Patent Publication Number: US-2021162413-A1

Title: Microfluidic immunoassays

Description:
BACKGROUND 
     Immunoassays are biochemical tests that detect and measure the presence or concentration of a macromolecule or a small molecule in a solution using an antibody or an antigen. The molecule or molecules detected by the immunoassay, the “analyte,” may comprise a protein or other kinds of molecules. Immunoassays are often utilized to identify and measure analytes in biological liquids, such as serum or urine, for medical or research purposes. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a top sectional view illustrating portions of an example microfluidic immunoassay platform. 
         FIG. 2  is a flow diagram of an example immunoassay method. 
         FIG. 3A  is a top sectional view illustrating portions of an example microfluidic immunoassay platform. 
         FIG. 3B  is a side sectional view of the platform of  FIG. 3A . 
         FIG. 4A  is a top sectional view illustrating portions of an example microfluidic immunoassay platform. 
         FIG. 4B  is a side sectional view of the platform of  FIG. 4A . 
         FIG. 5  is a top sectional view illustrating portions of an example microfluidic immunoassay platform. 
         FIG. 6  is a schematic diagram illustrating an example set of functionalized structures. 
         FIG. 7  is a flow diagram of an example method for forming an immunoassay platform and using the immunoassay platform. 
         FIG. 8A  is a top sectional view illustrating portions of an immunoassay platform during deposition of a first set of functionalized structures as part of forming the immunoassay platform. 
         FIG. 8B  is a top sectional view illustrating portions of the immunoassay platform of  FIG. 8A  during deposition of a second set of functionalized structures as part of forming the immunoassay platform. 
         FIG. 8C  is a top sectional view illustrating portions of the immunoassay platform of  FIG. 8B  during movement of an analyte-containing fluid through the first set of functionalized structures and the second set of functionalized structures. 
         FIG. 9A  is a top sectional view illustrating portions of an immunoassay platform during deposition of a first set of functionalized structures as part of forming the immunoassay platform. 
         FIG. 9B  is a top sectional view illustrating portions of the immunoassay platform of  FIG. 9A  during deposition of a second set of functionalized structures as part of forming the immunoassay platform. 
         FIG. 9C  is a top sectional view illustrating portions of the immunoassay platform of  FIG. 9B  during deposition of a third set of functionalized structures has part of forming the immunoassay platform. 
         FIG. 10A  is a top sectional view illustrating portions of an immunoassay platform during deposition of a first set of functionalized structures as part of forming the immunoassay platform. 
         FIG. 10B  is a top sectional view illustrating portions of the immunoassay platform of  FIG. 10A  during deposition of a second set of functionalized structures as part of forming the immunoassay platform. 
         FIG. 10C  is a top sectional view illustrating portions of the immunoassay platform of  FIG. 10B  during deposition of a third set of functionalized structures has part of forming the immunoassay platform. 
         FIG. 11  is a top sectional view illustrating portions of an example microfluidic immunoassay platform. 
     
    
    
     Throughout the drawings, identical reference numbers designate similar, but not necessarily identical, elements. The figures are not necessarily to scale, and the size of some parts may be exaggerated to more clearly illustrate the example shown. Moreover, the drawings provide examples and/or implementations consistent with the description; however, the description is not limited to the examples and/or implementations provided in the drawings. 
     DETAILED DESCRIPTION OF EXAMPLES 
     Disclosed herein are example microfluidic immunoassay platforms and methods that facilitate efficient and economical immunoassays. The disclosed immunoassay platforms and methods may provide high throughput with the ability to multiplex a greater number of analytes in a single test. The disclosed immunoassay platforms and methods provide such immunoassay multiplexing with a proportional lower degree of complexity and cost. 
     In contrast to immunoassays that are carried out utilizing magnetic functionalized beads, the disclosed immunoassay platforms and methods may offer greater flexibility in that the disclosed platforms and methods may be utilized with a larger variety of functionalized structures, such as non-magnetic beads and pillars. In contrast to immunoassays that are carried out with magnetic functionalized beads in a container or well plate, the disclosed immunoassay platforms and methods may be carried out on a platform having an electrically driven fluid actuator which controllably moves the solution containing the at least one analyte through and across the functionalized structures. In contrast to other immunoassays, the disclosed immunoassay platforms and methods may omit the step of sorting different assay beads after analyte binding when multiplexing different analytes. 
     The disclosed immunoassay platforms and methods utilize an electrically driven fluid actuator on a substrate to move a fluid containing at least one analyte along a channel through sets of functionalized structures. A “functionalized” structure is a structure that has been treated with a binding agent that binds to a specific molecule in a solution that may contain a complex mixture of molecules. In some implementations, the binding agent may be an antibody that binds to an epitope of an antigen analyte. In other implementations, the binding agent may be an antigen that binds to an antibody analyte. In some implementations, the binding agent, whether an antibody or an antigen, is chemically linkable to a detectable label. Such labels may emit radiation, produce a color change in a solution, fluoresce under light or, when induced, emit light. Such labels facilitate the detection of a bound analyte to measure the presence or concentration of the analyte in a solution, or other characteristics of the analyte. 
     The functionalized structures may have a variety of forms. In one implementation, the functionalized structure may comprise magnetic beads. In another implementation, the functionalized structures may comprise non-magnetic beads. In yet another implementation, the functionalized structures may comprise posts or pillars. The size and shape of the individual functionalized structures may be varied on a single platform to provide different immunoassay testing characteristics within or across the platform. The number, layout or arrangement, and density of the functionalized structures may be varied on a single platform to provide different immunoassay testing characteristics within or across the platform. 
     As will be appreciated, examples provided herein may be formed by performing various microfabrication and/or micromachining processes on a substrate to form and/or connect structures and/or components. Substrates forming the various fluidic components may comprise a silicon-based wafer or other such similar materials used for microfabricated devices (e.g., glass, gallium arsenide, quartz, sapphire, metal, plastics, etc.). Examples may comprise microfluidic channels, fluid actuators, and/or volumetric chambers. Microfluidic channels and/or chambers may be formed by performing etching, microfabrication processes (e.g., photolithography), or micromachining processes in a substrate. Accordingly, microfluidic channels and/or chambers may be defined by surfaces fabricated in the substrate of a microfluidic device. In some implementations, microfluidic channels and/or chambers may be formed by an overall package, wherein multiple connected package components combine to form or define the microfluidic channel and/or chamber. 
     In some examples described herein, at least one dimension of a microfluidic channel and/or capillary chamber may be of sufficiently small size (e.g., of nanometer sized scale, micrometer sized scale, millimeter sized scale, etc.) to facilitate pumping of small volumes of fluid (e.g., picoliter scale, nanoliter scale, microliter scale, milliliter scale, etc.). For example, some microfluidic channels may facilitate capillary pumping due to capillary force. In addition, examples may couple at least two microfluidic channels to a microfluidic output channel via a fluid junction. 
     The electrically driven fluid actuator used to drive or move the solution containing an analyte through and across the functionalized structures may enhance binding kinetics. In other words, the electrically driven fluid actuator may improve the ability of the analyte in the solution to come into contact with and bind to the binding agents on the surfaces of the functionalized structures. The electrically driven fluid actuator controls the rate at which the solution is moved through and across the functionalized structures. The electrically driven fluid actuator facilitates the provision of immunoassays on a single platform or chip, facilitating the provision of lab-on-chips. 
     The fluid actuator on the platform used to displace fluid through and across the functionalized structures, also on the platform, may comprise a thermal resistive fluid actuator, a piezo-membrane based actuator, and electrostatic membrane actuator, mechanical/impact driven membrane actuator, a magnetostrictive drive actuator, and electrochemical actuator, and external laser actuators (that form a bubble through boiling with a laser beam), other such microdevices, or any combination thereof. 
