Patent Publication Number: US-2022219162-A1

Title: Lateral-flow assay device having flow constrictions

Description:
CROSS REFERENCE TO RELATED APPLICATION(S) 
     This application claims priority under applicable portions of 35 U.S.C. § 119 to U.S. Patent Application Ser. No. 62/034,825, filed Aug. 8, 2014 and entitled: LATERAL-FLOW ASSAY DEVICE HAVING FLOW CONSTRICTIONS, the entire contents of which are herein incorporated by reference. 
    
    
     TECHNICAL FIELD 
     This application relates to the field of clinical diagnostics and more specifically to lateral-flow assay devices. 
     BACKGROUND 
     The use of diagnostic assays is very well known for the diagnosis, treatment and management of many diseases. In that regard, different types of diagnostic assays have been developed to simplify the detection of various analytes in clinical samples such as blood, serum, plasma, urine, saliva, tissue biopsies, stool, sputum, skin or throat swabs and tissue samples or processed tissue samples. These assays are frequently expected to provide a fast and reliable result, while being easy to use and inexpensive to manufacture. 
     One common type of disposable assay device includes a sample addition zone or area for receiving the liquid sample, at least one reagent zone (also known as a conjugate zone), a reaction zone (also known as a detection zone), and optionally an absorbing zone. These zones can be arranged in order along a fluid passage or channel. These assay devices, commonly known as lateral test strips, can employ a porous material, e.g., nitrocellulose, defining a path for fluid capable of supporting capillary flow. Examples include those devices shown in U.S. Pat. Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660, all of which are incorporated herein by reference in their entireties. 
     The sample addition zone of these assay devices frequently includes a porous material, capable of absorbing the liquid sample, and, when separation of blood cells is required, also effective to trap the red blood cells. Examples of such materials are polymeric membrane filters or fibrous materials, such as paper, fleece, or tissue, comprising e.g., cellulose, wool, glass fiber, asbestos, synthetic fibers, polymers, or mixtures of the same. 
     Another type of lateral-flow assay device is defined by a non-porous substrate having a plurality of upwardly extending microposts (also referred to as “micropillars” or “projections”). The microposts are defined dimensionally and in terms of their spacing to produce capillary flow when a liquid is introduced. Examples of such devices are disclosed in U.S. Pat. No. 8,025,854B2, WO 2003/103835, WO 2005/089082, WO 2005/118139 and WO 2006/137785, all of which are incorporated by reference herein in their entireties. 
     A known non-porous assay device of the above type is shown in  FIG. 1 . The lateral-flow assay device  1  has at least one sample addition zone  2  configured to receive a sample  101 , graphically represented using a teardrop shape. The sample  101  can include, e.g., a bodily fluid or other fluid to be tested for an analyte. The lateral-flow assay device  1  also includes a reagent zone  3 , at least one detection zone  4 , and at least one wicking zone  5 , each disposed on a common substrate  9 . The zones  2 ,  3 ,  4 ,  5  are aligned along a defined fluid flow path  64  by which the sample  101  or a portion thereof flows from the sample addition zone  2  to the wicking zone  5  under the influence of capillary pressure provided between ones of a plurality of microposts  7 . Capture elements, such as antibodies, can be supported in the detection zone  4 , these elements being capable of binding to an analyte of interest, the capture elements being deposited on the device, e.g., by coating. The term “element” is not limited to atoms, i.e., chemical elements of the periodic table, but can also refer to molecules, e.g., of ionically or covalently-bonded atoms, or other chemical compounds or biological substances. In addition, a labeled conjugate material, also capable of participating in reactions that will enable determination of the concentration of the analyte, is separately deposited on the device in the reagent zone  3 , wherein the conjugate material carries a label for detection in the detection zone  4  of the lateral-flow assay device  1 . 
     The conjugate material is gradually dissolved as the sample  101  flows through the reagent zone  3 , forming a conjugate plume of dissolved labeled conjugate material and sample  101  that flows downstream along the defined fluid flow path  64  of the lateral-flow assay device  1  to the detection zone  4 . As the conjugate plume flows into the detection zone  4 , the conjugated material will be captured by the capture elements such as via a complex of conjugated material and analyte (e.g., as in a “sandwich” assay) or directly (e.g., as in a “competitive” assay). Unbound dissolved conjugate material will be swept past the detection zone  4  and into the wicking zone  5 . 
