Patent Publication Number: US-2018029037-A1

Title: Microfluidic Metering of Fluids

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional application of U.S. Ser. No. 14/463,865, filed Aug. 20, 2014, which claims benefit of priority from U.S. Provisional Application Ser. No. 61/869,373, filed on Aug. 23, 2013. 
    
    
     TECHNICAL FIELD 
     This document relates to methods and materials involved in metering fluids. For example, this document provides microfluidic channels configured to precisely meter small volumes of samples and/or reagents, which can be used in microfluidic systems for diagnosing one or more disease conditions. 
     BACKGROUND 
     In parts of the world, diseases such as HIV infection (and various stages of the disease), syphilis infection, malaria infection, and anemia are common and debilitating to humans, particularly to pregnant women. For example, nearly 3.5 million pregnant women are HIV-infected, and nearly 700,000 babies contract HIV from their mothers each year. These infant HIV infections can be prevented by identifying and treating mothers having HIV. In addition, nearly 20% of pregnant women in developing countries are infected with syphilis, leading to more than 500,000 infant stillbirths and deaths each year. Nearly 10,000 women and 200,000 infants die each year from malaria during pregnancy, and nearly 45% of pregnant women in developing countries suffer from anemia as a result of, for example, worm infections, parasites, and/or nutritional deficiencies. Anemia can adversely affect a pregnant woman&#39;s chance of surviving post-partum hemorrhage and stunt infant development. About 115,000 maternal deaths and 500,000 infant deaths have been associated with anemia in developing countries. 
     SUMMARY 
     This document provides devices and methods for metering fluids. Assays on small amounts of sample can require precise metering of small volumes of sample and required reagents. Additionally, some assays rely upon the exclusion of air from an assay chamber. In some cases, the devices and methods provided herein can deliver a precise volume of one or more fluids. In some cases, the devices and methods provided herein can deliver multiple fluids to a common channel without the presence of air bubbles along the interface between fluids. 
     A device for metering fluids provided herein, in some cases, includes a metering channel being defined between a metering inlet and a metering outlet, a loading channel having a loading inlet and intersecting the metering channel at a loading-metering intersection point, and an outflow channel having an outflow outlet and intersecting the metering channel at a metering-outflow intersection point. The metering channel can define a volume of fluid to be metered between the metering-outflow intersection point and the loading-metering intersection point. The inlets and outlets of the devices and systems provided herein can, in some cases, include valves to control the flow of fluids into and out of said devices. In some cases, the metering-outflow intersection point and/or the loading-metering intersection point can include a capillary-stop geometry to restrict fluid from heading down particular paths (e.g., when fluid is flowing due to capillary action). 
     A device for metering fluids provided herein, in some cases, includes a plurality of metering channels each having a metering inlet and each intersecting at least one of the other metering channels at one or more metering-metering intersection points, an outflow channel having an outflow outlet and intersecting a first of said plurality of metering channel at a metering-outflow intersection point, and a loading channel having a loading inlet and intersecting a second of said plurality of metering channel at a loading-metering intersection point. Each metering channel can define a volume of fluid to be metered between the two of the intersection points. The inlets and outlets of the devices and systems provided herein can, in some cases, include valves to control the flow of fluids into and out of said devices. The metering-outflow intersection point, the loading-metering intersection point, and/or the one or more metering-metering intersection points can each have a capillary-stop geometries, which can restrict fluid from heading down particular paths (e.g., when fluid is flowing due to capillary action). 
     A method for metering fluids provided herein, in some cases, includes delivering fluids in sequence to fill the metering channel with a metered fluid and a loading channel with a loading fluid followed by pushing the fluids out of the channels. 
     In some cases, filling the metering channel can include opening a metering inlet valve and a metering outlet valve, closing the other valves, and pumping or pulling the metered fluid into the metering channel. For example, by having the other valves closed, pressure within other channels can prevent the metered fluid from flowing into the other channels. In some cases, filling the metering channel can include delivering a metered fluid to a metering inlet such that the metered fluid is wicked by capillary action through the metering channel. For example, the metering channel can be a microfluidic channel having a hydrophilic surface. In some cases, intersection points and/or the metering outlet can have capillary-stop geometries such that wicked fluid is not wicked into other channels or past the metering outlet. In some cases, a combination of valves, capillary-stop geometries, pumping, and wicking can be used to fill the metering channel without a substantial volume of metered fluid being delivered into intersecting channels provided herein. 