     In some implementations, electrically driven fluid actuator may comprise an inertial pump formed up on the platform that pumps fluid through and across the functionalized structures. In one implementation, the inertial pump may push fluid through and across the functionalized structures. In another implementation, the inertial pump may displace fluid so as to draw fluid through and across the functionalized structures. 
     As used herein, an inertial pump corresponds to a fluid actuator and related components disposed in an asymmetric position in a fluid channel, where an asymmetric position of the fluid actuator corresponds to the fluid actuator being positioned less distance from a first end of the fluid channel as compared to a distance to a second end of the fluid channel. Accordingly, in some examples, a fluid actuator of an inertial pump is not positioned at a mid-point of a fluid channel. The asymmetric positioning of the fluid actuator in the fluid channel facilitates an asymmetric response in fluid proximate the fluid actuator that results in fluid displacement when the fluid actuator is actuated. Repeated actuation of the fluid actuator causes a pulse-like flow of fluid through the fluid channel. 
     In some examples, an inertial pump includes a thermal actuator having a heating element (e.g., a thermal resistor) that may be heated to cause a bubble to form in a fluid proximate the heating element. In such examples, a surface of a heating element (having a surface area) may be proximate to a surface of a fluid channel in which the heating element is disposed such that fluid in the fluid channel may thermally interact with the heating element. In some examples, the heating element may comprise a thermal resistor with at least one passivation layer disposed on a heating surface such that fluid to be heated may contact a topmost surface of the at least one passivation layer. Formation and subsequent collapse of such bubble may generate flow of the fluid. As will be appreciated, asymmetries of the expansion-collapse cycle for a bubble may generate such flow for fluid pumping, where such pumping may be referred to as “inertial pumping.” 
     In other examples, the fluid actuator(s) forming an inertial pump or used to eject fluid through an ejection orifice or nozzle may comprise piezo-membrane based actuators, electrostatic membrane actuators, mechanical/impact driven membrane actuators, magnetostrictive drive actuators, electrochemical actuators, external laser actuators (that form a bubble through boiling with a laser beam), other such microdevices, or any combination thereof. In some implementations, the fluid actuators may displace fluid through movement of a membrane (such as a piezo-electric membrane) that generates compressive and tensile fluid displacements to thereby cause inertial fluid flow. 
     As will be appreciated, the fluid actuator forming the inertial pump may be connected to a controller, and electrical actuation of the fluid actuator by the controller may thereby control pumping of fluid. Actuation of the fluid actuator may be of relatively short duration. In some examples, the fluid actuator may be pulsed at a particular frequency for a particular duration. In some examples, actuation of the fluid actuator may be 1 microsecond (μs) or less. In some examples, actuation of the fluid actuator may be within a range of approximately 0.1 microsecond (μs) to approximately 10 milliseconds (ms). In some examples described herein, actuation of the fluid actuator includes electrical actuation. In such examples, a controller may be electrically connected to a fluid actuator such that an electrical signal may be transmitted by the controller to the fluid actuator to thereby actuate the fluid actuator. Each fluid actuator of an example microfluidic device may be actuated according to actuation characteristics. Examples of actuation characteristics include, for example, frequency of actuation, duration of actuation, number of pulses per actuation, intensity or amplitude of actuation, phase offset of actuation. 
     In other implementations, the electrically driven fluid actuator may be part of a fluid ejector that ejects droplets of fluid, creating a low pressure are sub-atmospheric pressure that draws fluid through and across the functionalized structures. In some implementations, the sub-atmospheric pressure may be ⅓ of an atmosphere. For example, in one implementation, fluid ejector may comprise a thermal resistor that vaporizes the adjacent fluid to create a bubble that displaces adjacent liquid to eject at least one drop of the liquid through an adjacent orifice, creating a low pressure that draws fluid through and across the functionalized structures. 
     Disclosed herein is an example microfluidic immunoassay platform that may include a substrate, a microfluidic channel in the substrate, a first set of functionalized structures along the channel, a second set of functionalized structures along the channel and an electrically driven fluid actuator contained on the substrate to move fluid containing at least one analyte along the channel through the first set of functionalized structures and through the second set of functionalized structures. 
     Disclosed herein is an example immunoassay method that includes providing a first set of functionalized structures and a second set of functionalized structures along a channel of a substrate and moving a fluid containing an analyte along the channel with an electrically driven fluid actuator contained on the substrate. 
     Disclosed herein is an example method for forming and using an immunoassay platform. The method comprises moving a first fluid containing a first set of functionalized beads along a channel in a substrate to deposit the first set of functionalized beads along the channel, moving a second fluid containing a second set of functionalized beads along the channel in the substrate to deposit the second set of functionalized beads along the channel and moving a third fluid containing at least one analyte through the first set of functionalized beads along the channel and through the second set of functionalized beads along the channel. 
       FIG. 1  schematically illustrates portions of an example microfluidic immunoassay platform  20 . Platform  20  facilitates efficient and economical immunoassays. Platform  20  may provide high throughput with the ability to multiplex a greater number of analytes in a single test. Platform  20  may provide such immunoassay multiplexing with a proportional lower degree of complexity and cost. 
     In contrast to immunoassays that are carried out utilizing magnetic functionalized beads, platform  20  may offer greater flexibility in that platform  20  may be utilized with a larger variety of functionalized structures, such as non-magnetic beads and pillars. In contrast to immunoassays that are carried out with magnetic functionalized beads in a container or well plate, platform  20  may be carried out on a platform having an electrically driven fluid actuator which controllably moves the solution containing the at least one analyte through and across the functionalized structures. In contrast to other immunoassays, platform  20  may omit the step of sorting different assay beads after analyte binding when multiplexing different analytes. Platform  20  comprises substrate  22 , microfluidic channel  24 , sets  30 - 1  and  30 - 2  (collectively referred to as sets  30 ) of functionalized structures, and electrically driven fluid actuator  40 . 
     Substrate  22  comprises at least one layer of material forming a foundation or base of platform  20 . Substrate  22  may comprise a silicon-based wafer or die or a wafer or die from other such similar materials used for microfabricated devices (e.g., glass, gallium arsenide, plastics, etc.). In one implementation in which a detectable label is linked to an analyte bound to the functionalized structures or to the functionalized structures, at least portion  23  (shown by broken lines) of substrate  22  proximate or adjacent to the functionalized structures may be sufficiently translucent or transparent to facilitate optical detection of the detectable labels or their properties. For example, in some implementations, substrate  22  may completely surround channel  24 , wherein portion  23  of the substrate  22  adjacent to channel  24  is transparent to facilitate optical sensing of florescence or luminescence of the detectable labels/markers/tags that become linked to the bound analyte or functionalized structures. In one such implementation, the portion  23  of substrate  22  that is transparent may be formed from a transparent glass material. In some implementations, the entirety of substrate  22  may be formed from a transparent material. In other implementations, portion  23  of substrate  22  may comprise at least one window or opening through which the detectable labels physically coupled to the functionalized structures or the bound analyte(s) may be optically detected. 
     In some implementations, the material or materials forming substrate  22  may be optically opaque, wherein the detectable labels chemically linked to the target analyte are detected following washing of the targeted analyte from the functionalized structures. For example, in one implementation, the analyte or analytes that have been bound to the functionalized structures of set  30 - 2  may be first washed with a first wash solution and then analyzed, wherein the analyte or analytes that have been bound to the functionalized structures of set  30 - 1  may be subsequently washed with a second wash solution and then analyzed. Such analysis may involve the detection of detection labels that have been chemically linked to the analyte either before the solution was passed along channel  24 , after the analyte has bound to the functionalized structures, during the washing of the analyte from the functionalized structures, or after the analyte has been washed from the functionalized structures. 
     Microfluidic channel  24  is formed or extends within substrate  22 . Although illustrated as being linear, microfluidic channel  24  may be curved or branched or have a serpentine path. Microfluidic channel  24  contains sets  30  and directs fluid, the solution containing the at least one analyte, through, around and across sets  30  of functionalized structures. 