     An instrument such as that disclosed in U.S. 2006/0289787A1, U.S. 2007/0231883A1, U.S. Pat. Nos. 7,416,700 and 6,139,800, all incorporated by reference in their entireties herein, is configured to detect the bound conjugated material in the detection zone  4 . Common labels include fluorescent dyes that can be detected by instruments which excite the fluorescent dyes and incorporate a detector capable of detecting the resulting fluorescence. In the foregoing devices and in the conduction of assays, the resulting level of signal in the detection zone is read using a suitable detection instrument after the conjugate material has been dissolved and the sample  101  and unbound conjugate material have reached and subsequently filled the wicking zone  5  of the lateral-flow assay device  1 . 
     In a typical point of care (POC) lateral flow assay format, it is desirable to remove unbound conjugate materials to lower background signal and improve assay accuracy. In some assays, fluid of the sample  101  continues to flow through the detection zone  4  after all the dissolved conjugate passes the detection zone  4 . In this way, the flowing sample  101  removes unbound conjugate materials. However, endogenous interferents may be present in the sample  101  that may interfere with assay results (e.g., hemoglobin, bilirubin of a particular patient). For these assays, wash fluid separate from the sample  101  can be applied to remove the interferent from the detection zone  4  or other parts of the detection channel. Moreover, some assays involve pre-mixing the conjugate material with the sample  101  prior to addition of the mix to the sample addition zone  2  to obtain a longer incubation time. For these types of assays, since the sample  101  is mixed with the conjugate, a wash fluid is applied to remove unbound conjugate from the detection zone  4 . In these and other embodiments, wash fluid can be formatted or designed to provide an acceptable wash. Accordingly, adding wash fluid is necessary for some selected assays in, e.g., a POC lateral flow format. 
     However, adding wash reagent is a challenge in various prior lateral-flow assay devices. The wash fluid is to flow in the gaps between pillars (or in the pores of a porous structure, such as cellulose acetate). However, since flow resistance in gaps or pores is much larger than outside of the pillar matrix (or porous) structure, wash fluid cannot be “pushed” into the fluid flow path  64  (the pillar matrix) to accomplish the wash. Wash fluid has to be “pulled” into the gaps between pillars or pores of a porous material by the capillary pressure. There is therefore a need for assay devices and ways of using assay devices that are more compatible and usable with various wash fluids. 
     BRIEF DESCRIPTION 
     According to one aspect, there is provided a lateral-flow assay device comprising:
         a) a substrate having a sample addition zone and a wash addition zone disposed along a fluid flow path through which a sample flows under capillary action in a downstream direction away from the sample addition zone and towards the wash addition zone, wherein the fluid flow path is configured to receive a wash fluid in the wash addition zone;   b) at least one hydrophilic surface arranged in the wash addition zone; and   c) one or more flow constriction(s) spaced apart from the fluid flow path and arranged to define, with the at least one hydrophilic surface, a reservoir configured to retain the wash fluid by formation of a meniscus between the hydrophilic surface and the one or more flow constriction(s); wherein the fluid flow path is configured to draw the wash fluid from the reservoir by capillary pressure.       

     According to another aspect, there is provided apparatus for analyzing a fluidic sample, the apparatus comprising:
         a) at least one assay device including a sample addition zone and a wash addition zone disposed along a fluid flow path;   b) a sample-metering mechanism configured to selectively apply the fluidic sample to the sample addition zone;   c) a wash-metering mechanism configured to selectively apply a wash fluid to the wash addition zone, wherein the wash addition zone includes one or more flow constriction(s) spaced apart from the fluid flow path to form a meniscus in the applied wash fluid;   d) at least one measurement device; and   e) a controller configured to operate each of the sample-metering mechanism, wash-metering mechanism, and at least one measurement device in accordance with a predetermined timing protocol in order to determine at least one characteristic of the applied fluidic sample, wherein the controller operates the wash-metering mechanism after operating the sample-metering mechanism.       

     According to still another aspect, there is provided a method of displacing a fluidic sample in a fluid flow path of an assay device, the method comprising: 
     dispensing the fluidic sample from a sample supply onto a sample addition zone of the assay device, wherein the dispensed fluidic sample travels along the fluid flow path of the assay device; and 
     dispensing a wash fluid from a wash-fluid supply onto a wash addition zone of the assay device downstream of the sample addition zone along the fluid flow path so that a meniscus is formed in the dispensed wash fluid by at least one flow constriction of the assay device, wherein the fluid-flow path draws dispensed wash fluid out of a reservoir defined at least partly by the meniscus and the drawn wash fluid displaces at least some of the fluidic sample in the fluid-flow path. 