     In some cases, filling the loading channel can include opening the loading inlet valve and one of the outlet valves (e.g., a loading outlet valve), closing the other valves, and pumping or pulling the loading fluid into the loading channel. For example, by having the other valves closed, pressure within other channels can prevent the loading fluid from flowing into an intersecting metering channel. In some cases, filling the loading channel can include delivering a loading fluid to a loading inlet such that the loading fluid is wicked by capillary action through the loading channel. For example, the loading channel can be a microfluidic channel having a hydrophilic surface. In some cases, a loading-metering intersection point and/or a loading outlet can have capillary-stop geometries such that wicked fluid is not wicked into an intersecting metering channel or past the loading outlet. In some cases, a combination of valves, capillary-stop geometries, pumping, and wicking can be used to fill the loading channel without a substantial volume of loading fluid being delivered into an intersecting metering channel. 
     The metering channel and the loading channel can be filled in either order. Excess fluids can exit the metering outlet or the loading outlet. Although the metered and loading fluids form an interface at the loading-metering intersection point, the microfluidic geometry at the loading-metering intersection point can limit mixing of the fluids at the loading-metering intersection point. The fluids can be pushed out of the arrangement by closing a metering inlet and a metering outlet, and delivering fluid through the loading inlet to push loading fluid through the loading-metering intersection point to push metered fluid through the metering-outflow intersection point, through the outflow channel, and thus through the outflow outlet. For example, a fluid (e.g., additional loading fluid) can be pumped through the loading inlet valve. The volume of the metered fluid delivered through the outflow outlet valve is defined by the geometry of the metering channel between the loading-metering intersection point and the metering-outflow intersection point. 
     In some cases, the loading channel does not include a loading outlet. In cases where the loading channel does not include a loading outlet, the loading channel can be filled with the loading fluid prior to filling the metering channel with the metered fluid. In cases where the loading channel does not include a loading outlet, the metering outlet or an outflow outlet can be opened and loading fluid pumped or pulled into the loading channel until excess loading fluid passes through the loading-metering intersection point into the metering channel. Excess loading fluid in the metering channel can be removed from the metering channel when the metering channel is filled with metered fluid, which would push excess loading fluid out of the metering outlet. 
     In some cases, a method of metering fluids provided herein includes metering multiple fluids. In some cases, a diagnostic device provided herein can require a precise metering of a biological sample (e.g., blood) and precise metering of a reagent. For example, an assay may require a precise metering of one or more staining reagents and/or a washing reagent. A method of metering multiple fluids can include filling multiple metering channels with different metered fluids, each metering channel having a metering inlet and intersecting at least one of the other metering channels, filling a loading channel with a loading fluid, the loading channel intersecting a first metering channel at a loading-metering intersection point, and delivering metered amounts of different metered fluids in succession through an outflow channel that intersects a second metering channel at a metering-outflow intersection point by delivering a fluid (e.g., additional loading fluid) through the loading inlet. 
     The methods and devices provided herein can provide a reliable and inexpensive method to meter small amounts of fluid precisely. The methods and devices provided herein also can provide a train of metered fluids in a single channel. In some cases, interfaces between fluids in a train of fluids can be substantially free of air bubbles. For example, in some cases, diagnostic assays can require the introduction of sample and/or reagent into an assay chamber without the presence of air. Air bubbles can lodge in a channel and alter flow patterns, trap fluids behind them, strip captured cells off the walls of a channel, interfere with imaging if the assay relies in it, or a combination thereof. Devices and systems provided herein can manage air bubbles in one or more of the channels included therein by having geometries that have high surface tension and by ensuring laminar in the channels, such that bubbles stick together and follow the flow past intersections. 
     The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description and drawings, and from the claims. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         FIGS. 1A-1D  depict a first example of an arrangement of microfluidic channels and illustrate how that arrangement can be used to precisely meter a predetermined amount of a metered fluid. 
         FIGS. 2A-2D  depict a second example of an arrangement of microfluidic channels and illustrate how that arrangement can be used to precisely meter a predetermined amount of a metered fluid. 
