Abstract:
A system and method for controlling fluid flow within a microchannel includes a fluid circuit comprising a fluid outlet well and one or more fluid inlet wells, all in communication with a microchannel. A negative pressure differential is applied to the outlet well and fluid flow from an inlet well into the microchannel is controlled by opening or closing the inlet well to atmospheric pressure. To stop fluid flow from the inlet well, a negative pressure differential may be applied to the inlet well to equalize pressure between the inlet and outlet wells. By sequentially opening and closing different inlet wells to atmosphere, controlled amounts of different reagents can be serially introduced into the microchannel.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of application Ser. No. 12/164,992, filed Jun. 30, 2008, now U.S. Pat. No. 8,122,901, which is incorporated herein by reference in its entirety. 
    
    
     BACKGROUND 
     1. Field of the Invention 
     This invention relates to systems and methods for performing microfluidic assays. More specifically, the invention relates to systems and methods for controlling flow through a microchannel. 
     2. Discussion of the Background 
     The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (“PCR”) is perhaps the most well known of a number of different amplification techniques. 
     PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies to be detected and analyzed. In principle, each cycle of PCR could double the number of copies. In practice, the multiplication achieved after each cycle is always less than 2. Furthermore, as PCR cycling continues, the buildup of amplified DNA products eventually ceases as the concentrations of required reactants diminish. For general details concerning PCR, see Sambrook and Russell,  Molecular Cloning—A Laboratory Manual  (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000);  Current Protocols in Molecular Biology , F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley &amp; Sons, Inc., (supplemented through 2005) and  PCR Protocols A Guide to Methods and Applications , M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990). 
     Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the accumulation of products over time allows one to determine the efficiency of the reaction, as well as to estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR see  Real - Time PCR: An Essential Guide , K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004). 
     Several different real-time detection chemistries now exist to indicate the presence of amplified DNA. Most of these depend upon fluorescence indicators that change properties as a result of the PCR process. Among these detection chemistries are DNA binding dyes (such as SYBR® Green) that increase fluorescence efficiency upon binding to double stranded DNA. Other real-time detection chemistries utilize Foerster resonance energy transfer (FRET), a phenomenon by which the fluorescence efficiency of a dye is strongly dependent on its proximity to another light absorbing moiety or quencher. These dyes and quenchers are typically attached to a DNA sequence-specific probe or primer. Among the FRET-based detection chemistries are hydrolysis probes and conformation probes. Hydrolysis probes (such as the TaqMan® probe) use the polymerase enzyme to cleave a reporter dye molecule from a quencher dye molecule attached to an oligonucleotide probe. Conformation probes (such as molecular beacons) utilize a dye attached to an oligonucleotide, whose fluorescence emission changes upon the conformational change of the oligonucleotide hybridizing to the target DNA. 
     Commonly-assigned, co-pending U.S. application Ser. No. 11/505,358, entitled “Real-Time PCR in Micro-Channels,” the disclosure of which is hereby incorporated by reference, describes a process for performing PCR within discrete droplets flowing through a micro-channel and separated from one another by droplets of non-reacting fluids, such as buffer solution, known as flow markers. 
     Devices for performing in-line assays, such as PCR, within microchannels include microfluidic chips having one or more microchannels formed within the chip are known in the art. These chips utilize a sample sipper tube and open ports on the chip topside to receive and deliver reagents and sample material (e.g., DNA) to the microchannels within the chip. The chip platform is designed to receive reagents at the open ports—typically dispensed by a pipetter—on the chip top, and reagent flows from the open port into the microchannels, typically under the influence of a vacuum applied at an opposite end of each microchannel. The DNA sample is supplied to the microchannel from the ports of a micro-port plate via the sipper tube, which extends below the chip and through which sample material is drawn from the ports due to the vacuum applied to the microchannel. 
     In some applications, it is desirable that fluids from all of the top-side open ports flow into the microchannel, and, in other applications, it will be desirable that fluid flow from one or more, but less than all, of the top-side open ports. Also, to introduce different reagents into the microchannel via a sipper tube—typically extending down below the microchip—it is necessary to move the sipper tube from reagent container to reagent container in a sequence corresponding to the desired sequence for introducing the reagents into the microchannel. This requires that the processing instrument for performing in-line assays within the microfluidic channel of a microchip include means for effecting relative movement between the sipper tube and the different reagent containers. In addition, sipper tubes, which project laterally from a microchannel, are extremely fragile, thereby necessitating special handling, packaging, and shipping. 