     Various aspects advantageously provide an effective supply of the wash fluid to the fluid flow path, even in the face of variations in the rate of wash-fluid delivery or the volume of wash fluid delivered. Various aspects advantageously restrict the wash fluid from flowing outside the pillar (or other porous) structures of the fluid flow path. Various aspects advantageously effectively restrict the flow of the sample through the fluid flow path, which can improve the accuracy of assays. 
     These and other features and advantages of various embodiments, variations, and modifications will be readily apparent from the following Detailed Description, which should be read in conjunction with the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of a known lateral-flow assay device; 
         FIG. 2  is a plan view of another known lateral-flow assay device; 
         FIG. 3  illustrates a plan view of a lateral-flow assay device made in accordance with at least one embodiment; 
         FIG. 4  is a plan view of details of a wash addition zone of a lateral-flow assay device according to an exemplary embodiment; 
         FIG. 5  is a front elevational section along the line V-V in  FIG. 4  and shows flow constrictions according to an exemplary embodiment; 
         FIG. 6  is a side elevational section along the line VI-VI in  FIG. 4  and shows wash fluid ingress into a fluid flow path according to an exemplary embodiment; 
         FIGS. 7 and 8  are plan views of exemplary groove configurations in wash addition areas according to various embodiments; 
         FIGS. 9 and 10  are elevational sections of an exemplary lateral-flow assay device according to various embodiments and illustrate stages in which fluid fills an internal volume of the assay device; 
         FIG. 11A  is a sectioned perspective of a lateral-flow assay device according to various aspects; 
         FIG. 11B  is an elevational section along the line XIB-XIB in  FIG. 11A ; 
         FIG. 12  is an elevational section of an exemplary lateral-flow assay device illustrating effects of contact angle; 
         FIG. 13  is an elevational section of an exemplary lateral-flow assay device illustrating stages in which fluid fills an internal volume of the lateral-flow assay device; 
         FIG. 14  is an elevational section of another exemplary lateral-flow assay device; 
         FIGS. 15-27  are perspectives of components of lateral-flow assay devices according to various aspects; 
         FIGS. 28-30  are graphical representations of photographs of stages in an experimental test of an exemplary lateral-flow assay device according to various aspects; 
         FIGS. 31-33  are graphical representations of photographs of stages in another experimental test of an exemplary lateral-flow assay device according to various aspects; 
         FIGS. 34-36  are graphical representations of photographs of stages in yet another experimental test of an exemplary lateral-flow assay device according to various aspects; 
         FIG. 37  is a schematic of an apparatus for analyzing a fluidic sample according to at least one exemplary embodiment, and related components; 
         FIG. 38  shows a flowchart illustrating an exemplary method for displacing a fluidic sample in a fluid flow path of an assay device; and 
         FIG. 39  is a high-level diagram showing components of a data-processing system in accordance with various embodiments. 
     
    
    
     DETAILED DESCRIPTION 
     The following description relates to certain embodiments for a wash addition area design for a lateral-flow assay device. It will be readily apparent that the embodiments described herein are intended to be merely exemplary and therefore numerous other variations and modifications are possible. In addition, several terms are used throughout the following discussion such as “first”, “second”, “above”, “below”, “top”, “bottom”, “lateral” and the like for purposes of providing a suitable frame of reference in regard to the accompanying drawings. To that end, these terms should not be regarded as being overly restrictive in terms of the scope of the described apparatus and methods, unless otherwise specifically indicated herein. 
     It should further be noted that the accompanying drawings are not necessarily presented to scale and therefore no narrowing interpretation should be made in terms of dimensions that have been depicted. 
     As used in this specification and the appended claims, the singular forms “a”, “an” and “the” are intended to further include plural referents unless the context clearly dictates otherwise. 
     The term “about” as used in connection with a numerical value throughout the description and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. The interval governing this term is preferably ±30%. 
