Patent Publication Number: US-8124030-B2

Title: Microfluidic device having regulated fluid transfer between elements located therein

Description:
REFERENCE TO RELATED APPLICATIONS 
     This Application is a U.S. National Stage filing under 35 U.S.C. X371 of International Application No. PCT/US2008/063086 filed on May 8, 2008, which claims priority of U.S. Provisional Patent Application No. 60/916,774 filed on May 8, 2007. The contents of the aforementioned applications are incorporated by reference as if set forth fully herein. Priority to the aforementioned application is hereby expressly claimed in accordance with 35 U.S.C. 119, 120, 365 and 371 and any other applicable statutes. 
    
    
     FIELD OF THE INVENTION 
     The field of the invention generally relates to microfluidic devices. More specifically, the field of the invention relates to microfluidic devices that are spun or rotated about an axis to effectuate fluid flow and/or transfer. 
     BACKGROUND OF THE INVENTION 
     Microfluidic devices are becoming increasingly more important in both research and commercial applications. Microfluidic devices, for example, are able to mix and react reagents in small quantities, thereby minimizing reagent costs. These same microfluidic devices also have a relatively small size or “footprint,” thereby saving on laboratory space. For example, microfluidic devices are increasingly being used in clinical applications. Finally, because of their small scale, microfluidic devices are able to quickly and cost effectively synthesize products which can later be used in research and/or commercial applications. 
     In one type of microfluidic device, various microfluidic features such as channels, chambers, reservoirs, and the like are formed in a disk-shaped device. The disk may include, for instance, a Compact Disk (CD) having microfluidic features formed therein. This disk is then rotated about an axis or rotation (typically the center of the disk) to effectuate movement of fluid from one location to another. Rotation of the disk generally causes the flow of fluid to move toward the edges of the device. There is a need in these types of devices to regulate or modulate the flow of fluid from one location to another. In prior designs, there was no means to stop or otherwise affect fluid transfer once it had been initiated. This poses several problems including the possibility of cross-contamination when fluids from one reservoir or chamber backflow into other chambers or reservoirs. This is significant because as disk-based devices start to incorporate multiple processes like cell lysis, washing, and purification on a single disk, the chance of cross-contamination increases. In addition, in prior designs there is the possibility of fluids leaking out of vent holes located within the disk structure. 
     For example, in U.S. Pat. No. 6,319,469, each reaction chamber is vented to an air displacement channel located over each reaction chamber. If this venting strategy is used in a configuration where a first chamber is connected to an output chamber located radially outward of the first chamber, fluid transfer occurs from the first chamber to the output chamber. However, assuming a slower flow rate out of the output chamber (e.g., because of the presence of downstream valve, filter, channel restriction and/or microbeads), fluid accumulates in the output chamber and if the output chamber vent is located below the level of the liquid in the first chamber, liquid will leak out of this vent. In another possible configuration, where two chambers are independently connected to an output chamber, if the vent of the output chamber is located above the level of the two input chambers, there is a possibility of backflow into the upstream-located input chambers. 
     There thus is a need for a device and method that is capable of regulating fluid flow between the various features and elements contained in disk-based microfluidic devices. Such a device should permit the regulation of flow between various chambers or elements without the use of cumbersome and expensive mechanical or electrical valves. In particular, there is a need for a disk design that incorporates the ability to prevent cross-contamination between different chambers that have one or more common channels or outlets. 
     SUMMARY OF THE INVENTION 
     According to one embodiment of the invention, a microfluidic device includes a substrate configured for rotation about an axis, the substrate having a first chamber disposed therein. The microfluidic device includes an output chamber disposed in the substrate and located radially outward of the first chamber. A vent hole is provided that is operatively connected to the output chamber. The first chamber includes a fluid transfer channel in communication with the output chamber and a ventilation channel in communication with output chamber, wherein the ventilation channel is coupled to a radially inward portion of the first chamber. 