         FIG. 3  depicts an example of a capillary stop. 
         FIGS. 4A-4C  depict a third example of an arrangement of microfluidic channels and illustrate how that arrangement can be used to precisely meter a predetermined amount of a metered fluid. 
         FIG. 5  depict an example of an assay card used to meter blood and reagent into an assay chamber. 
         FIGS. 6A-6F  depict a fourth example of an arrangement of microfluidic channels and illustrate how that arrangement can be used to precisely meter a predetermined amount of a first metered fluid and a second metered fluid. 
     
    
    
     Like reference symbols in the various drawings indicate like elements. 
     DETAILED DESCRIPTION 
     This document provides methods and devices related to metering precise amounts of fluid. In some cases, the methods and devices provided herein relate to diagnosing one or more disease conditions (e.g., HIV infections, syphilis infections, malaria infections, anemia, gestational diabetes, and/or pre-eclampsia). As described herein, a biological sample can be collected from a mammal (e.g., pregnant woman) and analyzed using a kit including a metering device provided herein to determine whether or not the mammal has any of a group of different disease conditions. In the case of a device that diagnoses multiple disease conditions, the analysis for each disease condition can be performed in parallel such that the results for each condition are provided at essentially the same time. In some cases, the methods and devices provided herein can be used outside a clinical laboratory setting. For example, the methods and devices provided herein can be used in rural settings outside of a hospital or clinic. Any appropriate mammal can be tested using the methods and materials provided herein. For example, dogs, cats, horses, cows, pigs, monkeys, and humans can be tested using a diagnostic device or kit provided herein. 
     The methods and devices provided herein can provide precise metering of small volumes of blood and/or reagents for tests that determine whether or not the mammal has one or more disease conditions. In some cases, methods and devices provided herein can repeatedly deliver a predetermined volume of fluid with a deviation of not more than 5% (e.g., not more than 4%, not more than 3%, not more than 2%, not more than 1%, or not more than 0.5% deviation). The deviation of a device or method provided herein can be assessed by metering ten consecutive volumes of fluid including a reporter molecule (e.g., a fluorescent additive or radiolabel such as tritium), using a signal from the reporter molecule to determine an average volume of each metered fluid (e.g., using a plate-reader), and determining the maximum deviation from that average volume and dividing that maximum deviation by the average volume to determine the deviation. In some cases, an average volume of metered fluid can be determined using Karl Fisher analysis. In some cases, methods and devices provided herein can be arranged to meter a predetermined volume of fluid of 500 μL or less (e.g., 250 μL or less, 100 μL or less, 75 μL or less, 50 μL or less, 25 μL or less, 10 μL or less, or 5 μL or less). In some cases, methods and devices provided herein can be arranged to meter a predetermined volume of fluid of between 0.5 μL and 500 μL with a maximum plus or minus deviation of 5%, a predetermined volume of fluid of between 1 μL and 250 μL with a maximum plus or minus deviation of 4%, a predetermined volume of fluid of between 2 μL and 100 μL with a maximum plus or minus deviation of 3%, a predetermined volume of fluid of between 5 μL and 50 μL with a maximum plus or minus deviation of 2%, or a predetermined volume of fluid of between 8 μL and 20 μL with a maximum plus or minus deviation of 1%. 
     In some cases, the methods and devices provided herein can deliver multiple fluids through a common channel (e.g., an outflow channel) in sequence. In some cases, multiple fluids delivered sequentially through a common channel can be precisely metered. In some cases, methods and devices provided herein can meter one or more fluids through a common channel without creating air bubbles at the interface of the one or more metered fluids and fluids coming thereafter. For example, methods and devices provided herein can deliver blood and one or more reagents sequentially through a common channel towards an assay chamber without air bubbles being introduced into the common channel. In some cases, air bubbles can lodge in the channels and alter flow patterns, trap fluids behind them that then can&#39;t be washed out, strip captured cells off the walls of a channel, interfere with imaging if the assay relies in it, or a combination thereof. In some cases, a devices and systems provided herein include geometries that promote laminar flow such that bubbles tend to stick together and flow past intersections. 
     Methods and devices provided herein can use a geometry of an arrangement of channels to meter the volume of one or more fluids, which can be achieved without a need to form a vacuum. In some cases, methods and devices provided herein can provide a train of fluids without forming air bubbles between each fluid. In some cases, methods and devices provided herein can precisely meter fluids without relying on the precision of pumps. 