     SUMMARY 
     Aspects of the invention are embodied in a method of controlling fluid flow within a microfluidic circuit including an inlet port through which fluid is introduced into the circuit, at least one microchannel for fluid flow in fluid communication with the inlet port, and an outlet port in fluid communication with the microchannel. The method comprises causing fluid flow from the inlet port into the microchannel by applying a first pressure to the outlet port and opening the inlet port to a second pressure higher than the first pressure, such as atmospheric pressure, and then stopping the fluid flow from the inlet port by closing the inlet port to the second pressure and applying the first pressure to the inlet port. 
     Further aspects of the invention are embodied in a system for controlling microfluidic flow. The system comprises a microfluidic circuit including at least one inlet port through which fluid is introduced into the circuit, at least one microchannel for microfluidic flow in fluid communication with the inlet port, and an outlet port in fluid communication with the microchannel. The system further includes at least one pressure source in communication with the outlet port and a valve mechanism operatively associated with each inlet port and in communication with the pressure source. The valve mechanism is adapted to selectively connect the inlet port (1) to a first pressure generated by the pressure source or (2) to a second pressure higher than the first pressure. 
     According to further aspects of the invention, the system includes a controller adapted to cause the pressure source to apply the first pressure to the outlet port and to cause the valve mechanism to open the inlet port to the second pressure to cause fluid to flow from the inlet port into the microchannel. After the inlet port has been open to the second pressure for a predetermined period of time, the controller causes the valve mechanism to close the inlet port to the second pressure and open the inlet port to the pressure source to stop flow from the inlet port into the microchannel. 
     Further aspects of the invention are embodied in a method for sequentially introducing predetermined amounts of different reaction fluids into a microchannel. The method comprises the steps of providing a microfluidic circuit including a microchannel, a plurality of inlet ports in fluid communication with the microchannel and through which different reaction fluids are introduced into the microchannel, and an outlet port in fluid communication with the microchannel. A negative pressure differential is applied to the outlet port, and a predetermined amount of reaction fluid is sequentially introduced into the microchannel from each of the inlet ports by sequentially opening each inlet port to higher pressure for a predetermined period of time while the other inlet ports are closed to cause a predetermined amount of fluid to flow from that inlet port into the microchannel, and, after the predetermined period of time, stopping fluid flow from that inlet port by closing that inlet port to the higher pressure and applying the negative pressure differential to that inlet port for period of time to equalize the pressure between the inlet port and the inlet of the microchannel, and then shutting off the valve to the inlet port. 
     The above and other aspects and embodiments of the present invention are described below with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments of the present invention. In the drawings, like reference numbers indicate identical or functionally similar elements. 
         FIG. 1  is a schematic view of a microfluidic chip and flow control system embodying aspects of the present invention. 
         FIG. 2  is a schematic view of an alternative embodiment of a microfluidic chip and flow control system embodying aspects of the present invention. 
         FIG. 3  is a schematic view of a second alternative embodiment of a microfluidic chip and flow control system embodying aspects of the present invention. 
         FIG. 4  is a flow chart illustrating steps of performing a sequential, multiplex assay within a micro channel in accordance with aspects of the present invention. 
         FIG. 5  shows time history profiles of the flows of DNA, polymerase, assay primers, and the resulting sample test stream within a microchannel. 
         FIG. 6  shows time history profiles of intermittent application of negative pressure and atmospheric pressure to a fluid input well of a microfluidic chip to achieve flow metering. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     As used herein, the words “a” and “an” mean “one or more.” Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. 
     A system for microfluidic flow embodying aspects of the present invention is shown in  FIG. 1 . The system includes a microfluidic circuit which, in the illustrated embodiment, is carried on a microfluidic chip  10 . Microfluidic chip  10  includes inlet ports  12 ,  14 ,  16 , a microchannel  20  that is in fluid communication with the inlet ports  12 ,  14 ,  16 , and an outlet port  18  also in fluid communication with the microchannel  20 . The embodiment shown in  FIG. 1  is exemplary; the microfluidic circuit may include more or less than three inlet ports and may include more than one microchannel in communication with some or all of the inlet ports. The microfluidic circuit may also include more than one outlet port. Fluid is introduced into the circuit through the fluid inlet ports  12 ,  14 , and  16 . Fluid may be provided to the fluid inlet ports in any appropriate manner known in the art. Or, alternatively, fluid may be provided to the fluid inlet ports by means of a fluid-containing cartridge coupled to each port in a fluid-communicating manner as described in commonly assigned U.S. patent application Ser. No. 11/850,229 “Chip and cartridge design configuration for performing microfluidic assays”, the disclosure of which is hereby incorporated by reference. 