     In terms of defining certain of the terms that follow, the term “analyte” is used as a synonym of the term “marker” and intended to minimally encompass any chemical or biological substance that is measured quantitatively or qualitatively and can include small molecules, proteins, antibodies, DNA, RNA, nucleic acids, virus components or intact viruses, bacteria components or intact bacteria, cellular components or intact cells and complexes and derivatives thereof. 
     The term “sample” herein means a volume of a liquid, solution or suspension, intended to be subjected to qualitative or quantitative determination of any of its properties, such as the presence or absence of a component, the concentration of a component, etc. Typical samples in the context of the present invention as described herein are human or animal bodily fluids such as blood, plasma, serum, lymph, urine, saliva, semen, amniotic fluid, gastric fluid, phlegm, sputum, mucus, tears, stool, etc. Other types of samples are derived from human or animal tissue samples where the tissue sample has been processed into a liquid, solution, or suspension to reveal particular tissue components for examination. The embodiments of the present invention are applicable to all bodily samples, but preferably to samples of whole blood, urine or sputum. 
     In other instances, the sample can be related to food testing, environmental testing, bio-threat or bio-hazard testing, etc. This represents only a small example of samples that can be used in the present invention. 
     As described herein, determinations based on lateral flow of a sample and the interaction of components present in the sample with reagents present in the device or added to the device during the procedure, and detection of such interaction, either quantitatively or qualitatively, may be for any purpose, such as diagnostic purposes. Such tests are often referred to as “lateral flow assays”. 
     Examples of diagnostic determinations include, but are not limited to, the determination of analytes, also called markers, specific for different disorders, e.g., chronic metabolic disorders, such as blood glucose, blood ketones, urine glucose (diabetes), blood cholesterol (atherosclerosis, obesity, etc.); markers of other specific diseases, e.g., acute diseases, such as cardiac coronary infarct markers (e.g., troponin I, troponin-T, NT-proBNP), markers of thyroid function (e.g., determination of thyroid stimulating hormone (TSH)), markers of viral infections (e.g., the use of lateral flow immunoassays for the detection of specific viral antibodies), etc. 
     Yet another important field of assays is the field of companion diagnostics in which a therapeutic agent, such as a drug, is administered to an individual in need of such a drug. An appropriate assay is then conducted to determine the level of an appropriate marker to determine whether the drug is having its desired effect. Alternatively, assay devices as described herein can be used prior to administration of a therapeutic agent to determine if the agent will help the individual in need. 
     Yet another important field of assays is that of drug tests, for easy and rapid detection of drugs and drug metabolites indicating drug abuse. Exemplary assays include the determination of specific drugs and drug metabolites in a urine or other sample. 
     The term “lateral-flow assay device”, as discussed herein, refers to any device that receives fluid, such as at least one sample, such as a bodily fluid sample, and includes at least one laterally disposed fluid transport or flow path along which various stations or sites (zones) are provided for supporting various reagents, filters and the like through which sample traverses under the influence of capillary or other applied forces and in which lateral flow assays are conducted for the detection of at least one analyte of interest. 
     The terms “automated clinical analyzer”, “clinical diagnostic apparatus” or “clinical analyzer,” as discussed herein, refer to any apparatus enabling the scheduling and processing of various analytical test elements, including lateral-flow assay devices, as discussed herein, and in which a plurality of test elements can be initially loaded for processing. Such apparatus can include a plurality of components or systems configured for loading, incubating and testing/evaluating a plurality of analytical test elements in automated or semi-automated fashion and in which test elements are automatically dispensed from at least one contained storage supply, such as a cartridge, without user intervention. 
     The term “testing apparatus” refers to any device or analytical system that enables the support, scheduling and processing of lateral-flow assay devices. A testing apparatus can include an automated clinical analyzer or clinical diagnostic apparatus such as a bench, table-top or main frame clinical analyzer, as well as point of care and other suitable devices. For purposes of this application, the testing apparatus may include a plurality of components or systems for loading, testing, or evaluating at least one lateral-flow assay device, including detection instruments for detecting the presence of at least one detectable signal of the assay device. 
     The terms “zone”, “area” and “site” are interchangeably used in the context of this description, examples and claims to define parts of a fluid flow path on an assay device, either in prior art devices or according to an embodiment described herein, including devices in which a sample is first applied to the device and then subsequently directed. The term “reaction” is used to refer to any interaction that takes place between components of a sample and reagent(s) on or in the substrate, or between two or more components present in the sample. The term “reaction” is in particular used to define a reaction taking place between an analyte and a reagent as part of the qualitative or quantitative determination of the analyte. 