     Microfluidic channel  24  may be formed by performing etching, microfabrication processes (e.g., photolithography), or micromachining processes in substrate  22 . Accordingly, channel  22  may be defined by surfaces fabricated in the substrate of a microfluidic device. In some implementations, microfluidic channel  22  may be formed by an overall package, wherein multiple connected package components combine to form or define the microfluidic channel. 
     In some examples described herein, at least one dimension of a microfluidic channel and/or capillary chamber may be of sufficiently small size (e.g., of nanometer sized scale, micrometer sized scale, millimeter sized scale, etc.) to facilitate pumping of small volumes of fluid (e.g., picoliter scale, nanoliter scale, microliter scale, milliliter scale, etc.). For example, some microfluidic channels may facilitate capillary pumping due to capillary force. In addition, examples may couple at least two microfluidic channels to a microfluidic output channel via a fluid junction. 
     Sets  30  comprise different groupings of individual functionalized structures. Each of the functionalized structures is a structure that has been treated with a binding agent that binds to a specific molecule in a solution that may contain a complex mixture of molecules. In some implementations, the binding agent may be an antibody that binds to an epitope of an antigen analyte. In other implementations, the binding agent may be an antigen that binds to an antibody analyte. In some implementations, the binding agent, whether an antibody or an antigen, is chemically linkable to a detectable label. Such labels may emit radiation, produce a color change in a solution, fluoresce under light or, when induced, emit light. Such labels facilitate the detection of a bound analyte to measure the presence or concentration of the analyte in a solution. 
     The individual functionalized structures of sets  30  may have a variety of forms. In one implementation, the functionalized structures may comprise magnetic beads. In another implementation, the functionalized structures may comprise non-magnetic beads. In such implementations, each individual structure/bead of the sets of functionalized structures has a diameter of less than or equal to 10 μm. In yet another implementation, the functionalized structures may comprise posts or pillars. In some implementations, the functionalized structures may comprise a mixture of at least one of magnetic beads, non-magnetic beads and pillars. In one implementation, each individual set  30  is homogenous, wherein each of the individual functionalized structures has the same size and shape and wherein the arrangement and density of functionalized structures is uniform across the set  30 . In one implementation, sets  30 - 1  and  30 - 2  are different with respect to one another in at least one characteristic other than relative location. For example, in one implementation, the individual functionalized structures of set  30 - 1  may have a different size and/or shape as compared to those functionalized structures of set  30 - 2 . In one implementation, the functionalized structures of set  30 - 1  may have a different number, arrangement or layout, and/or density as compared to the functionalized structures of set  30 - 2 . In one implementation, the functionalized structures of set  30 - 1  may have a different size and/or shape as well as at least one of a different number, arrangement or density as compared to the functionalized structures of set  30 - 2 . In one implementation, sets  30  may have similar individual functionalized structures, but wherein sets  30  have different densities and/or layout of the individual functionalized structures. 
     In yet other implementations, each of sets  30 - 1  and  30 - 2  may be heterogeneous in that each of sets  30  has a mixture or combination of different sized and shaped functionalized structures, wherein the mixture of functionalized structures of set  30 - 1  is different than the mixture of functionalized structures of set  30 - 2 . For example, in one implementation, set  30 - 1  may have types A and B of functionalized structures while set  30 - 2  has types C and D of functionalized structures, wherein each of types A, B, C and D of functionalized structures are different from one another with respect to at least one of the size, shape, density, number, layout, mixture or combination ratios and binding agents. In one implementation, set  30 - 1  may have type A and B of functionalized structures will set  30 - 2  has types B and C of functionalized structures. In one implementation, sets  30  may have similar combinations of different types of functionalized structures, but wherein sets  30  have different relative numbers of the different types of functionalized structures. For example, set  30 - 1  may have X % of type A functionalized structure and Y % of a type B functionalized structure while set  30 - 2  has R % of the type A functionalized structure and T % of the type B functionalized structure, wherein the variables X and Y are different than the variables R and T, respectively. 
     In one implementation, the functionalized structures of sets  30  may be functionalized, may be provided with binding agents, or combinations of binding agents, that are similar to one another. In another implementation, the functionalized structure sets  30  may be functionalized with different binding agents or with different combinations of different binding agents that bind to different predefined or preselected analytes. 
     By varying at least one of the size, shape, density, layout, mixture or combination ratios and binding agents amongst the sets  30 , platform  20  may be customized so as to focus on a target analyte or a group of target analytes within or across a single platform. Although platform  20  is illustrated as having two sets  30  in series along channel  24 , in other implementations, platform  20  may comprise a greater number of sets  30  in series along channel  24 , wherein each of the sets  30  is different from the others. In some implementations, platform  20  may comprise sets of functionalized structures along channel  24  that are similar to one another, but that are spaced from one another along channel  24 . In one implementation, platform  20  may comprise two similar sets of functionalized structures spaced by an intervening set of functionalized structures that is different than the two similar sets of functionalized structures, in at least one of individual functionalized structure shape/size and/or in at least one of functionalization (selected binding agents), number, layout and/or density of functionalized structures. 
     Fluid actuator  40  comprises at least one fluid actuator that moves a solution containing (or possibly containing) at least one target analyte through and across sets  30  of functionalized structures. Fluid actuator  40  is directly formed upon substrate  22  and is electrically driven. Fluid actuator  40  may incorporate electrical switches or transistors, formed in, on, or mounted to substrate  22 , which control the actuation of fluid actuator  40 . 
     The fluid actuator  40  on the platform  20  used to displace fluid through and across the sets  30  of functionalized structures, also on the platform  20 , may comprise a thermal resistive fluid actuator, a piezo-membrane based actuator, and electrostatic membrane actuator, mechanical/impact driven membrane actuator, a magnetostrictive drive actuator, and electrochemical actuator, and external laser actuators (that form a bubble through boiling with a laser beam), other such microdevices, or any combination thereof. 
     In the example illustrated, fluid actuator  40  is illustrated as an inertial pump formed up on the platform that pumps fluid through and across the sets  30  of functionalized structures. In one implementation, the inertial pump may push fluid through and across the functionalized structures. In another implementation, as shown by broken lines, platform  20  may additionally or alternatively include fluid actuator  40 ′ which forms an inertial pump or ejection pump that draws fluid through and across the functionalized structures. 
     In some examples, the fluid actuator(s)  40 ,  40 ′ forming an inertial pump includes a thermal actuator having a heating element (e.g., a thermal resistor) that may be heated to cause a bubble to form in a fluid proximate the heating element. In such examples, a surface of a heating element (having a surface area) may be proximate to a surface of a fluid channel in which the heating element is disposed such that fluid in the fluid channel may thermally interact with the heating element. In some examples, the heating element may comprise a thermal resistor with at least one passivation layer disposed on a heating surface such that fluid to be heated may contact a topmost surface of the at least one passivation layer. Formation and subsequent collapse of such bubble may generate flow of the fluid. As will be appreciated, asymmetries of the expansion-collapse cycle for a bubble may generate such flow for fluid pumping, where such pumping may be referred to as “inertial pumping.” 
     In other examples, the fluid actuator(s)  40 ,  40 ′ forming an inertial pump and an ejection pump, respectively, may comprise piezo-membrane based actuators, electrostatic membrane actuators, mechanical/impact driven membrane actuators, magnetostrictive drive actuators, electrochemical actuators, external laser actuators (that form a bubble through boiling with a laser beam), other such microdevices, or any combination thereof. In some implementations, the fluid actuators  40 ,  40 ′ may displace fluid through movement of a membrane (such as a piezo-electric membrane) that generates compressive and tensile fluid displacements to thereby cause inertial fluid flow. 