     The microfluidic chip  10  may be formed from glass, silica, quartz, or plastic or any other suitable material. 
     Fluid is collected from the microchannel  20  through the fluid outlet  18  and may be deposited in any appropriate waste reservoir, such as, for example, a chip as described in the commonly assigned U.S. patent application Ser. No. 11/850,229. 
     Fluid movement through the circuit is generated and controlled by means of a negative pressure differential applied between the outlet port  18  and one or more of the inlet ports  12 ,  14 ,  16 . Application of a negative pressure differential between the outlet port  18  and one or more of the inlet ports  12 ,  14 ,  16  will cause fluid flow from the inlet port(s), through the microchannel  20  and to the outlet port  18 . A pressure differential can be generated by one or more pressure sources, such as negative pressure source  22 , which, in one embodiment, may comprise a vacuum pump. In the illustrated embodiment, pressure differentials between the outlet port  18  and the inlet ports  12 ,  14 ,  16  is controlled by means of pressure control valves controlling pressure at each of the inlet ports  12 ,  14 ,  16  and the outlet port  18 . 
     More specifically, a pressure control valve  30  is arranged in communication with the pressure source  22  and the outlet port  18 . Similarly, a pressure control valve  24  is arranged in communication with the inlet port  12 , a pressure control valve  26  is arranged in communication with the inlet port  14 , and a pressure control valve  28  is arranged in communication with the inlet port  16 . Arrangements having more than three inlet ports would preferably have a pressure control valve associated with each inlet port. In the illustrated embodiment of  FIG. 1 , valves  24 ,  26 ,  28  are three-way valves which may selectively connect each associated inlet port  12 ,  14 ,  16 , respectively, to either atmospheric pressure, represented by the circled letter “A”, or an alternative pressure source, which may be the negative pressure source  22 . That is, in the illustrated embodiment, valve  24  is in communication pressure source  22  via pressure line  32  and is in communication with inlet port  12  via pressure line  34 . Valve  26  is in communication with pressure source  22  via pressure line  36  and is in communication with inlet port  14  via pressure line  38 . Valve  28  is in communication with pressure source  22  via pressure line  40  and is in communication with inlet port  16  via pressure line  42 . Valve  30  is connected via pressure line  44  to the pressure source  22  and by pressure line  46  to outlet port  18 . In the illustrated embodiment, valve  30  is also a three-way valve for selectively connecting the outlet port  18  to either atmospheric pressure, indicated by the circled “A”, or to the pressure source  22 . 
     Pressure source  22  and valves  24 ,  26 ,  28 ,  30  may be controlled by a controller  50 . Controller  50  is connected via a control line  52  to the pressure source  22 , via a control line  54  to the valve  24 , via a control line  56  to valve  26 , via a control line  58  to valve  28 , and via a control line  60  to valve  30 . Controller  50  may also be connected to one or more of the various components wirelessly or by other means known to persons of ordinary skill in the art. Controller  50  may comprise a programmed computer or other microprocessor. 
     As mentioned above, fluid flow from an inlet port  12 ,  14 , and/or  16  through the microchannel  20  and to the outlet port  18  is generated by the application of a negative pressure differential between the outlet port  18  and one or more of the inlet ports. More specifically, to generate a fluid flow from inlet port  12 , a negative pressure is applied to the outlet port  18  by connecting the negative pressure source  22  to the outlet port  18  via the control valve  30  and pressure lines  44  and  46 . Inlet port  12  is opened to atmospheric pressure by valve  24 . This creates the negative pressure differential between the outlet port  18  and the inlet port  12 . Assuming that fluid flow from other inlet ports is not desired while fluid is flowing from the inlet port  12 , inlet port  14  is closed to atmospheric pressure by valve  26  and inlet port  16  is closed to atmospheric pressure by valve  28 . To stop fluid flow from the inlet port  12 , valve  24  is activated (e.g., via the controller  50 ) to close off the inlet port  12  to atmospheric pressure. To rapidly stop the flow of fluid from the inlet port  12 , it may be desirable to connect the inlet port  12  to the negative pressure source  22  via the control valve  24  for a period of time sufficient to equalize the pressure between the inlet port  12  and the inlet of the microchannel, and then shut off control valve  24  to maintain this pressure equilibrium. 