     The terms “substrate” or “support” refers to the carrier or matrix to which a sample is added, and on or in which the determination is performed, or where the reaction between analyte and reagent takes place. 
     The term “detection” and “detection signal” refers herein to the ability to provide a perceivable indicator that can be monitored either visually and/or by machine vision such as a detection instrument (e.g., a fluorimeter, reflectometer or other suitable device). 
     Referring to  FIG. 2 , there is shown one version of a lateral-flow assay device  20  including a planar substrate  40  which can be made from a moldable plastic or other suitable non-porous material. Further details of this and related devices are described below and in U.S. Patent Application Publication No. 2014/0141527 A1, entitled “Quality/Process Control of a Lateral-flow assay device Based on Flow Monitoring,” which is incorporated herein by reference in its entirety. 
     The substrate  40  is defined by a top surface  44 , which is further defined by a fluid flow path  64 . The fluid flow path  64  includes a plurality of discrete areas or zones in spaced relation to one another including a sample addition zone  48 , a reagent zone  52 , a plurality of detection zones  56  located in a detection channel  55  (for clarity, only one detection zone  56  is shown) and a receiving or wicking zone  60 . According to this design, each of the above-noted zones are fluidly interconnected with one another in linear fashion along at least one defined fluid flow path  64  and in which a plurality of microposts  7 ,  FIG. 1 , are disposed within at least one of the zones and/or the fluid flow path  64 , the microposts  7  extending upwardly from either the lower surface of the fluid flow path  64  or the discrete zones defined on the lateral-flow assay device  20 . 
     The microposts  7  are preferably dimensioned to induce lateral capillary flow, wherein the microposts  7  preferably include a height, diameter and/or center to center spacing to induce fluidic flow along the at least one fluid flow path. In one version thereof, the microposts  7  can be sufficiently dimensioned so as to induce capillary flow as a so-called “open” structure without the need for additional structure (i.e., side walls, cover or lid) or the application of any externally applied forces. According to this specific design, a defined fluid flow path  64  is created, extending from the sample addition zone  48  to the wicking zone  60 . The illustrated fluid flow path  64  extends substantially in a straight-line fashion between the sample addition zone  48  and the wicking zone  60 . In other configurations, the fluid flow path  64  can include one or more lateral bends or turns. 
     As noted and in various embodiments, the defined fluid flow path  64  is at least partially open, or entirely open. As noted above and by “open” what is meant is that there is no lid or cover which is maintained at a distance that would contribute to capillary flow. Thus a lid, if present as physical protection for the fluid flow path  64  and the lateral-flow assay device  20 , is not required to contribute to the capillary flow in the flow path. According to this specific design, a hydrophilic layer  70  can be directly applied to the top of the microposts  7  in the wicking zone  60  in order to increase fluid flow in the lateral-flow assay device  20  and in which a plurality of vents  72  can be defined in the hydrophilic layer  70 . The hydrophilic layer  70  can include a plastic backer tape (not shown) and a hydrophilic adhesive (not shown) on the side of the backer tape arranged to face the fluid flow path  64 . In various examples, a flow promoter  57  is arranged in the fluid flow path  64  bridging the edge of the hydrophilic layer  70  to promote flow under the hydrophilic layer  70  placed over the wicking zone  60 . 
     Various examples of flow promoters, mixers, flow restrictors, and other structures useful for controlling flow in the fluid flow path  64  are described in U.S. Patent Application Ser. No. 62/035,083, filed Aug. 8, 2014, the disclosure of which is incorporated herein by reference in its entirety. That application describes examples of size and shape characteristics of the sample addition zones  48  according to various aspects, features in the reagent zone  52  to effect more efficient dissolution according to various aspects, a curved portion of the fluid flow path  64  configured to mix fluid passing through the fluid flow path  64  according to various aspects, and features in the wicking zone  60  including flow promoters similar to the flow promoter  57  according to various aspects. 
     An open lateral flow path is described including the defined microposts  7 , for example, in the following published applications: WO 2003/103835, WO 2005/089082; WO 2005/118139; WO 2006/137785; and WO 2007/149042, all of which are incorporated by reference in their entireties. The extending microposts  7  have a height, diameter and a distance or distances between the microposts  7  such that lateral capillary flow of an applied fluid, such as plasma, preferably human plasma, in the zone having the microposts  7  is achieved. These relationships are discussed in U.S. Pat. No. 8,821,812, which is incorporated by reference in its entirety. 