     As will be appreciated, the fluid actuators  40 ,  40 ′ forming the inertial pump and the ejection pump, respectively, may be connected to a controller, and electrical actuation of the fluid actuator by the controller may thereby control pumping of fluid. Actuation of the fluid actuator may be of relatively short duration. In some examples, the fluid actuator may be pulsed at a particular frequency for a particular duration. In some examples, actuation of the fluid actuator may be 1 microsecond (μs) or less. In some examples, actuation of the fluid actuator may be within a range of approximately 0.1 microsecond (μs) to approximately 10 milliseconds (ms). In some examples described herein, actuation of the fluid actuator includes electrical actuation. In such examples, a controller may be electrically connected to a fluid actuator such that an electrical signal may be transmitted by the controller to the fluid actuator to thereby actuate the fluid actuator. Each fluid actuator of an example microfluidic device may be actuated according to actuation characteristics. Examples of actuation characteristics include, for example, frequency of actuation, duration of actuation, number of pulses per actuation, intensity or amplitude of actuation, phase offset of actuation. 
     In other implementations, the electrically driven fluid actuator  40 ′ may be part of a fluid ejector  42 ′ that ejects droplets of fluid, creating a low pressure or negative pressure that draws fluid through and across sets  30  of the functionalized structures. For example, in one implementation, fluid ejector  42 ′ may comprise fluid actuator  40 ′ in the form of a thermal resistor that vaporizes the adjacent fluid to create a bubble that displaces adjacent liquid to eject at least one drop of the liquid through an adjacent orifice  44 ′, creating a low pressure or sub-atmospheric pressure that draws/pulls fluid through and across the sets  30  of the functionalized structures. 
       FIG. 2  is a flow diagram of an example immunoassay method  100  that facilitates efficient and economical immunoassays. Method  100  may provide high throughput with the ability to multiplex a greater number of analytes in a single test. Method  100  may provide such immunoassay multiplexing with a proportional lower degree of complexity and cost. 
     In contrast to immunoassays that are carried out utilizing magnetic functionalized beads, method  100  may offer greater flexibility in that method  100  may be utilized with a larger variety of functionalized structures, such as non-magnetic beads and pillars. In contrast to immunoassays that are carried out with magnetic functionalized beads in a container or well plate, method  100  may be carried out on a platform having an electrically driven fluid actuator which controllably moves the solution containing the at least one analyte through and across the functionalized structures. In contrast to other immunoassays, method  100  may omit the step of sorting different assay beads after analyte binding when multiplexing different analytes. Although method  100  is described in the context of being carried out with platform  20 , it should be appreciated that method  100  may likewise be carried out with any of the following described microfluidic immunoassay platforms or with similar microfluidic immunoassay platforms. 
     As indicated by block  104 , first and second sets  30  of functionalized structures are provided along a channel  24  of a substrate, such as substrate  22 . In one implementation, sets  30  are in series along channel  24 . 
     As indicated by block  108 , a fluid or solution containing an analyte (or potentially containing an analyte) is moved along the channel  24  with an electrically driven fluid actuator  40  and/or  40 ′ contained on the substrate  22 . The rate at which the solution is to move the through and across the first and second sets of functionalized structures may be controlled to enhance binding of the at least one target analyte (if present) to the functionalized structures. 
     Thereafter, the fluid discharged from channel  24  may be analyzed to identify the at least one analyte that may have been bound within channel  24  to the functionalized structures sets  30 . In some implementations, the analyte bound to the sets  30  of functionalized structures may be washed and removed from channel  24 , wherein the wash fluid or solution containing the previously bound analyte or analytes may be analyzed. In one implementation, the wash fluid or solution is selective, removing and carrying away either the analyte bound to the functionalized structures of set  30 - 1  or the analyte bound to the functionalized structures of set  30 - 2 . For example, in one implementation, the analyte or analytes that have been bound to the functionalized structures of set  30 - 2  may be first washed with a first washed solution and then analyzed, wherein the analyte or analytes that have been bound to the functionalized structures of set  30 - 1  may be subsequently washed with a second wash solution and then analyzed. Such analysis may involve the detection of detection labels that have been chemically linked to the analyte either before the solution was passed along channel  24 , after the analyte has bound to the functionalized structures, during the washing of the analyte from the functionalized structures, or after the analyte has been washed from the functionalized structures. In some implementations, the at least one analyte may be detected while in a bound state to either of the sets  30 . 
       FIGS. 3A and 3B  schematically illustrate portions of an example microfluidic immunoassay platform  220 .  FIG. 3A  is a top sectional view while  FIG. 3B  is a side sectional view of platform  220 . Platform  220  is similar to platform  20  described above except that platform  220  is specifically illustrated as comprising sets  230 - 1  and  230 - 2  of functionalized structures  250 - 1  and  250 - 2  (collectively referred to as sets  230  and structures  250 ), respectively. Those remaining components of platform  220  which correspond to components of platform  20  are numbered similarly. 
     Functionalized structures  250  comprise columns, posts, or pillars. Functionalized structures  250 - 1  have diameters that are larger than the diameters of functionalized structures  250 - 2 . Set  230 - 1  has a first number of structures  250 - 1  while set  230 - 2  has a second number, larger than the first number, of structures  250 - 2 . Set  230 - 1  has a first density of structures  250 - 1  while set  230 - 2  has a second density of structures  250 - 2 , larger than the first density of structures  250 - 1 . The different sizes of structures  250  as well the different number and density of structures  250  as between sets  230 - 1  and  230 - 2  causes solution or fluid containing or potentially conveying an analyte to have different flow characteristics through and across sets  230 - 1  and  230 - 2 . In other implementations, sets  230  may have similar structures  250  with similar numbers, diameters and densities. In the example illustrated, structures  250  extend a full height of channel  24 , increasing likelihood of contact between the analyte in the fluid and the outer surface of the structure  250 . In other implementations, structure  250  may have a height less than the height of channel  24 . 
     As shown by  FIG. 3B , structures  250 - 1  of set  230 - 1  each have an outer circumferential surface which is functionalized with a first binding agent  252 - 1 . Structures  250 - 2  of set  230 - 2  each have an outer circumferential surface functionalized with a second binding agent  252 - 2 , different than the first binding agent  252 - 1 . The different binding agents  252  are chosen so as to bind to different analytes. As described above, in one implementation, the binding agents may comprise different antibodies. In another implementation, the binding agent may comprise different antigens. 
     In use, fluid actuator  40  moves a solution containing or potentially containing different target analytes along channel  24  through and across functionalized structures  250 - 1  and  250 - 2 . Due to the different binding agents of the different sets  230 , different analytes are bound to functionalized structures  250 - 1  as compared to functionalized structures  250 - 2 . The different bound analytes may then be analyzed. In one implementation, the different bound analytes are subsequently washed from their respective sets  230 - 1 ,  230 - 2  and analyzed. In one implementation, two separate washing steps are carried out to separately remove the different analytes bound to the different sets  230 . In one implementation, distinct detectable labels are chemically linked to the distinct analytes to distinguish between the analytes in a single wash solution. For example, a first detectable label that chemically links to the first analyte but not a second analyte may be used to identify the first analyte while a second detectable label that chemically links to the second analyte but not the first analyte may be used to identify the second analyte. The detectable labels facilitate detection and analysis of the presence and/or concentration of the analytes that were in the initial solution. In one implementation, the fluid actuator  40  may be additionally used to pump the different analyte washing fluids through and across structures  250  to controllably remove or release the bound analytes. 
       FIGS. 4A and 4B  schematically illustrate portions of an example microfluidic immunoassay platform  320 .  FIG. 4A  is a top sectional view while  FIG. 4B  is a side sectional view of platform  320 . Platform  320  is similar to platform  20  described above except that platform  320  is specifically illustrated as comprising sets  330 - 1  and  330 - 2  of functionalized structures  350 - 1  and  350 - 2  (collectively referred to as sets  330  and structures  350 ), respectively. Those remaining components of platform  320  which correspond to components of platform  20  are numbered similarly. 