     A predetermined volume of fluid can be introduced into the microchannel  20  from any of the inlet ports  12 ,  14 , and  16 —assuming the flow rate generated by the pressure differential between the outlet port  18  and the applicable inlet port is known—by maintaining the pressure differential for a period of time which, for the generated flow rate, will introduce the desired volume of fluid into the microchannel  20 . Maintaining the pressure differential can be effected by proper control of the pressure control valves associated with the inlet ports and the outlet port. 
     Activation and timing of the control valve  24  may be controlled by the controller  50 . 
     To then generate fluid flow from the inlet port  14 , valve  26  is activated (e.g., by controller  50 ) to open inlet port  14  to atmospheric pressure while negative pressure is applied to the outlet port  18 , thus creating the negative pressure differential between the outlet port  18  and the inlet port  14 . Fluid flow from the inlet port  14  is stopped by activating valve  26  to close inlet port  14  to atmospheric pressure, and, to rapidly stop flow from the inlet port  14 , valve  26  opens the inlet port  14  to the negative pressure source  22  for a period of time sufficient to equalize the pressure between the inlet of the microchannel and the inlet port  14 , and then shut off valve  26  to maintain this pressure equilibrium. 
     Similarly, to generate fluid flow from the inlet port  16 , valve  28  is activated (e.g., by controller  50 ) to open inlet port  16  to atmospheric pressure while negative pressure is applied to the outlet port  18 , thus creating the negative pressure differential between the outlet port  18  and the inlet port  16 . Fluid flow from the inlet port  16  is stopped by activating valve  28  to close inlet port  16  to atmospheric pressure, and, to rapidly stop flow from the inlet port  16 , valve  28  opens the inlet port  16  to the negative pressure source  22  for a period of time sufficient to equalize the pressure between the inlet of the microchannel and the inlet port  16 , and then shut off valve  28 . 
       FIGS. 2 and 3  show alternative arrangements for controlling the pressure differential between an outlet port and one or more of the inlet ports of a microfluidic circuit.  FIG. 2  shows a system similar to that shown in  FIG. 1  except that each inlet port  12 ,  14 ,  16  is coupled to two two-way valves as opposed to a single three-way valve. More specifically, inlet port  12  is coupled to a first two-way valve  24   a  for selectively connecting the inlet port  12  to the pressure source  22  via pressure lines  32  and  62 . Inlet port  12  is also coupled to a second two-way valve  24   b  for selectively connecting the inlet port  12  to atmospheric pressure “A” via pressure line  64 . 
     Similarly, inlet port  14  is coupled to a first two-way valve  26   a  for selectively connecting port  14  to the pressure source  22  via pressure lines  36  and  66  and to a second two-way valve  26   b  for selectively connecting the inlet port  14  to atmospheric pressure via pressure line  68 . Inlet port  16  is coupled to a first two-way valve  28   a  for selectively connecting the inlet port  16  to the pressure source  22  via pressure lines  40  and  70  and to a second two-way valve  28   b  for selectively connecting the inlet port  16  to atmospheric pressure via pressure line  72 . 
     In the system shown in  FIG. 2 , outlet port  18  is coupled to two-way valve  76  for selectively connecting the outlet port  18  to the pressure source  22  via pressure lines  44  and  46 . 
     Controller  50  controls the negative pressure source  22  via control line  52 , controls two-way valve  76  via control line  60 , controls two-way valve  24   a  via control line  72 , and controls two-way valve  24   b  via control line  74 . Controller  50  is also linked to valves  26   a ,  26   b ,  28   a , and  28   b  for controlling those valves, but the control connections between the controller  50  and the respective valves are not shown in  FIG. 2  so as to avoid unnecessarily cluttering the Figure. 
       FIG. 3  shows an alternative arrangement of the system embodying aspects of the present invention. In the embodiment of  FIG. 3 , each inlet port  12 ,  14 ,  16  is coupled to a three-way valve for selectively connecting the port either to pressure source #1  22 , or pressure source #2  80 . More specifically, inlet port  12  is coupled to valve  82  configured to selectively connect the inlet port  12  to pressure source #1  22  via pressure lines  88 ,  90 , and  100  or to pressure source #2  80  via pressure lines  96 ,  98 , and  100 . Inlet port  14  is coupled to valve  84  configured to selectively connect inlet port  14  to the pressure source #1  22  via pressure lines  90  and  102  or to pressure source #2  80  via pressure lines  96  and  102 . Inlet port  16  is coupled to pressure valve  86  configured to selectively couple port  16  to pressure source #1  22  via pressure lines  90 ,  92  and  104  or to pressure source #2  80  via pressure lines  96 ,  94  and  104 . Outlet port  18  is coupled to valve  122  for selectively connecting outlet port  18  to pressure source #1  22  via pressure lines  106  and  46 . 