     In another aspect, a microfluidic device includes a substrate configured for rotation about an axis, the substrate having a first start chamber disposed therein. The microfluidic device includes an output chamber disposed in the substrate and located radially outward of the start chamber. A fluid transfer channel connects the first start chamber to the output chamber. A ventilation channel connects the output chamber to the first start chamber, the ventilation channel connecting at one end to a radially inward portion of the first start chamber and at an opposing end to a junction point on the output chamber. The device includes a vent hole operatively connected to the output chamber. The junction between the ventilation channel and the output chamber is located radially outward with respect to the level of fluid in the start chamber. 
     In another aspect of the invention, a method of regulating fluid flow in a microfluidic device includes providing a substrate configured for rotation about an axis, the substrate having first and second chambers disposed therein containing a liquid, the substrate further including an output chamber disposed in the substrate and located radially outward of the first and second chambers, the output chamber being operatively coupled to the first chamber via a fluid transfer channel and a ventilation channel, the output channel further being operatively coupled to the second chamber via a fluid transfer channel and a ventilation channel, the substrate also including a vent hole operatively connected to the output chamber, the substrate also including a output channel coupled to the output chamber. Rotation of the substrate at a first, low rotational speed transfers liquid from the first chamber to the output chamber but rotation of the substrate at the first, low rotational speed does not transfer fluid from the second chamber to the output chamber. Rotation of the substrate at a second, high rotational speed transfers liquid from the second chamber to the output chamber. According to the method, the substrate is then rotated at the first, low rotational speed to transfer liquid from the output chamber to the output channel. This last reduction in rotational speed primes the siphoning channel allowing fluid to exit the output chamber. The substrate may then be rotated at a higher rotational speed to empty the second chamber. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  illustrates a schematic representation of a centrifugal microfluidic system formed on a rotationally driven substrate according to one embodiment. 
         FIG. 2  illustrates one exemplary embodiment of a substrate formed as a multi-layer structure. 
         FIG. 3A  is a photographic image of a system like that disclosed in  FIG. 1  that is spun at 100 rpm. 
         FIG. 3B  is a photographic image of a system like that disclosed in  FIG. 1  that is spun at 1500 rpm. 
         FIG. 3C  is a photographic image of a system like that disclosed in  FIG. 1  that is spun at 1500 rpm. 
         FIG. 3D  is a photographic image of a system like that disclosed in  FIG. 1  that is spun at 100 rpm. 
         FIG. 3E  is a photographic image of a system like that disclosed in  FIG. 1  that is spun at 1500 rpm. 
         FIG. 3F  is a photographic image of a system like that disclosed in  FIG. 1  that is spun at 1500 rpm. 
         FIG. 3G  is a photographic image of a system like that disclosed in  FIG. 1  that is spun at 1500 rpm. 
     
    
    
     DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS 
       FIG. 1  illustrates a centrifugal microfluidic device  2  according to one embodiment. The microfluidic device  2  may include a number of microfluidic features  4  disposed in a substrate  6 . Microfluidic feature  4  includes such structures as chambers, channels, vents, inlets, outlets, and other structures commonly found in microfluidic devices. The microfluidic feature  4  illustrated in  FIG. 1  may, in reality, be smaller or larger than depicted in  FIG. 1 . Generally, the size of the feature  4  is not critical and there is no limitation on the size of the substrate  6 . Further, the microfluidic devices  2  contemplated herein may include several or multiple different (or the same) microfluidic features  4  populated around the periphery of substrate  6 . 
     As seen in  FIG. 1 , the microfluidic feature  4  includes multiple start chambers  12 A,  12 B (e.g., reservoirs) located within the substrate  6 . In one aspect, the substrate  6  may include a polymer-based material that is formed into a circular or disk shape such as, for example, in the form of a compact disk (CD). As explained in detail below, the substrate  6  may be made of multiple layers of a polycarbonate material arranged in a sandwich-type arrangement to create the various microfluidic features. The substrate  6  is configured to be rotatable about an axis of rotation  16 . As seen in  FIG. 1 , the axis of rotation  16  may coincide with an aperture  18  or hole located through the substrate  6  that is dimensioned to receive a rotatable spindle or drive shaft (not shown). As explained below, during operation of the microfluidic device  4 , the substrate  6  is rotated is cyclical fashion about the axis of rotation  16 . This may be accomplished from a rotating spindle or drive shaft that interfaces with the substrate  6  via the aperture  18 . For example, drive systems found in commercially available DVD or CD players may be employed to provide the rotation motion to the substrate  6 . Such devices are known to those skilled in the art and are not explained herein. 