     Functionalized structures  350  comprise spheres or beads. In one implementation, each individual structure/bead of the sets of functionalized structures has a diameter of less than or equal to 10 μm. Functionalized structures  350 - 1  have diameters that are smaller than the diameters of functionalized structures  350 - 2 . Set  330 - 1  has a first number of structures  350 - 1  while set  330 - 2  has a second number, smaller than the first number, of structures  350 - 2 . Set  330 - 1  has a first density of structures  350 - 1  while set  330 - 2  has a second density of structures  350 - 2 , less than the first density of structures  350 - 1 . The different sizes of structures  350  as well the different number and density of structures  350  as between sets  330 - 1  and  330 - 2  causes solution or fluid containing or potentially conveying an analyte to have different flow characteristics through and across sets  330 - 1  and  330 - 2 . In other implementations, sets  330  may have similar structures  350  with similar numbers, diameters and densities. 
     As shown by  FIG. 4B , structures  350 - 1  of set  330 - 1  each have an outer surface which is functionalized with a first binding agent  352 - 1 . Structures  350 - 2  of set  330 - 2  each have an outer surface functionalized with a second binding agent  352 - 2 , different than the first binding agent  352 - 1 . The different binding agents  352  are chosen so as to bind to different analytes. As described above, in one implementation, the binding agents may comprise different antibodies. In another implementation, the binding agent may comprise different antigens. 
     In use, fluid actuator  40  moves a solution containing or potentially containing different target analytes along channel  24  through and across functionalized structures  350 - 1  and  350 - 2 . Due to the different binding agents of the different sets  330 , different analytes are bound to functionalized structures  350 - 1  as compared to functionalized structures  350 - 2 . The different bound analytes may then be analyzed. In one implementation, the different bound analytes are subsequently washed from their respective sets  330 - 1 ,  330 - 2  and analyzed. In one implementation, two separate washing steps are carried out to separately remove the different analytes bound to the different sets  330 . In one implementation, distinct detectable labels are chemically linked to the distinct analytes to distinguish between the analytes in a single wash solution. For example, a first detectable label that chemical links to the first analyte but not a second analyte may be used to identify the first analyte while a second detectable label that chemical links to the second analyte but not the first analyte may be used to identify the second analyte. The detectable labels facilitate detection and analysis of the presence and/or concentration of the analytes that were in the initial solution. In one implementation, the fluid actuator  40  may be additionally used to pump the different analyte washing fluids through and across structures  350  to controllably remove or release the bound analytes. 
       FIG. 5  is a top sectional view illustrating portions of an example microfluidic immunoassay platform  420 . Like the above described microfluidic immunoassay platforms, platform  420  facilitates efficient and economical immunoassays. Platform  420  may provide high throughput with the ability to multiplex a greater number of analytes in a single test. Platform  420  may provide such immunoassay multiplexing with a proportional lower degree of complexity and cost. Platform  420  facilitates multiple immunoassays in parallel with one another. Microfluidic immunoassay platform  420  comprises substrate  22 , sample supply  423 , microfluidic channels  424 - 1 ,  424 - 2 ,  424 - 3 ,  424 - 4  (collectively referred to as channels  424 ). Substrate  22  is described above. Sample supply  423  comprises a volume, passage or slot through which a sample or solution is supplied, the sample or solution potentially containing at least one analyte being targeted for identification or analysis. Sample supply  423  supplies the sample or solution to each of channels  424 . 
     Each of channels  424  is similar to channel  24  described above. Each of channels  424  contains a series of sets of functionalized structures through which solution from supply  423  is moved by at least one fluid actuator. Channel  424 - 1  contains sets  430 - 1 A,  430 - 1 B and  430 - 1 C (collectively referred to as sets  430 - 1 ) of functionalized structures  450 - 1 A,  450 - 1 B and  450 - 1 C (collectively referred to as structures  450 - 1 ), respectively. Sets  430 - 1 A,  430 - 1 B and  430 - 1 C are spaced along channel  424 - 1  and retained against downstream movement, in the direction indicated by arrow  425 , by filters  460 - 1 A,  460 - 1 B and  460 - 1 C, respectively. Filter  460 - 1 A forms passages therethrough that are sized and spaced so as to impede the passage of functionalized structures  450 - 1 A while allowing structures  450 - 1 B and  450 - 1 C to pass. Filter  460 - 1 B forms passages therethrough that are sized and spaced so as to impede the passage of functionalized structures  450 - 1 B while allowing structures  450 - 1 C to pass. Filter  460 - 1 C forms passages therethrough that are sized and spaced so as to impede the passage of functionalized structures  450 - 1 C (as well as structures  450 - 1 B and  450 - 1 A) while allowing the liquid or fluid carrying the functionalized structures to pass. Filter  460 - 1 A is located along channel  424 - 1  between sets  430 - 1 A and  430 - 1 B. Filter  460 - 1 B is located along channel  424 - 1  between sets  430 - 1 B and  430 - 1 C.  2460 - 1 C is located downstream of filters  460 - 1 A in  460 - 1 B. In one implementation, each of filters  460  may comprise pillars spaced to provide the filtering openings or passages. In another implementation, filters  460  may comprise screens or other structures which provide the noted filtering. 
     In the example illustrated, functionalized structures  450 - 1  each comprise non-magnetic beads having functionalized surfaces. In one implementation, each individual structure/bead of the sets  430  of functionalized structures has a diameter of less than or equal to 10 μm. Structures  450 - 1 A are larger than structures  450 - 1 B, which are larger than structures  450 - 1 C. In one implementation, the different structures  450 - 1 A,  450 - 1 B and  450 - 1 C are differently functionalized, having different analyte binding agents. In other implementations, structures  450 - 1 A,  450 - 1 B, and  450 - 1 C are functionalized in a similar fashion with a similar binding agent or agents. 
     Channel  424 - 1  receives a solution containing analytes or potentially containing analytes as pumped by fluid actuator  440 - 1 . Actuator  440 - 1  comprises an electrically driven fluid actuator and is similar to actuator  40  described above. In one implementation, actuator  440 - 1  forms an inertial pump that pumps a sample or solution along channel  424 - 1  through each of sets  430 - 1 . As a solution containing or potentially carrying analytes flows through sets  430 - 1 , analytes within the solution bind to the binding agents of the functionalized structures  450 - 1 . In implementations where each of sets  430 - 1  have differently functionalized surfaces with different binding agents, different analytes are captured or retained by each of the different sets  430 - 1 . 
     Channel  424 - 2  is connected to sample supply  423 . Channel  424 - 2  retains sets  430 - 2 A,  430 - 2 B and  430 - 2 C (collectively referred to as sets  430 - 2 ) of functionalized structures  450 - 2 A,  450 - 2 B and  450 - 2 C (collectively referred to as functionalized structures  450 - 2 ), respectively. Functionalized structures  450 - 2  comprise non-magnetic beads having functionalized surfaces. In the example illustrated, the different sets  430 - 2  of functionalized structures  450 - 2  are differently sized with structures  450 - 2 A being larger than structures  450 - 2 B, which are larger than structures  450 - 2 C. In the example illustrated, each of the different sets  430 - 2  of functionalized structures  450 - 2  are differently functionalized, having different binding agents. In other implementations, at least two, and in one implementation, all three of sets  430  have functionalized structures  450 - 2  which are similarly functionalized. In one implementation, sets  430 - 2 A,  430 - 2 B, and  430 - 2 C are functionalized similar to sets  430 - 1 A,  430 - 1 B, and  430 - 1 C, respectively, providing verification and direct comparison of the results from channels  424 - 1  and  424 - 2 . In another implementation, sets  430 - 2  may be functionally differently than sets  430 - 1 , facilitating the detection of the presence and/or concentration of different analytes in the solution supplied to supply  423 . 
