment of the invention which makes it possible to remove steam (or other vapor) generated by a heating process by a slight air flow caused by air being passed in and out of the microfluidic circuit by a cross channel. FIG. 6 shows microfluidic circuit  45  with one inlet  46  leading to reaction chamber  47  with one outlet  48 . Near outlet  48  are two cross channels  49  and  50 , which function as air ducts. Air flows into the system via first air duct  49 , driven by positive pressure from pressure regulation device  55 . The air from air duct  49  crosses main microchannel  51  between reaction chambers  47  and  52  in the vicinity of reaction chamber  47 , and is drawn out of the system via second air duct  50 , due to negative pressure from pressure regulation device  56 . Outlet duct  53  of chamber  52  and first and second air ducts  49  and  50 , respectively, extend to the end of substrate  21 , where they can be connected to individually. In addition to pressure regulation devices  55  and  56  on first and second air ducts  49  and  50 , a third pressure regulation device  57  may be provided on outlet duct  53  to regulate overall system pressure. 
     EXAMPLE 3 
     A sample can be concentrated by accelerating evaporation. This can be accomplished by passing the dry gas into the system via downstream air ducts, and removing the gas at the same rate via an air duct closest to the sample that is to be concentrated, for example through the use of a circuit of the type shown in FIG.  6 . 
     EXAMPLE 4 
     Many processes in organic chemistry involve purification of organic molecules by suspending a mixture in a solvent and allowing the solvent to evaporate. As the solvent evaporates the molecule of interest will precipitate out. Such is the case in DNA purification by ethanol precipitation. 
     This can be accomplished by maintaining the solvent mixture in a microfluidic evaporation chamber connected to one or more air ducts. The air duct is connected to a vacuum pump and the internal system pressure is slowly lowered to allow for accelerated and controlled evaporation on a micro scale. 
     Although the above examples illustrate substrates containing only a single microfluidic circuit, and microfluidic circuits containing only a single inlet and one, two or three outlets, it will be appreciated that the principal of the invention can be applied to devices in which multiple microfluidic circuits are formed on a single substrate. Moreover, the inventive air flow and pressure regulation method may be applied to microfluidic circuits having larger numbers of inlets and outlets, larger numbers of microchannels, reaction chambers, and air displacement ducts, and greater complexity of interconnections between these structures. While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims. 
     LIST OF REFERENCES 
     R. C. Anderson et al.,  Microfluidic Biochemical Analysis System, IEEE Transducers &#39; 97, page 477-80, 1997. 
     U.S. Pat. No. 5,897,842 
     U.S. Pat. No. 5,856,174 
     U.S. Pat. No. 5,375,979 
     WO9722825A1 
     WO9939120A1 
     All publications, patents and patent applications cited herein are incorporated by reference.