       FIGS. 1A-1D  illustrates one basic approach.  FIG. 1A  depicts a first example of an arrangement  100  of microfluidic channels prior to introduction of fluid. The arrangement includes a metering channel  110  having a metering inlet P 2  and a metering outlet P 5 . Metering channel  110  intersects a loading channel  120  and an outflow channel  150 . Outflow channel  150  and metering channel  110  intersect at a metering-outflow intersection point  112 . The portion of the metering channel  110  between the metering-outflow intersection point  112  and the metering outlet P 5  forms a metering waste channel  118 . Loading channel  120  and metering channel  110  intersect at a loading-metering intersection point  114 . The portion of metering channel  110  between the metering-outflow intersection point  112  and the loading-metering intersection point  114  defines the metering portion of a metering channel  110 . Accordingly, the geometry of metering channel  110  between metering-outflow intersection point  112  and loading-metering intersection point  114  determines the volume of the fluid metered. As shown, loading channel  120  can include a loading inlet P 1 , a loading waste channel  128 , and a loading outlet P 3 . Outflow channel  150  can include an outflow outlet P 6 . 
     Each of inlets and outlets P 1 , P 2 , P 3 , P 5 , and P 6  can include a valve, which can be used to control the flow of fluid past each inlet or outlet. In some cases, valves at inlets and outlets P 1 , P 2 , P 3 , P 5 , and P 6  can be opened and closed to control the flow of fluids therethrough. In some cases, capillary-stop geometry can be used at inlets and outlets P 1 , P 2 , P 3 , P 5 , and P 6  to prevent the flow of fluid past the inlet or outlet due to wicking of the fluid, but allow for the fluid to be pumped there through. In each arrangement provided herein, each inlet or outlet can include a valve, capillary-stop geometry, or a combination thereof to control the flow of fluid there through. 
     In some cases, arrangement  100  can include air prior to the introduction of fluids. Fluids can push the air out as they fill the channels. In some cases, ambient air can be evacuated prior to the introduction of fluids. In some cases, an inert gas (e.g. Nitrogen, Argon) can be within the arrangement  100  prior to the introduction of fluids. 
       FIG. 1B  depicts a first step where loading inlet P 1  and loading outlet P 3  permit for fluid flow there through and inlets and outlets P 2 , P 5 , and P 6  restrict the flow of fluid, as indicated by the shading in  FIG. 1B . A loading fluid  126  is introduced through loading inlet P 1  to fill loading channel  120  with loading fluid  126 . Excess amounts of loading fluid  126  exit loading channel  120  through loading outlet P 3 , thus the specific volume of the loading fluid  126  introduced into the loading channel  120  does not matter as long as it is sufficient to fill the volume of the loading channel  120 . Microfluidic geometry of loading channel  120  and metering channel  110  at loading-metering intersection point  114  can limit the flow of loading fluid  126  into metering channel  110 . 
       FIG. 1C  depicts a second step where metering inlet P 2  and metering outlet P 5  permit for fluid flow there through and inlets and outlets P 1 , P 3 , and P 6  restrict the flow of fluid, as indicated by the shading in  FIG. 1C . A metered fluid  116  is introduced through the metering inlet P 2  to fill metering channel  110  with metered fluid  116 . Excess amounts of metered fluid  116  exit metering channel  110  through metering outlet P 5 , thus the specific volume of metered fluid  116  introduced into the metering channel  110  does not matter as long as it is sufficient to fill the volume of the metering channel  110 . Microfluidic geometry of channels  110 ,  120 , and  150  at metering-outflow intersection point  112  and loading-metering intersection point  114 , optionally along with the closing of valves at inlets and outlets P 1 , P 3 , and P 6  or the use of capillary-stop geometries, can limit the flow of the metered fluid  116  into loading channel  120  or outflow channel  150 . In some cases, the order of introduction of metered fluid  116  and loading fluid  126  into metering channel  110  and loading channel  120  can be reversed. The successive introduction of the metered fluid  116  into the metering channel  110  and loading fluid  126  into the loading channel  120  can create a bubble free interface between the two fluids at the loading-metering intersection point. 