     Still referring to  FIG. 1 , the microfluidic feature  4  includes an output chamber  22  or reservoir that is located radially outward with respect to the two start chambers  12 A,  12 B. As used herein, radially outward is a relative term meant to indicate that the particular location is located further away from the axis of rotation  16  of the substrate  6 . Similarly, radially inward is meant to indicate that the particular location is located closer to the axis of rotation  16  of the substrate  6 . The output chamber  22  may include an outlet  24  that is coupled to an output channel  26 . As explained below, during certain conditions, fluid contained in the output chamber  22  is permitted to leave the output chamber  22  via the outlet  24  and into the output channel  26 . The output channel  26  (or outlet  24 ) may be further coupled to other microfluidic features  4  for further operations. For example, in one aspect of the invention, a first start chamber  12 A is configured to hold a fluid (not shown) that is a lysing agent. The second start chamber  12 B is configured to hold a fluid (not shown) that is a washing agent. Many other configurations are contemplated. For example, one chamber  12 A,  12 B may hold a lysing agent while another chamber  12 A,  12 B may hold a washing or purification agent. Reagents or samples from a patient (e.g., bodily fluid such as plasma or some constituent of a bodily fluid) may also be contained in the chambers  12 A,  12 B. Generally, any liquid which is able to flow through the various microfluidic features  4  may be used. 
     Still referring to  FIG. 1 , each chamber  12 A,  12 B includes a respective fluid transfer channel  30 A,  30 B that connects respective first and second reservoirs  12 A,  12 B to the output chamber  22 . As shown in  FIG. 1 , the fluid transfer channels  30 A,  30 B connect to the output chamber  22  at opposing side locations on the output chamber  22 .  FIG. 1  also illustrates respective ventilation channels  34 A,  34 B that connect the first and second reservoirs  12 A,  12 B to the output chamber  22 . As seen in  FIG. 1 , the ventilation channels  34 A,  34 B connect to each chamber  12 A,  12 B at a top or a radially inward location. Opposing ends of the ventilation channels  34 A,  34 B connect to the top or radially inward portion of the output chamber  22  as seen in  FIG. 1  (as opposed to radially outward portion of output chamber  22 ). The ventilation channel  34 A that couples the first chamber  12 A to the output chamber  22  includes a radially inward bend portion  36 . The bend portion  36  is needed for the siphon valve aspect of the invention which prevents fluid from leaving the output chamber  22 . Still referring to  FIG. 1 , the device  2  includes a vent hole  38  or the like that is in fluidic communication via channel  40  or the like with respect to the output chamber  22 . The vent hole  38  is located radially inward with respect to the junction between the ventilation channels  34 A,  34 B and the output chamber  22 . The vent hole  38  is bidirectional in that air can pass into or out of vent depending on the rotational state of the substrate  6 . For example, air can leave the vent hole  38  when the device  2  is the state illustrated in  FIG. 3C  whereby some fluid enters channel  40 . Conversely, air can enter the vent hole  38  in the state illustrated in  FIG. 3D  when the substrate  6  is rotated at a slow rotational speed. Air enters channel  40  and output chamber  22 . 