     In addition to optimizing the above-mentioned height, diameter and a distance or distances, the above-noted microposts  7  may be given a desired chemical, biological or physical functionality, e.g. by modifying the surface of the microposts  7  for purposes, for example, of the reagent zone(s)  52  and detection zone(s)  56  of the lateral-flow assay device  20 . In one embodiment, the microposts  7  have a height in the interval of about 15 to about 150 μm, preferably about 30 to about 100 μm, a diameter of about 10 to about 160 μm, preferably 40 to about 100 μm, and a gap or gaps between the microposts  7  of about 3 to about 200 μm, preferably 5 to 50 μm or 10 to about 50 μm from each other. The fluid flow path  64  between the sample addition zone  48  and the wicking zone  60  may have a length of about 5 to about 500 mm, preferably about 10 to about 100 mm, and a width of about 0.3 to about 10 mm, preferably about 0.3 to about 3 mm, preferably about 0.5 to 1.5 mm. The microposts  7 , according to this device design, are substantially cylindrical in terms of their configuration and cross section. However, their specific design of the microposts  7  can also easily be varied to those of different shapes (e.g., rhombic, hexagonal, etc) and sizes to augment flow, as well as to filter materials. 
     Still referring to  FIG. 2 , the sample addition zone  48  can receive a fluid sample  101 ,  FIG. 1 , from a liquid dispenser, such as a pipette or other suitable device. The sample is typically deposited onto the top of the sample addition zone  48 . In various embodiments, a filter material (not shown) is placed within the sample addition zone  48  to filter particulates from the sample or to filter blood cells from blood so that plasma can travel through the lateral-flow assay device  20 . In these embodiments, the sample is typically deposited onto the filter material. 
     The sample then flows, e.g., via capillary action of the microposts, to the reagent zone  52 , which can include reagent(s) useful in the reaction, e.g., binding partners such as antibodies or antigens for immunoassays, substrates for enzyme assays, probes for molecular diagnostic assays, or auxiliary materials such as materials that stabilize the integrated reagents, materials that suppress interfering reactions, and the like. Generally, one of the reagents useful in the reaction bears a detectable signal as discussed herein. In some cases, the reagents may react with the analyte directly or through a cascade of reactions to form a detectable signal such as a colored or fluorescent molecule. In one preferred embodiment, the reagent zone  52  includes conjugate material. The term “conjugate” means any moiety bearing both a detection element and a binding partner. 
     For purposes of this description, a detection element is an agent which is detectable with respect to its physical distribution and/or the intensity of the signal it delivers, such as but not limited to luminescent molecules (e.g., fluorescent agents, phosphorescent agents, chemiluminescent agents, bioluminescent agents and the like), colored molecules, molecules producing colors upon reaction, enzymes, radioisotopes, ligands exhibiting specific binding and the like. The detection element also referred to as a label is preferably chosen from chromophores, fluorophores, radioactive labels and enzymes. Suitable labels are available from commercial suppliers, providing a wide range of dyes for the labeling of antibodies, proteins and nucleic acids. There are, for example, fluorophores spanning practically the entire visible and infrared spectrum. Suitable fluorescent or phosphorescent labels include for instance, but are not limited to, fluorosceins, Cy3, Cy5 and the like. Suitable chemiluminescent labels include but are not limited to luminol, cyalume and the like. 
     Similarly, radioactive labels are commercially available, or detection elements can be synthesized so that they incorporate a radioactive label. Suitable radioactive labels include but are not limited to radioactive iodine and phosphorus; e.g.,  125 I and  32 P. 
     Suitable enzymatic labels include but are not limited to horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase and the like. Two labels are “distinguishable” when they can be individually detected and preferably quantified simultaneously, without significantly disturbing, interfering or quenching each other. Two or more labels may be used, for example, when multiple analytes or markers are being detected. 
     The binding partner is a material that can form a complex that can be used to determine the presence of or an amount of an analyte. For example, in a “sandwich” assay, the binding partner in the conjugate can form a complex including the analyte and the conjugate and that complex can further bind to another binding partner, also called a capture element, integrated into the detection zone  56 . In a competitive immunoassay, the analyte will interfere with binding of the binding partner in the conjugate to another binding partner, also called a capture element, integrated into the detection zone  56 . Example binding partners included in conjugates include antibodies, antigens, analyte or analyte-mimics, protein, etc. 