       FIG. 1D  depicts a third step where loading inlet P 1  and outlet P 6  permit for fluid flow there through and inlets and outlets P 2 , P 3 , and P 5  restrict the flow of fluid, as indicated by the shading in  FIG. 1D . An additional amount of loading fluid  126  can be introduced through the loading inlet P 1  to push loading fluid  126  in loading channel  120  into metering channel  110  at loading-metering intersection point  114 , which thus pushes metered fluid  116  in metering channel  110 , between the two intersection points  112  and  114 , into outflow channel  150  at metering-outflow intersection point  112 , and out of the outflow outlet P 6 . The volume of the metered fluid  116  pushed into the outflow channel  150  and through outflow outlet P 6  is dictated by the geometry between the two intersection points  112  and  114 . In some cases, the fluid introduced into loading channel  120  and used to thus push the fluids into the outflow channel  150  can be a different fluid than the loading fluid. 
     In some cases, the loading channel can intersect the metering channel, but not have a loading outlet.  FIGS. 2A-2D  depict a second example of an arrangement  200  of microfluidic channels where the arrangement  200  differs from the arrangement  100  depicted in  FIGS. 1A-1D  due to the arrangement  200  lacking a loading outlet P 3 . In the first step depicted in  FIG. 2B , when loading fluid  126  is introduced into loading channel  120 , excess amounts  129  of loading fluid  126  travel into metering channel  110  at the loading-metering intersection point  114 . As shown in  FIG. 2B  with the shading, the loading inlet P 1  and the metering outlet P 5  can permit the flow of fluid there through and the metering inlet P 2  and the outflow outlet P 6  restrict the flow of fluid therethrough during the filling of loading channel  120  with loading fluid  126 . Excess loading fluid  129  in the metering channel  110  can then be pushed out of metering channel  110  through metering outlet P 5  when metering channel  110  is filled with metered fluid  116  in a second step, as illustrated in  FIG. 2C . Excess amounts of metered fluid  116  also exit metering outlet P 5 . The capillary-stop geometry at two intersection points  112  and  114  and the closing of loading inlet P 1  and the outflow outlet P 6  during the filling of metering channel  110  limits the flow of fluid into loading channel  120  or outflow channel  150 . In a third step illustrated in  FIG. 2D , an additional amount of loading fluid  126  (or a different fluid) is introduced into loading channel  120  to push loading fluid  126  into metering channel  110 , which pushes a predetermined volume of metered fluid  116  in the metering channel  110  between the two interaction points  112  and  114  into outflow channel  150  at the metering-outflow intersection point  112 . 
     As discussed above, the flow of fluid through inlets and outlets P 1 , P 2 , P 3 , P 5 , and P 6  can be controlled using valves and/or capillary stop geometries. An example of a capillary stop is shown in  FIG. 3 . A flow fo fluid  380  can advance down a channel  310  by capillary action (e.g., wicking). A capillary stop  313  can be formed by having sharp angles at a widening point  350 , which will stop the flow of fluid past the capillary stop  313  by capillary action. Fluid flow past the widening point  350  can be achieved by supplying pressure to the system  300  to pump the fluid flow  380  past the capillary stop  313 . In this way, a capillary stop  313  can act similar to a valve in a device, system, or method provided herein. 
       FIGS. 4A-4C  depict another arrangement  400  and method for metering a fluid. As shown in  FIGS. 4A-4C , the system can include capillary stop geometry  113  at an intersection metering-outflow intersection point  112 . As shown in  FIG. 4A , a metered fluid can enter metering inlet P 5  and flow via capillary action towards metering outlet P 2 . A valve at loading inlet P 1  can be closed, as indicated by the shading, which can inhibit a flow of metering fluid into loading channel  120 . A capillary stop  113  at the metering-outflow intersection point  112  can inhibit metering fluid from entering outflow channel  150  despite outflow outlet P 6  remaining open. As shown in  FIG. 4B , a loading fluid can enter loading inlet P 1  and flow through metering outlet P 2 . Loading channel  120  can also be filled via capillary action. A valve at metering inlet P 5  can be closed to inhibit a flow of loading fluid through the metering channel  110  towards metering inlet P 5 . Capillary stop  113  can provide a hold strong enough to prevent the metering fluid from being pushed into outflow channel  150 . A metered amount of metered fluid in the metering channel between a loading-metering intersection point  114  and a metering-outflow intersection point  112  can then be pumped past capillary stop  113  by closing a valve at metering inlet P 5  and a valve at metering outlet P 2  and pumping addition loading fluid through loading inlet P 1 . Pressure from the pumping of loading fluid into the loading inlet P 1  can overcome the capillary stop and allow metering fluid to enter outflow channel  150 . 