       FIG. 2  illustrates one exemplary construction of the microfluidic device  2 . As seen in  FIG. 2 , the substrate  6  is made of three (3) polycarbonate disks  50 ,  52 ,  54  and two (2) intermediate pressure sensitive adhesive layers  56 ,  58 . The layout of the various microfluidic features  4  may be designed using conventional design software such as, for instance, SOLIDWORKS or AUTOCAD. A computer numerically controlled (CNC) milling machine mills the various features and cuts the polycarbonate disks  50 ,  52 ,  54 . Alignment holes may be drilled in one or more the disks  50 ,  52 ,  54  at the same location so that each disk  50 ,  52 ,  54  may be properly oriented in the radial direction when the composite structure is formed. One of the disks  50  acts as a cover disk. For instance, the cover disk  50  may be made from 0.6 mm thick polycarbonate. The middle disk  52  may also be made from polycarbonate although the thickness is typically greater than the cover disk  50 . For instance, the middle disk  52  may have a thickness of around 3.175 mm. The middle disk  52  contains the various chambers  12 A,  12 B,  22 . The bottom disk  54  may also be formed from polycarbonate and has a thickness like that of cover disk  50  (e.g., 0.6 mm). 
     The pressure sensitive adhesive layers  56 ,  58  may include 100 μm thick sheets of double-sided adhesive film. For example, the pressure sensitive adhesive layers  56 ,  58  may be obtained from FLEXcon Corporation, located at 1 FLEXcon Industrial Park, Spencer, Mass. 01562-2642. Exemplary pressure sensitive adhesive layers  56 ,  58  include FLEX mount DFM 200 clear V-95 available from FLEXcon. The various designs/features in the pressure sensitive adhesive layers  56 ,  58  may be created using software-based design tools. The instructions may then loaded into a roll-feed cutter plotter (e.g., using SignGo software available from Wissen UK Inc. Ltd., United Kingdom). For example, a Western Graphtec CR2000-60 (Santa Ana, Calif.) roll-feed cutter plotter may be used to cut features in the pressure sensitive adhesive layers  56 ,  58 . Channel features are cut in one pass though the top release film and the middle adhesive layer, but not through the bottom supporting release film. 
     The top disk  50  includes any vent holes including vent hole  38 . The middle disk  52 , which is thicker, contains the chambers such as chambers  12 A,  12 B, and output chamber  22 . The channels such as the fluid transfer channels  30 A,  30 B, the ventilation channels  34 A,  34 B, and the output channel  26  are formed in the upper adhesive layer  50 . The width of the various channels, e.g., channels  26 ,  34 A,  34 B,  34 A, and  34 B are typically less than 1 mm. To form the final composite structure, the pressure sensitive adhesive layers  56 ,  58  are placed between the disks  50 ,  52 ,  54  in alignment (using alignment holes) and the entire stack is then bonded together. It should be understood that the dimensions given above are illustrative only and other dimensions may work in accordance with the inventive concepts described herein. 
     By incorporating the ventilation channels  34 A,  34 B along with the common vent hole  38  in the microfluidic device  2 , when the substrate  6  is rotationally driven about the axis of rotation  16 , regulated flow between the start chambers  12 A,  12 B and the output chamber  22  can occur. In particular, fluid may be able to flow into the output chamber  22 , where mixing may occur between the fluids initially contained in the respective start chambers  12 A,  12 B without fear of cross contamination of the “virgin” start chambers  12 A,  12 B. For example, if the ventilation channels  34 A,  34 B and the common vent hole  38  were removed from the device  2 , the fluid contained in the output chamber  22  (which may include a mixture of fluid from chambers  12 A,  12   b ) could flow in reverse or retrograde fashion to contaminate the liquid contained in start chambers  12 A,  12 B. For instance, assume that ventilation channels  34 A,  34 B and the common vent hole  38  were omitted from the device  2 , and that start chamber  12 A was filled with lysate or lysis material and start chamber  12 B was filled with a wash or an elution material with each chamber  12 A,  12 B having respective vent holes (not shown). In this situation, wash material from chamber  12 B may enter the output chamber  22  and flow back to the other start chamber  12 A, thereby contaminating start chamber  12 A with wash. Similarly, lysate or lysis material from chamber  12 A may enter the output chamber  22  and flow back to the other start chamber  12 B, thereby contaminating start chamber  12 B with lysate or lysis material. The present invention avoids this cross-contamination problem through the use of fluid regulation via ventilation channels  34 A,  34 B, and common vent hole  38 . 