     Channel  424 - 2  includes filter  460 - 2 C. Filter  460 - 2 C is similar to filter  460 - 1 C described above. Filter  460 - 2 C has openings sized so as to impede the passage of functionalized structures  450 - 2 C of set  430 - 2 C. As a result, filter  460 - 2 C blocks the passage of all functionalized structures  450 - 2  upstream. As shown by  FIG. 5 , structures  450 - 2 C are stacked against filter  460 - 2 C. structures  450 - 2 B are stacked against structures  450 - 2 C. structures  450 - 2 A are stacked against structures  450 - 2 B. In the example illustrated, functionalized structures  450 - 2  are stacked in an order from largest to smallest in the downstream direction  425 . Such an order may facilitate the flow of solution along channel  424 - 2  to the smallest functionalized structures  450 - 2 C. In other implementations, the size order of sets  430 - 2  may have other arrangements, such as smallest to largest in the downstream direction or non-ordered size progression. 
     Similar to channel  424 - 1 , channel  424 - 2  receives solution containing analytes (or potentially containing analytes), that is pumped by fluid actuator  440 - 2 . Fluid actuator  440 - 2  is similar to fluid actuator  440 - 1 . In the example illustrated, fluid actuator  440 - 2  comprises an inertial pump that moves fluid through channel  424 - 2  in the downstream direction as indicated by arrow  425 . In one implementation, fluid actuator  440 - 2  comprises a thermal resistor. 
     Channel  424 - 3  is similar to channel  424 - 1  except that fluid is moved through channel  424 - 3  by fluid actuator  440 - 3  and orifice  442 - 3 , downstream of the sets  430 - 3 A,  430 - 3 B and  430 - 3 C (collectively referred to as sets  430 - 3 ) of functionalized structures  450 - 3 A,  450 - 3 B and  450 - 3 C (collectively referred to as structures  450 - 3 ), respectively. Fluid actuator  440 - 3  and orifice  442 - 3  cooperate to form a fluid ejector  444 - 3 . Although fluid ejector  444 - 3  is illustrated as being provided at an end of a closed end channel  424 - 3 , in other implementations, channel  424 - 3  may continue further downstream of the fluid ejector  444 - 3 . The fluid ejector  444 - 3  ejects droplets of fluid through orifice  442 - 3  so as to draw fluid from supply  423  through and across each of the sets  430 - 3 . 
     Functionalized structures  450 - 3  comprise non-magnetic beads having functionalized surfaces. In the example illustrated, the different sets  430 - 3  of functionalized structures  450 - 3  are differently sized with structures  450 - 3 A being larger than structures  450 - 3 B, which are larger than structures  450 - 3 C. In the example illustrated, each of the different sets  430 - 3  of functionalized structures  450 - 3  are differently functionalized, having different binding agents. In other implementations, at least two, and in one implementation, all three of sets  430 - 3  have functionalized structures  450 - 3  which are similarly functionalized. In one implementation, sets  430 - 3 A,  430 - 3 B and  430 - 3 C are functionalized similar to sets  430 - 1 A,  430 - 1 B and  430 - 1 C, respectively, providing verification and direct comparison of the results from channels  424 - 1  and  424 - 3 . In another implementation, sets  430 - 3  may be functionally differently than sets  430 - 1 , facilitating the detection of the presence and/or concentration of different analytes in the solution supplied to supply  423 . Although sets  430 - 3  are illustrated as being retained along channel  424 - 3  by filters  460 - 3 A,  460 - 3 B and  460 - 3 C, in other implementations, sets  430 - 3  may be retained in a fashion similar to that shown with respect to channel  424 - 2 , wherein filters  460 - 1 A and  460 - 1 B are omitted such that set  430 - 3 B stacks against set  430 - 3 C and set  430 - 3 A stacks against set  430 - 3 B. 
     Microfluidic channel  424 - 4  is similar to microfluidic channel  424 - 2 . Channel  424 - 4  is similar to channel  424 - 1  except that fluid is additionally moved through channel  424 - 4  by fluid actuator  440 - 4 B and orifice  442 - 4 , downstream of the sets  430 - 4 A,  430 - 4 B, and  430 - 4 C (collectively referred to as sets  430 - 4 ) of functionalized structures  450 - 4 A,  450 - 4 B, and  450 - 4 C (collectively referred to as structures  450 - 4 ), respectively. Fluid actuator  440 - 4 B and orifice  442 - 4  cooperate to form a fluid ejector  444 - 4 . Although fluid ejector  444 - 4  is illustrated as being provided at an end of a closed end channel  424 - 4 , in other implementations, channel  424 - 4  may continue further downstream of the fluid ejector  444 - 4 . The fluid ejector  444 - 4  ejects droplets of fluid through orifice  442 - 4  so as to draw or pull fluid from supply  423  through and across each of the sets  430 - 4 . In the example illustrated, movement of fluid along channel  424  is further facilitated by fluid actuator  440 - 4 A, which is similar to fluid actuators  440 - 1  or  440 - 2 . In some implementations, fluid actuator  440 - 4 A may be omitted. 
     Functionalized structures  450 - 4  comprise non-magnetic beads having functionalized surfaces. In the example illustrated, the different sets  430 - 4  of functionalized structures  450 - 4  are differently sized with structures  450 - 4 A being larger than structures  450 - 4 B, which are larger than structures  450 - 4 C. In the example illustrated, each of the different sets  430 - 4  of functionalized structures  450 - 4  are differently functionalized, having different binding agents. In other implementations, at least two, and in one implementation, all three of sets  430 - 4  have functionalized structures  450 - 4  which are similarly functionalized. In one implementation, sets  430 - 4 A,  430 - 4 B, and  430 - 4 C are functionalized similar to sets  430 - 1 A,  430 - 1 B, and  430 - 1 C, respectively, providing verification and direct comparison of the results from channels  424 - 1  and  424 - 4 . In another implementation, sets  430 - 4  may be functionalized differently than sets  430 - 1 , facilitating the detection of the presence and/or concentration of different analytes in the solution supplied by supply  423 . Although sets  430 - 4  are illustrated as being retained along channel  424 - 4  by filter  460 - 4 C, wherein upstream sets are stacked against one another as described above with respect to sets  430 - 2 , in other implementations, sets  430 - 4  may be retained in a fashion similar to that shown with respect to sets  430 - 1  or  430 - 3  as described above with individual filters retaining and spacing the different sets along channel  424 - 4 . 
       FIG. 6  illustrates portions of an example set  530  of functionalized structures  550  in the form of beads, such as non-magnetic beads. The functionalized structures  550  are each functionalized with capture elements in the form of antibodies  570 . In other implementations, capture elements may be in the form of antigens. A solution containing an analyte or potentially containing an analyte, such as an antigen, is directed through and across functionalized structures  550 .  FIG. 6  illustrates an example where the solution contains the analyte  572 , which results in the analyte  572  binding to the capture elements, antibodies  570 .  FIG. 6  further illustrates conjugated second antibodies  574 , which are bound to the analyte/antigen  572  and to which detection labels  576 , such as florescence, have been linked. Such functionalized structures  550  and the attached analyte, conjugated secondary antibodies, and detection labels, may be subsequently washed to separate the captured analyte and coupled detection labels for analysis. In some implementations, the linking of the detection labels and conjugated secondary antibodies may take place after the originally captured analyte has been washed from the functionalized structures  550 . 
       FIG. 7  is a flow diagram of an example method  600  for forming and using a microfluidic immunoassay platform, such as any of the platforms described above.  FIGS. 8A, 8B and 8C  are sectional views illustrating the carrying out of method  600  to form microfluidic immunoassay platform  320  described above. Method  600  facilitates efficient and economical forming of an immunoassay platform. 