     In some cases, devices provided herein include diagnostic devices and kits, which can employ the methods provided herein. In some cases, the devices and kits provided herein can be microfluidic diagnostic devices and/or kits. In some cases, the outflow outlet valve leads into a microfluidic assay chamber. For example, referring back to  FIGS. 1A-1D , arrangement  100  can, in some cases, be used to deliver a metered quantity of a biological sample (e.g., blood) and a reagent (e.g., a lysing reagent) to a microfluidic assay chamber. 
       FIG. 5  also depicts an arrangement of channels as part of a microfluidic diagnostic device  500 , have an inlet  501  for receiving biological sample and a reservoir  502  for holding a reagent. For example, the microfluidic diagnostic device  500  can be designed to determine a CD4 +  count for a subject, the biological sample can be blood including CD4 +  cells, and the reagent can be a lysing reagent. A biological sample metering channel  510  can include a metering inlet P 52  and a metering outlet P 53 , which can both include valves. A reagent loading channel  520  having a loading inlet P 51  can intersect biological sample metering channel  510  at a loading-metering intersection point  514 . An outflow channel  550  having an outflow outlet P 54  can intersect biological sample metering channel  510  at a metering-outflow intersection point  512 . Outflow channel  550  leads to a microfluidic assay chamber  560 , which includes capture molecules  562  supported on a substrate  564  and electrodes  570 , which form part of a testing circuit  580 . Microfluidic assay chamber  560  can also include a plurality of microfluidic components such as reactors, pumps, check valves, reservoirs, channels, sensors, and heaters to enable diagnostic device to detect medical conditions from a biological sample. 
     In use, blood can be delivered through valve P 52  to fill biological sample metering channel  510  by opening valves at P 52  and P 53 , closing a valve at P 51 , and using capillary action to allow the blood to flow into biological sample metering channel  510 . Excess blood can flow through waste channel  518  and past valve P 53 . A capillary stop at the metering-outflow intersection point  512  can resist a flow of blood into outflow channel  550 . Lysing reagent can be delivered from reservoir  502  through a valve at loading inlet P 51  to fill reagent loading channel  520  by opening valves at P 51  and P 53 , closing valves P 52 , and using a capillary action to allow the lysing reagent to flow into reagent loading channel  520 . Excess lysing reagent can flow through waste channel  518  and past valve P 53 . The blood and the lysing reagent can form a bubble free interface at loading-metering intersection point  514 . Because the blood in biological sample metering channel  510  and the lysing reagent in reagent loading channel  520  do not appreciably mix, the lysing reagent does not lyse the CD4+ cells in the blood. The filling of reagent loading channel  520  with lysing reagent and the filling of metering channel  510  with blood can occur in any desired order. In some cases, a microfluidic diagnostic device can provide a measured amount of a biological sample, followed by a binding solution, followed by a wash solution, followed by a measured lysing reagent. 
     After filling biological sample metering channel  510  with blood and reagent loading channel  520  with lysing reagent, a train of blood and lysing reagent can be delivered to microfluidic assay chamber  560  by opening valves P 51  and P 54 , closing valves P 52  and P 53 , and using a force (e.g., a pump) to deliver additional lysing reagent from reagent reservoir  502  past valve P 51 . In some case, an external device, including a controller, can receive the microfluidic diagnostic device  500  and apply pressure to reagent reservoir  502  to push lysing reagent into the reagent loading channel  520 . Capture molecules  562  on substrate  564  can be adapted to capture CD4 +  cells  16 . Blood, with CD4 +  cells  16  left behind, thus moves out of the microfluidic assay chamber  560 . Lysing reagent follows the blood into microfluidic assay chamber  560  to lyse the CD4 +  cells  16  left behind in microfluidic assay chamber  560 . A bubble free interface between the lysing reagent and the blood, however, can eliminate the opportunity for air bubbles to form around captured CD4 +  cells in microfluidic assay chamber  560 , which might prevent the lysing of those cells within the assay chamber. As the CD4 +  cells are lysed, circuit  580  and electrodes  570  within microfluidic assay chamber  560  can be used to determine a change in current, impedance, or conductance in microfluidic assay chamber  560 , which can be used to determine a number of CD4 +  cells in the sample. Precise metering of the blood can allow for a precise number of cells being metered into the microfluidic assay chamber, thus a precise CD4 +  count for a subject can be calculated from detected changes in current, impedance, or conductance. 