     For a design without flow regulation, fluid transfer will occur from start chamber  12 B to output chamber  22 , thence to chamber  44  and output channel  26 . However, assuming a slower flow rate through chamber  44  (for instance, if chamber  44  were full of microbeads), fluid accumulates in chamber  44  and backs up into output chamber  22 . Given the reference frame of radially inward as “higher” and radially outward as “lower,” the fluid will continue to rise higher until it reaches the same level as the starting fluid in start chamber  12 B, as that fluid will have higher “potential energy” if it is higher and continue to drain out of start chamber  12 B. Given the large volume of start chamber  12 B compared with chamber  44  and output chamber  22 , it is easy to see that output chamber  22  will completely overflow, leaking fluid out of venting hole  38 . If vent hole  38  were moved “higher,” the fluid would backflow in retrograde fashion through siphon valve  36  back into start chamber  12 A, causing cross-contamination. It should also be noted that fluid cannot be trapped in chamber  44  by siphon valve  26 , since there is no guarantee that the fluid level will remain below the bend portion in output channel  26 . Therefore, the regulation of the fluid level in output chamber  22  at the level of the junction of ventilation channel  34 B and output chamber  22  is useful not only in preventing cross-contamination into start chamber  12 A, but also to ensure that the siphon valve in output channel  26  is not surpassed. 
     The microfluidic design described herein uses the ventilation channels  34 A,  34 B to equilibrate the respective levels of fluid in the respective start chamber  12 A,  12 B depending on the rotational speed of the substrate  6 . For instance, with respect to the first start chamber  12 A and the first ventilation channel  34 A, upon rotation of the substrate  6  at sufficient volume to fill the output chamber  22 , an equilibrium level will be reached when the negative pneumatic pressure in the start chamber  12 A equals the pressure of the fluid that is above the fluid level in the output chamber  22  (i.e., fluid level in the first ventilation channel  34 A is the same height as fluid level in the first chamber  12 A as seen in  FIG. 3C ). When this equilibrium level is reached, fluid flow from the start reservoir  12 A to the output chamber  22  ceases. Fluid flow from the start chamber  12 A to the output chamber  22  only resumes once the fluid level in output chamber  22  is below the junction of the output chamber  22  and the first ventilation channel  34 A. In this regard, the flow rates of output chamber  22  and the start chamber  12 A is modulated by the first ventilation channel  34 A and the common vent hole  38 . In a similar fashion, the flow rate of the output chamber  22  and the start chamber  12 B is modulated by the second ventilation channel  34 B. The junction between the ventilation channel  34 A and the output chamber  22  is designed to be radially-outward with respect to the level of fluid contained in the start chamber  12 A. Because fluid flows from the radially-inward to the radially-outward direction, regulation of the fluid level at a radially-outward location prevents fluid transfer in the reverse direction. 
     The microfluidic device  2  is thus a self-regulating microfluidic system in which a number of microfluidic elements or features (e.g., reservoirs, chambers, channels and the like) may be employed on a single substrate  6  and connected to each other by ventilation channels  34 A,  34 B and fluid transfer channels  30 A,  30 B. The self-regulating system is thus able to avoid the problems of cross-contamination. The system accomplishes this regulation by negative feedback whereby excess fluid (which passes into the ventilation channels  34 A,  34 B) will stop fluid transfer from the starting chambers  12 A,  12 B to the output chamber  22 . The system and device  2  described herein has applications for integrated centrifugal microfluidic sample preparation, cellular and chemical analysis, clinical, and medical diagnosis applications. 