     Controller  50  controls pressure source #1  22  via control line  52  and controls pressure source #2  80  via control line  110 . Controller  50  also controls pressure valve  120  via control line  118 , pressure valve  82  via control line  116 , pressure valve  84  via control line  114 , and pressure valve  86  via control line  112 . 
     To generate fluid flow from inlet port  12 , control valve  120  is activated (e.g., by controller  50 ) to connect outlet port  18  to pressure source #1  22 , and control valve  82  is activated to connect inlet port  12  to pressure source #2  80 . The pressure generated by pressure source #2  80  is preferably greater than the pressure generated by pressure source #1  22 . Thus, a negative pressure differential is created between outlet port  18  and inlet port  12 . Inlet ports  14  and  16  are connected, by valves  84  and  86 , respectively, to pressure source #1  22  for a period of time to equalize the pressure between the inlet port and the inlet of the microchannel, and then shut off valves  84  and  86  to maintain an established pressure, so there is no pressure differential between inlet ports  14  and  16  and the inlet of the microchannel and thus no fluid flow from inlet ports  14  and  16  to outlet port  18 . To stop fluid flow from inlet port  12 , control valve  82  is activated to connect inlet port  12  to pressure source #1  22  to equalize the pressure between the outlet port  18  and the inlet port  12  and then shut off control valve  82 . 
     To generate fluid flow from inlet port  14 , control valve  84  is activated to connect inlet port  14  to pressure source #2  80  to create a negative pressure differential between outlet port  18  and inlet port  14 . Valves  82  and  86  to inlet ports  12  and  16  are closed off, so there is no pressure differential between inlet ports  12  and  16  and inlet of the microchannel and thus no fluid flow from inlet ports  12  and  16  to outlet port  18 . To stop fluid flow from inlet port  14 , control valve  84  is activated to connect inlet port  14  to pressure source #1  22  to equalize the pressure between the inlet of the microchannel and the inlet port  14 , and then valve  84  is shut off to maintain this pressure equilibrium. 
     To generate fluid flow from inlet port  16 , control valve  86  is activated to connect inlet port  16  to pressure source #2  80  to create a negative pressure differential between outlet port  18  and inlet port  16 . Inlet ports  12  and  14  are connected, by valves  82  and  84 , respectively, to pressure source #1  22  for a period of time to equalize the pressure between the inlet port and the inlet of the microchannel, and then shut off valves  84  and  86  to maintain an established pressure, so there is no pressure differential between inlet ports  12  and  14  and outlet port  18  and thus no fluid flow from inlet ports  12  and  14  to outlet port  18 . To stop fluid flow from inlet port  16 , control valve  86  is activated to connect inlet port  16  to pressure source #1  22  to equalize the pressure between the outlet port  18  and the inlet port  16 , and then shut off control valve  86 . 
     As an alternative arrangement, three-way valves  82 ,  84 ,  86  could each be replaced by two two-way valves for selectively connecting each associated inlet port with pressure source #1  22  or pressure source #2  80 . 
     Suitable valves for use in the present invention include two-way and three-way solenoid valves by IQ Valves Co., Melbourne, Fla. and The Lee Company, Westbrook, Conn. 
     The systems shown in  FIGS. 1 ,  2  and  3  can be utilized in a process for performing PCR within discreet droplets of assay reagents flowing through a microchannel and separated from one another by droplets of non-reacting fluids, such as buffer solution, as is described in commonly assigned, co-pending U.S. application Ser. No. 11/505,358. The process will be described with reference to  FIGS. 4 and 5 . 
       FIG. 4  is a flow chart illustrating the steps for performing PCR within discreet droplets flowing through a microchannel, and  FIG. 5  shows time history curves representing the flow of various materials through the channel. The process will be described with reference to the system shown in  FIG. 1 . It should be understood, however, that the process could also be performed with the systems of  FIG. 2  or  3  or a hybrid combination of the systems of  FIGS. 1 ,  2 , and  3 . 
     Referring to  FIG. 4 , at step  130  negative pressure is applied to the outlet port  18  and all of the inlet ports  12 ,  14 ,  16 , etc, by connecting the ports, via the associated valves, to negative pressure source  22 , and by shutting off the valves to inlets  12 ,  14 , and  16 . This is known as a stop condition as there is no pressure differential between the waste port and any inlet port, and thus no fluid flow into the microchannel  20 . 