     As the sample interacts with the reagent in the reagent zone  52 , the detection material begins to dissolve in which a resultant detectable signal is contained within the fluid flow, which is subsequently carried into the adjacent detection zone  56 . 
     Still referring to  FIG. 2 , the detection zone  56  is where any detectable signal can be read. In a preferred embodiment and attached to the microposts  7  in the detection zone  56  are capture elements. The capture elements can hold binding partners for the conjugate or complexes containing the conjugate, as described above. For example, if the analyte is a specific protein, the conjugate may be an antibody that will specifically bind that protein to a detection element such as fluorescence probe. The capture element could then be another antibody that also specifically binds to that protein. In another example, if the marker or analyte is DNA, the capture molecule can be, but is not limited to, synthetic oligonucleotides, analogues, thereof, or specific antibodies. Other suitable capture elements include antibodies, antibody fragments, aptamers, and nucleic acid sequences, specific for the analyte to be detected. A non-limiting example of a suitable capture element is a molecule that bears avidin functionality that would bind to a conjugate containing a biotin functionality. The detection zone  56  can include multiple detection zones. The multiple detection zones can be used for assays that include one or more markers. In the event of multiple detection zones, the capture elements can include multiple capture elements, such as first and second capture elements. The conjugate can be pre-deposited on the lateral-flow assay device  20 , such as by coating in the reagent zone  52 . Similarly, the capture elements can be pre-deposited on the lateral-flow assay device  20  on the detection zone  56 . Preferably, both the detection and capture elements are pre-deposited on the lateral-flow assay device  20 , or on the reagent zone  52  and the detection zone  56 , respectively. 
     Downstream from the detection zone  56  and along the fluid flow path  64  is the wicking zone  60 . The wicking zone  60  is an area of the lateral-flow assay device  20  with the capacity of receiving liquid sample and any other material in the flow path, e.g. unbound reagents, wash fluids, etc. The wicking zone  60  provides a capillary pressure to continue moving the liquid sample through and out the intermediate detection zones  56  of the lateral-flow assay device  20 . The wicking zone  60  and other zones of the herein described lateral-flow assay device  20  can include a porous material such as nitrocellulose, or alternatively can be a non-porous structure defined by the microposts  7 , as previously described. The wicking zone  60  can further include non-capillary fluid driving means, such as an evaporative heater or a pump. Further details of wicking zones as used in lateral-flow assay devices  20  according to the various embodiments are found in U.S. Pat. No. 8,025,854 and U.S. Patent Application Publication No. 2006/0239859 A1, both of which are incorporated herein by reference in their entireties. 
     Tests (assays) are typically completed when the last of the conjugate material has moved into the wicking zone  60  of the lateral-flow assay device  20 . At this stage, a detection instrument, such as a fluorimeter or similar device, is used to scan the detection zone  56 , the detection instrument being, e.g., incorporated within a portable (hand-held or bench top) testing apparatus. The detection instrument that can be used to perform the various methods and techniques described herein can assume a varied number of forms. For example, a mainframe clinical analyzer can be used to retain a plurality of lateral-flow assay devices as described in copending U.S. Patent Application Publication No. 2013/0330713 A1, the entire contents of which are herein incorporated by reference. In a clinical analyzer at least one detection instrument, such as a fluorimeter, can be provided, for example, in relation to an incubator assembly as a monitoring station in which results can be transmitted to a contained processor. 
     In various examples, the instrument can include a scanning apparatus that is capable of detecting fluorescence or fluorescent signals. Alternatively, an imaging apparatus and image analysis can also be used to determine, for example, the presence and position of at least one fluorescent fluid front of a lateral-flow assay device. According to yet another alternative version, infrared (IR) sensors could also be utilized to track the position of fluid position in the lateral-flow assay device. For instance, an IR sensor could be used to sense the ˜1200 nm peak that is typically associated with water in the fluid sample  101  to verify that sample had indeed touched off onto the substrate of the lateral-flow assay device. It should be readily apparent that other suitable approaches and apparatus capable of performing these techniques could be utilized herein. 