       FIGS. 3A-3G  illustrate photographic images taken of a microfluidic feature  4  like that disclosed in  FIG. 1 . The photographic images are taken at various angular velocities. Different colored fluids (water with food coloring) were loaded into the two starting chambers  12 A,  12 B with starting chamber  12 A containing a darker fluid. The device  2  was rotated at an initial angular velocity of 100 rpm. As seen in  FIG. 3A , in this initial condition there is fluid in the start chamber  12 B and none of its respective fluid in the output chamber  22 . The fluid transfer channel  30 B coupling the start chamber  12 B and the output chamber  22  acts as a capillary valve that prevents fluid transfer at low rotational speeds. At the low rotational speed, however, fluid from the staring chamber  12 A enters the fluid transfer channel  30 A. With reference to  FIGS. 3C-3G , the height or radial location of the junction between ventilation channel  34 B and output chamber  22  determines the fluid level in the output chamber  22  at which regulation occurs. In one aspect, this junction may be located radially outward with respect to the start chamber  12 B. Generally, the microfluidic feature  4  may be designed such that the fluid level in the output chamber  22  can be regulated at an arbitrary level with respect to the junction between fluid transfer channel  30 B and the output chamber  22 . 
       FIGS. 3A-3G  illustrate an optional chamber  44  that is located downstream of the output chamber  22  and upstream of the output channel  26 . In particular, fluid flows from the output chamber  22  to the chamber  44  and then to output channel  26 . The optional chamber may be used for solid phase nucleic acid extraction in which glass or silica beads are located in the chamber  44 . Solid phase extraction may be used to concentrate nucleic acid (e.g., DNA) from a sample for subsequent elution and processing. Typically, this involves: (1) adsorbing DNA to silica beads in the presence of chaotropic salts, (2) washing away contaminants with an alcohol solution, and (3) eluting DNA in a low-salt solution. Of course, it should be understood that chamber  44  is entirely optional. 
       FIG. 3B  illustrates the device  2  at a high rotational speed, namely, a rotational speed of 1500 rpm. As can be seen from  FIG. 3B , fluid is transferred from the start chamber  12 B to the output chamber  22 . Generally, any rotational speed capable of bursting the capillary valve located in the fluid transfer channel  30 B will suffice. Liquid will not pass a capillary valve so long as pressure at the meniscus is less than or equal to the capillary barrier pressure. A “burst frequency” is reached when the rotational frequency of the substrate  6  is such that the pressure at the meniscus exceeds the capillary barrier pressure. The burst frequency may be modified and altered depending on the particular construction and geometry of the fluid transfer channel  30 B. In some cases, this may be in excess of about 1000 rpm. In the state shown in  FIG. 3B , the fluid level in the output chamber  22  has not yet reached the level of the ventilation channel  34 B. Fluid that enters the output chamber  22  passes to chamber  44 . Outward flow from chamber  44  is blocked due to the siphon valve located at the output channel  26 . Once the chamber  44  is filled up (as seen in  FIG. 3C ), output chamber  22  fills up. In the illustrated device  2 , there are two separate siphon valves. A first siphon valve located in the bend portion  36  of the fluid transfer channel  30 A restricts flow from the first start chamber  12 A. A second siphon valve in the output channel  26  restricts flow from the output chamber  22 . 
       FIG. 3C  illustrates continued rotation of the device  2  at a rotational speed of 1500 rpm. In this image, the fluid level in the output chamber  22  surpasses the junction between the ventilation channel  34 B and the output chamber  22  and, consequently, liquid is drawn up the ventilation channel  34 B. This rise in the liquid level within the ventilation channel  34 B continues until an equilibrium level is reached between the pressure in the start chamber  12 B and the pressure of the fluid column in the ventilation channel  34 B. As seen in  FIG. 3C , this occurs when the upper level of level of the liquid in the ventilation channel  34 B is approximately equal to the level of liquid contained in the chamber  12 B. Again, because of the high rotational speed, there is no outflow from the output chamber  22  because, in this particular embodiment, the presence of the siphon valve located at the output channel  26  regulates fluid flow from chamber  44 . For example, this steady-state condition illustrated in  FIG. 3C  may be used to incubate chamber  44  with wash or elutant from start chamber  12 B. 