     In step  132 , the valve coupled to the DNA/buffer inlet port (e.g., valve  24  associated with inlet port  12 ) is switched from negative pressure to atmospheric pressure to generate a sample flow condition (i.e., a negative pressure differential between outlet port  18  and inlet port  12 ) as shown by the curve  162  in  FIG. 5 . Although not shown in  FIG. 4 , a valve coupled to a polymerase inlet port may also be switched from negative pressure to atmospheric pressure to generate a polymerase flow as shown by curve  164  in  FIG. 5 . The DNA/buffer mixture is combined into a common flow through the microchannel  20 . 
     In step  134 , a timer delay is implemented to fill the channels with the DNA/buffer (and optionally polymerase) mixture. 
     In step  136 , the valve coupled to a PRIMER1 inlet port (e.g., valve  26  associated with inlet port  14 ) is switched from negative pressure to atmospheric pressure to generate a primer flow condition into the microchannel  20  to be mixed with the sample flow stream. A timer delay that is proportional to the desired timer injection volume is implemented in step  138  to control the volume of PRIMER1 that flows into the mixture. In step  140 , the valve coupled to PRIMER1 inlet port is switched to the original condition, i.e., negative pressure with the valve shutting off, to stop primer flow, thereby generating the first portion of flow curve  166  (through clock interval  4 ) in  FIG. 5 . 
     A timer delay proportional to a desired spacer interleave is implemented in step  142 . This is a sample flow condition without primer flowing. 
     In step  144 , the valve coupled to the primer2 inlet port (e.g., valve  28  associated with inlet port  16 ) is changed from negative pressure with the valve shutting off to atmospheric pressure to generate a primer flow condition into the microchannel  20  to be mixed with the sample flow stream. A timer delay that is proportional to the desired injection volume of primer2 is implemented in step  146 . And, in step  148 , the valve coupled to the primer2 inlet port is switched back to the original, negative pressure with the valve in the shut off condition to stop the flow of primer2. Steps  144 ,  146 , and  148  generate the flow curve  168  shown in  FIG. 5 . 
     In step  150 , a primer injection sequence is repeated for additional primers and additional, discrete injections of previously-injected primers until the complete assay conditions are generated, thus generating flow curve  170 . The resulting sample test stream flow curve is designated by curve  172  in  FIG. 5  in which each “hump” in the curve represents a discrete volume of a primer mixed in the sample flow stream. A separate PCR (or other) assay can be performed in each discrete volume (or bolus) of sample/primer mixture. 
     In step  152 , PCR thermal cycling is performed on the flowing microfluidic stream thereby generating a PCR amplification reaction within each test bolus. In step  154 , a DNA thermal melt analysis is performed on the flowing microfluidic stream. And, in step  156 , a sequence of assay thermal melt data is generated for each test bolus for a multiplex assay performed within the microchannel  20 . 
     As shown in  FIG. 6 , any valve coupled to an inlet port can be operated in a pulse width modulated manner to regulate the volume of fluid injected at the inlet port. For example, as described above, a valve coupled to an inlet port can be set to a flow condition for a predetermined period of time corresponding to a desired volume of fluid to be injected into the microchannel. A smaller volume of fluid can be injected by having the valve coupled to the inlet port set to the flow condition for a shorter period of time. It may be desirable, however, to produce reaction droplets of a specified physical size and, thus, it may be desirable to have fluid flow from the inlet port for the specified period of time (and not the shorter time corresponding to the smaller volume). To produce a lower volume of fluid flow from an inlet port while maintaining the flow from the port for a specified period of time, the valve coupled to the port may be modulated between negative pressure and atmospheric pressure (or other higher pressure) over the desired flow period, as shown in curves  174  and  176  in  FIG. 6 . The resulting pressure at the inlet port is indicated by curve  180  in  FIG. 6 . The resulting reagent flow, as shown in curve  178  in  FIG. 6 , is a generally constant flow over the entire flow period at a flow rate that will result in a lower volume of fluid injected than if the inlet valve were kept open to atmospheric pressure for the entire flow period. 
     While various embodiments/variations of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments. Further, unless stated, none of the above embodiments are mutually exclusive. Thus, the present invention may include any combinations and/or integrations of the features of the various embodiments. 
     Additionally, while the processes described above and illustrated in the drawings are shown as a sequence of steps, this was done solely for the sake of illustration. Accordingly, it is contemplated that some steps may be added, some steps may be omitted, and the order of the steps may be re-arranged.