     As indicated by block  604  and shown by  FIG. 8A , a first fluid  621  containing a first set  330 - 2  of functionalized structures  350 - 2  (shown in  FIGS. 4A and 4B ), in the form of beads, is moved along a channel  24  of a substrate  22  to deposit the first set  330 - 2  along the channel  24 . In one implementation, the first fluid  621  may move through channel  24  by fluid actuator  40 . In one implementation, the set  330 - 2  may be stopped or retained by a filter, constriction, magnetization or other retention mechanism. 
     As indicated by block  608  and shown by  FIG. 8B , a second fluid  627  containing a second set  330 - 1  of functionalized structures  350 - 1  (shown in  FIGS. 4A and 4B ), in the form of beads, is moved along the channel  24  of substrate  22  to deposit the first set  330 - 1  upstream of set  330 - 2  along the channel  24 . In one implementation, the first fluid  621  may move through channel  24  by fluid actuator  40 . In one implementation, the second set  330 - 1  may be retained by a filter, constriction, magnetization or other retention mechanism. In another implementation, the second set  330 - 1  may be stacked against set  330 - 2 . 
     As indicated by block  610  and shown by  FIG. 8C , a third fluid, such as a solution or sample fluid or liquid  629  containing at least one analyte (or potentially containing an analyte for which the present fluid is being tested) is moved along channel  24  of substrate  22  through and across both of sets  330 - 1  and  330 - 2  of functionalized structures. The analyte is bound to the functionalized surfaces of the functionalized structures. For example, antibodies and antigens may bind to one another. In implementations where the sets  330 - 1  and  330 - 2  contain differently functionalized structures, different analytes my bind to the different functionalized structures of the different sets  330 . As described above, the bound analyte may then be detected to determine its presence and/or concentration either while within channel  24 , after the beads of sets  330  have been removed from channel  24 , or after the analytes have been washed from the beads of sets  330 . 
       FIGS. 9A, 9B and 9C  are top sectional views illustrating one example method  700  for forming at least a portion of a microfluidic immunoassay, such as channel  424 - 1  and its associated functionalized structures  450 - 1  as described above with respect to platform  420  in  FIG. 5 . As shown by  FIG. 9A , a source, supply  423 , is supplied with a first fluid  721 , in the form of a liquid, containing or suspending functionalized structures  450 - 1 C. Fluid actuator  440 - 1  is actuated to pump functionalized structures  450 - 1 C along channel  424 - 1  through filter  460 - 1 A and through filter  460 - 1 B. Due to their size relative to filter  460 - 1 C or the passages therethrough, filter  460 - 1 C blocks or impedes further downstream movement of functionalized structures  450 - 1 C, which results in structures  450 - 1 C collecting and grouping to form set  430 - 1 C (shown in  FIG. 9B ). 
     As shown by  FIG. 9B , supply  423  is supplied with a second fluid  723 , in the form of a liquid, containing or suspending functionalized structures  450 - 1 B. Fluid actuator  440 - 1  is actuated to pump functionalized structures  450 - 1 B along channel  424 - 1  through filter  460 - 1 A. Due to their size relative to filter  460 - 1 B or the passages therethrough, filter  460 - 1 B blocks or impedes further downstream movement of functionalized structures  450 - 1 B which results in structures  450 - 1 B collecting and grouping to form set  430 - 1 B (shown in  FIG. 9C ). 
     As shown by  FIG. 9C , supply  423  is supplied with a third fluid  725 , in the form of a liquid, containing or suspending functionalized structures  450 - 1 A. Fluid actuator  440 - 1  is actuated to pump functionalized structures  450 - 1 A along channel  424 - 1 . Due to their size relative to filter  460 - 1 A or the passages therethrough, filter  460 - 1 A blocks or impedes further downstream movement of functionalized structures  450 - 1 A which results in structures  450 - 1 A collecting and grouping to form set  430 - 1 A. Although each of sets  430 - 1  are illustrated as being formed through the supply of different volumes of fluid,  721 ,  723 ,  725  sequentially through channel  424 - 1 , in other implementations, at least two, and in one implementation all three of the sets  430 - 1  may be concurrently formed, wherein the solution supplied by supply  423  contains two or more of different functionalized structures  450 - 1 A,  450 - 1 B and  450 - 1 C, wherein the filters  460 - 1  separate and filter the functionalized structure to the different regions and different sets  430 - 1  along channel  424 . 
       FIGS. 10A, 10B and 10C  are top sectional views illustrating one example method  800  for forming at least a portion of a microfluidic immunoassay, such as channel  424 - 2  and its associated functionalized structures  450 - 2  as described above with respect to platform  420  in  FIG. 5 . As shown by  FIG. 10A , a source, supply  423 , is supplied with a first fluid  821 , in the form of a liquid, containing or suspending functionalized structures  450 - 2 C. Fluid actuator  440 - 2  is actuated to pump functionalized structures  450 - 2 C along channel  424 - 2 . Due to their size relative to filter  460 - 2 C or the passages therethrough, filter  460 - 2 C blocks or impedes further downstream movement of functionalized structures  450 - 2 C which results in structures  450 - 2 C collecting and grouping to form set  430 - 2 C. 
     As shown by  FIG. 10B , supply  423  is supplied with a second fluid  823 , in the form of a liquid, containing or suspending functionalized structures  450 - 2 B. Fluid actuator  440 - 2  is actuated to pump functionalized structures  450 - 2 B along channel  424 - 2  so as to stack against structures  450 - 2 C of set  430 - 2 C, which results in structures  450 - 2 B collecting and grouping to form set  430 - 2 B. 
     As shown by  FIG. 10C , supply  423  is supplied with a third fluid  825 , in the form of a liquid, containing or suspending functionalized structures  450 - 2 A. Fluid actuator  440 - 2  is actuated to pump functionalized structures  450 - 2 A along channel  424 - 1  so as to stack against structures  450 - 2 B of set  430 - 2 B, which results in structures  450 - 2 A collecting and grouping to form set  430 - 2 A. although each of method  700 ,  800  are illustrated utilizing fluid actuators  440 - 1  and  440 - 2 , respectively, to move the functionalized structures along channels  424 - 1  and  424 - 2 , respectively, in other implementations, fluid within such microfluidic channels may be moved by fluid ejectors, similar to fluid ejectors  444 - 3  or a combination of fluid actuators similar to fluid actuator  440 - 1 ,  440 - 2  and fluid ejectors similar to fluid ejector  444 - 3 . 
       FIG. 11  is a top sectional view illustrating portions of an example microfluidic immunoassay platform  920 . Similar to platform  420  described above, platform  920  comprises a multitude of microfluidic channels  924 - 1 ,  924 - 2 ,  924 - 3 ,  924 - 4 ,  924 - 5  and  924 - 6  (collectively referred to as channels  924 ) which receive, in parallel, a sample fluid or solution through sample supply  423 . In addition to supporting channels  924 , substrate  22  further supports a controller  990 . Platform may carry out method  100  described above. 
     Channel  924 - 1  is similar to channel  424 - 1  described above except that channel  924 - 1  additionally comprises fluid actuator  940 , orifice  942 , fluid actuator  970  and sensor  972 . Those remaining components of channel  924 - 1  which correspond to components of channel  424 - 1  are numbered similarly. Fluid actuator  940  and orifice  942  are located downstream of filter  460 - 1 C and cooperate to form a fluid ejector  944  which functions similarly to fluid ejector  444 - 3  described above. Fluid ejector  944  ejects droplets of fluid to draw or pull a solution containing analytes from supply  423  through and across sets  430 - 1 . In the example illustrated, the fluid ejector  944  is located at a blind and a closed end of channel  924 - 1 . As indicated by broken lines  973 , in other implementations, channel  924 - 1  may continue downstream of fluid ejector  944 . In the example illustrated, fluid ejector  944  and fluid actuator  440 - 1  may be alternately used or concurrently used to move fluid through channel  924 - 1 . As described above, fluid ejector  944  and fluid actuator  440 - 1  may be additionally used when populating channel  924 - 1  with functionalized structures  450 - 1  as described above with respect to method  700  and  800 . 