     It should be emphasized that the fluid that is in the output chamber  22  (which may contain a mixture of fluid from start chambers  12 A,  12 B) is prevented from returning or contaminating start chamber  12 B. Fluid transfer from the start chamber  12 A to the output chamber  22  is prevented because the fluid level in the output chamber  22  is regulated at a level below that of the fluid contained in the start chamber  12 A. Further, as seen in  FIG. 3C , the fluid that is contained in the output chamber  22  while able to partially flow upward in the ventilation channel  34 A, is unable to reach the start chamber  12 A. The fluid contained in the output chamber  22  is thus prevented from entering start chamber  12 A. Cross-contamination is prevented because fluid flow occurs in the radially-inward to radially-outward direction and the presence of the common vent hole  38  and the respective ventilation channels  34 A,  34 B are able to modulate flow from the respective start chambers  12 A,  12 B to the output chamber  22 . 
     Turning now to  FIG. 3D , a photographic image is taken of the microfluidic device  2  being rotated at low rotational speed of 100 rpm. At the lower rotational speed, the fluid contained in the microfluidic features  4  is now subject to a significantly reduced centrifugal force. While 100 rpm is illustrated, other low rotational speeds are contemplated such as those below about 200 rpm. The fluid level in the output chamber  22  is reduced because the pressure induced by centrifugal force has decreased, drawing air through the vent hole  38  (as seen by bubble in output chamber  22  in  FIG. 3D ). Further, the level of fluid within both ventilation channels  34 A,  34 B rises, leading the upper level of fluid in the chambers  12 A,  12 B to form a concave, meniscus-like surface in order to try to equalize the level of fluid with that in the ventilation channels  34 A,  34 B. Because of the reduction in the rotational speed of the substrate  6  from a high rotational speed to a low rotational speed, the siphon located in output channel  26  which regulates outflow from chamber  44  has been “primed.” Consequently, fluid outflow from the output chamber  22  via chamber  44  and output channel  26  is now permitted as seen by the passage of fluid down the output channel  26  in  FIG. 3D . 
     Turning now to  FIG. 3E , a photographic image of the microfluidic device  2  is illustrated after the rotational speed has been increased from the low 100 rpm rate to the higher 1500 rpm rate. Because the siphon has been primed, the flow of fluid from the chamber  12 B to the output chamber  22  and subsequently to the output channel  26  continues. The rate of fluid outflow from the output chamber  22  is matched by the transfer of fluid from the chamber  12 B to the output chamber  22 .  FIG. 3E  illustrates the dynamic interaction between the outflow of output chamber  22  and fluid transfer between start chamber  12 B and output chamber  22 . When the fluid level in the output chamber  22  drops below the junction between the ventilation channel  34 B and the output chamber  22  as a result of outflow through output channel  26 , the fluid in the ventilation channel  34 B drains out and air is permitted to enter the start chamber  12 B. Fluid flow between start chamber  12 B and output chamber  22  immediately resumes until the fluid level in the output chamber  22  again reaches the junction between the ventilation channel  34 B and the output chamber  22  and fluid is taken up ventilation channel  34 B. The dynamic interaction the junction between the ventilation channel  34 B and the output chamber  22  is illustrated by the presence of bubbles in ventilation channel  34 B as the result of the competing desires to uptake air and liquid. Further, as seen in  FIG. 3E , the fluid level is precisely located at the junction between the ventilation channel  34 B and the output chamber  22 , as compared to  FIG. 3C  in which the fluid level is shown as about halfway up  34 B. 
       FIG. 3F  illustrates a photographic image of the microfluidic device  2  rotated at 1500 rpm.  FIG. 3F  illustrates a state when start chamber  12 B has been completely drained, thereby permitting the fluid level in the output chamber  22  to fall below the junction of the ventilation channel  34 B and the output chamber  22 . Because there is no longer any inflow to the output chamber  22 , the dynamic interaction between start chamber  12 B and the output chamber  22  is no longer maintained.  FIG. 3G  illustrates transfer of fluid from the output chamber  22  to the chamber  44 , which empties via output channel  26 . 
     While embodiments of the present invention have been shown and described, various modifications may be made without departing from the scope of the present invention. The invention, therefore, should not be limited, except to the following claims, and their equivalents. Further, U.S. Provisional Patent Application No. 60/916,774 filed on May 8, 2007, to which this Application claims priority, is incorporated by reference as if set forth fully herein.