     Any number of fluids (e.g., samples and/or reagents) can be metered and combined using the mechanisms described in  FIGS. 1A-1D ,  FIGS. 2A-2D , and  FIGS. 4A-4C .  FIGS. 6A-6F  depict an exemplary arrangement that combines three different fluids.  FIGS. 6A-6F  depict an arrangement  600  including a first metering channel  610 , a second metering channel  620 , a loading channel  630 , and an outflow channel  650 . First metering channel  610  intersects the outflow channel  650  at a metering-outflow intersection point  612  and intersects second metering channel  620  at a metering-metering intersection point  614 . Second metering channel  620  intersects loading channel  630  at a loading-metering intersection point  622 . Intersection points  612 ,  614 , and  622  can each have capillary-stop geometry that guides fluids on the desired path. First metering channel  610  can include a first metering inlet P 4 , a first metering waste channel  618 , and a first metering outlet P 7 . Second metering channel  620  can include a second metering inlet P 2 , a second metering waste channel  628 , and a second metering outlet P 5 . Loading channel  630  can include a loading inlet P 1 , a loading waste channel  638 , and a loading outlet P 3 . 
     In a first step, as shown in  FIG. 6B , valves at loading inlet P 1  and loading outlet P 3  are open while the other valves at P 2 , P 4 , P 5 , P 7 , and P 8  are closed and a loading fluid  636  is pumped into loading channel  630 . In a second step, as shown in  FIG. 6C , valves at a second metering inlet P 2  and second metering outlet P 5  are open while the other valves at P 1 , P 3 , P 4 , P 7 , and P 8  are closed and a second metered fluid  326  is pumped into second metering channel  620 . In a third step, as shown in  FIG. 3D , valves at first metering inlet P 4  and first metering outlet P 7  are open while the other valves at P 1 , P 2 , P 3 , P 5 , and P 8  are closed and a first metered fluid  616  is pumped into first metering channel  610 . The filling of first metering channel  610 , second metering channel  620 , and loading channel  630  can occur in any order. For example, the filling of the metering channel can occur first, followed by the filling of second metering channel  620 , followed by the filling of loading channel  630 . 
     Once channels  610 ,  620 , and  630  are filled fluids  616 ,  626 , and  636 , respectively, each fluid can form a bubble free interface with an adjacent fluid at intersection points  614  and  622 . First and second metered fluids can then be delivered in a predetermined volume through the outflow channel by opening the loading inlet P 1  and the outflow outlet P 8  and closing the other valves P 2 , P 3 , P 4 , P 5 , and P 7 . An additional fluid  656  can be pumped through the loading inlet P 1  to push first metered fluid  616 , followed by second metered fluid  626 , followed by loading fluid  636  into the outflow channel  650  and through the outflow outlet P 8 . The volume of first metered fluid  616  passed into outflow channel  650  is determined by the geometry of first metering channel  610  between metering-outflow intersection point  612  and metering-metering intersection point  614 . The volume of second metered fluid  626  passed into outflow channel  650  is determined by the geometry of second metering channel  620  between metering-metering intersection point  614  and loading-metering intersection point  622 . In some cases, the volume of loading fluid  636  passed into the outflow channel  650  is determined by the geometry of loading channel  630  between loading inlet P 1  and loading-metering intersection point  622 . In some cases, fluid flow through arrangement  600  can be controlled by one or more capillary stops at one or more of the inlets, outlets, or intersection points. In some case, the additional fluid  656  used to push the fluids through the arrangement  600  can be the same as loading fluid  636 . In some case, additional fluid  656  used to push the fluids through the arrangement can be an inert fluid. 
     A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.