     Fluid actuator  970  comprise an electrically driven fluid actuator located downstream of filter  460 - 1 A, between filter  460 - 1 A and filter  460 - 1 B, between filter  460 - 1 A and the set  430 - 1 B of functionalized structures  450 - 1 B. Fluid actuator  970 , upon being actuated or electrically driven, moves fluid in an upstream direction, through filter  460 - 1 A and through set  430 - 1 A of functionalized structures  450 - 1 A, towards supply  423 . Fluid actuator  970 , when actuated, assists in dislodging the beads forming functionalized structures  450 - 1 A to assist in cleaning and unclogging debris from filter  460 - 1 A and from amongst functionalized structures  450 - 1 A. in some implementations, a fluid actuator similar to fluid actuator  970  may be additionally provided downstream of filter  460 - 1 B, between filter  460 - 1 B and set  430 - 1 C of functionalized structures  450 - 1 C. 
     In one implementation, fluid actuator  970  is located so as to form an inertial pump that, when actuated, pumps fluid in a direction upstream, towards supply  423 . In one implementation, fluid actuator  970  comprises a thermoresistive fluid actuator. In another implementation, fluid actuator  970  may comprise other types of fluid actuators as described above. 
     Sensor  972  comprises a device that senses the flow of fluid. In one implementation, sensor  972  comprises an impedance sensor. In another implementation, sensor  972  comprises other types of a flow sensor. Signals from sensor  972  are communicated to controller  990 . 
     Controller  990  receive signals from sensor  972  and based upon such signals, outputs control signals controlling the actuation of fluid actuator  970 . In one implementation, controller  990  comprises a non-transitory computer-readable medium that provides instructions for directing a processing unit or logic elements to control the actuation of fluid actuator  970 . In one implementation, controller  990  compares the sensed flow of fluid as indicated by sensor  972  against a predetermined threshold and actuates fluid actuator  970  to reverse flow fluid within channel  924 - 1  upon satisfaction of the predetermined threshold. In one implementation, the magnitude of the flow detected output by sensor  972  is utilized by controller  990  as a basis for controlling the frequency, duration, or force of reverse fluid actuation by fluid actuator  970 . Such reverse flow may occur while fluid actuators  940  and  440 - 1  are inactive. 
     Fluid channels  924 - 2 ,  924 - 3 , and  924 - 4  are each similar to fluid channel  924 - 1 . Each of fluid channels  924  comprises similar sets  430 - 1 A,  430 - 1 B, and  430 - 1 C of functionalized structures  450 - 1 A,  450 - 1 B, and  450 - 1 C, respectively. Each of fluid channels  924  comprises a reverse flow fluid actuator  970  and a sensor  972 , wherein controller  990  may control the actuation of fluid actuator  970  based upon signals from sensor  972 . Because a similar immunoassay or test is carried out across each of channels  424 , the verification or confirmation of results across multiple channels is achieved. 
     As shown by broken lines  975 , in lieu of each of channels  924  being supplied with a sample solution or fluid from a same reservoir supply  423 , each of channels  924  may alternatively be supplied with distinct solutions or fluids from distinct fluid sources  978 - 1  (S 1 ),  978 - 2  (S 2 ),  978 - 3  (S 3 ),  978 - 4  (S 4 ),  978 - 5  (S 5 ) and  978 - 6  (S 6 ) (collectively referred to as sources  978 ). Each of such sources  978  may supply a different solution or sample. In one implementation, sources  978  may provide the same samples, but wherein the samples have been diluted to different extents. In another implementation, each of sources  978  may provide a same sample, but wherein each sample has been provided with a different reagent, a different group of reagents or different concentrations of a reagent. In such implementations, the multiple similar channels  924 - 1 ,  924 - 2 ,  924 - 3 , and  924 - 4 , along with the different sources  978 , may provide test concordance. 
     Microfluidic channel  924 - 5  is similar to microfluidic channel  424 - 4  described above. In the example illustrated, channel  924 - 5  receives fluid from source  423 , the source that also supplies fluid to each of channels  924 - 1 ,  924 - 2 ,  924 - 3 , and  924 - 4 . In other implementations, channel  924 - 5  may alternatively receive a sample or solution for testing from a separate or distinct fluid source or supply  978 - 5 . 
     Microfluidic channel  924 - 6  receives a sample or supply of a solution from supply  423 . As indicated by broken lines, in other implementations, channel  924 - 6  may have a dedicated fluid source or fluid supply  978 - 6 . Microfluidic channel  924 - 6  contains a series or chain of functionalized structures  950 - 6  in the form of pillars having functionalized surfaces. Each of pillars forming functionalized structures  950 - 6  is similar to functionalized structures  250 - 1  described above. In the example illustrated, the pillars forming functionalized structures  950 - 6  comprise a single row of such structures. In other implementations, functionalized structures  950 - 6  may comprise a grid or array of such structures extending along channel  924 - 6 . Each of such functionalized structures  950 - 6  is functionalized in a similar fashion, having a similar binding agent or group of binding agents. 
     As further shown by  FIG. 11 , channel  924 - 6  additionally comprises fluid actuator  440 - 6 A and fluid actuator  440 - 6 B, between which structures  950 - 6  are sandwiched. Fluid actuator  440 - 6 A extends upstream of structure  950 - 6  and forms an inertial pump for pushing or pumping fluid through and across structures  950 - 6 . Fluid actuator  440 - 6 B extend proximate to orifice  442 - 6  and cooperates with orifice  442 - 6  to form a fluid ejector  444 - 6 . Ejector  444 - 6 , in response to control signals from controller  990 , ejects droplets of fluid so as to pull or draw fluid from supply  423  (or supply  978 - 6 ) through and across the chain of functionalized structures  950 - 6 . 
     Channel  924 - 6  and the series or chain of functionalized structures  950 - 6  facilitate a determination regarding a concentration of an analyte within a sample solution. In one implementation, the different extents to which an analyte has bound to the different functionalized structures  950 - 6  along the length of channel  924 - 6  is determined and, based upon this determination, the concentration of an analyte in the overall solution may be determined. A solution with a greater concentration of an analyte will result in a higher concentration of the analyte binding to the downstream functionalized structures  950 - 6 , whereas a solution with a lesser concentration of an analyte will result in a lower concentration of the analyte binding to the corresponding downstream functionalize structures  950 - 6 . 
     Although platform  920  is illustrated as comprising six microfluidic channels  924  having sets of functionalized structures, in other implementations, platform  920  may comprise a greater or fewer of such channels  924 . Additional or fewer channels similar to channel  924 - 1  may be provided. Likewise, additional channels similar to channels  924 - 5  and/or channel  924 - 6  may be provided. Likewise, although platform  420  is illustrated as comprising four microfluidic channels  424 , in other implementations, platform  420  may comprise a greater or fewer of such microfluidic channels  424 . Each of such channels may have any of the architectures shown. 
     Although the present disclosure has been described with reference to example implementations, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the claimed subject matter. For example, although different example implementations may have been described as including features providing one or more benefits, it is contemplated that the described features may be interchanged with one another or alternatively be combined with one another in the described example implementations or in other alternative implementations. Because the technology of the present disclosure is relatively complex, not all changes in the technology are foreseeable. The present disclosure described with reference to the example implementations and set forth in the following claims is manifestly intended to be as broad as possible. For example, unless specifically otherwise noted, the claims reciting a single particular element also encompass a plurality of such particular elements. The terms “first”, “second”, “third” and so on in the claims merely distinguish different elements and, unless otherwise stated, are not to be specifically associated with a particular order or particular numbering of elements in the disclosure.