     The microposts  7 ,  FIG. 1 , are preferably integrally molded into the substrate  40  from an optical plastic material such as ZEONOR®, such through an injection molding or embossing process. The width of the detection channel  55  in the fluid flow path  64  is typically on the order of about 0.5 mm to about 4 mm, and preferably on the order of about 2 mm. Other portions of the fluid flow path  64  according to various examples can have widths of less than about 0.5 mm, or on the order of about 0.5 mm to about 4 mm, or greater than about 4 mm. Widths of about 1 mm can also be used for the detection channel  55 , provided sufficient signal for a suitable detection instrument, such as a fluorimeter, can be read even if the reagent plume does not cover the entire width of the detection zone  56 . 
     Components of the lateral-flow assay devices (i.e., a physical structure of the device whether or not a discrete piece from other parts of the device) described herein can be prepared from copolymers, blends, laminates, metalized foils, metalized films or metals. Alternatively, device components can be prepared from copolymers, blends, laminates, metalized foils, metalized films or metals deposited one of the following materials: polyolefins, polyesters, styrene containing polymers, polycarbonate, acrylic polymers, chlorine containing polymers, acetal homopolymers and copolymers, cellulosics and their esters, cellulose nitrate, fluorine containing polymers, polyamides, polyimides, polymethylmethacrylates, sulfur containing polymers, polyurethanes, silicon containing polymers, glass, and ceramic materials. Alternatively, components of the device can be made with a plastic, elastomer, latex, silicon chip, or metal; the elastomer can comprise polyethylene, polypropylene, polystyrene, polyacrylates, silicon elastomers, or latex. Alternatively, components of the device can be prepared from latex, polystyrene latex or hydrophobic polymers; the hydrophobic polymer can comprise polypropylene, polyethylene, or polyester. Alternatively, components of the device can comprise TEFLON®, polystyrene, polyacrylate, or polycarbonate. Alternatively, device components are made from plastics which are capable of being embossed, milled or injection molded or from surfaces of copper, silver and gold films upon which may be adsorbed various long chain alkanethiols. The structures of plastic which are capable of being milled or injection molded can comprise a polystyrene, a polycarbonate, or a polyacrylate. In a particularly preferred embodiment, the lateral-flow assay devices are injection molded from a cyclic olefin polymer (COP), such as those sold under the name Zeonor®. Preferred injection molding techniques are described in U.S. Pat. Nos. 6,372,542, 6,733,682, 6,811,736, 6,884,370, and 6,733,682, all of which are incorporated herein by reference in their entireties. 
     Still referring to  FIG. 2 , the defined fluid flow path  64  of the lateral-flow assay device  20  or other lateral-flow assay devices described herein can include open or closed paths, grooves, and capillaries. In various embodiments, the fluid flow path  64  comprises a lateral flow path of adjacent ones of the microposts  7 ,  FIG. 1 , having a size, shape and mutual spacing such that capillary flow is sustained through the flow path. In one embodiment, the flow path is in a channel within the substrate  40  having a bottom surface and side walls. In this embodiment, the microposts  7  protrude from the bottom surface of the fluid flow path  64 . The side walls may or may not contribute to the capillary action of the liquid. If the sidewalls do not contribute to the capillary action of the liquid, then a gap can be provided between the outermost microposts  7  and the sidewalls to keep the liquid contained in the flow path defined by the microposts  7 . Preferably, the reagent that is used in the reagent zone  52  and the capture members or detection agent used in the detection zone  56  is bound directly to the exterior surface of the microposts  7  used in the herein described lateral-flow assay device  20 . 
       FIG. 3  illustrates a plan view of a lateral-flow assay device  300  in accordance with at least one embodiment. The lateral-flow assay device  300  includes the substrate  9  having the sample addition zone  2  and a wash addition zone  409 . The sample addition zone  2  and the wash addition zone  409  are disposed along the fluid flow path  64 , through which the sample  101 ,  FIG. 1 , flows under capillary action in a flow direction F (“downstream”) away from the sample addition zone  2  and towards the wash addition zone  409 . The fluid flow path  64  is configured to receive a wash fluid  301  (represented in phantom) in the wash addition zone  409 . For example, the lateral-flow assay device  300  can include a cover having an opening for passage of wash fluid, as discussed below. In another example, the fluid flow path  64  can be an open-channel flow path open to receipt of wash fluid from above. 
     The lateral-flow assay device  300  includes at least one hydrophilic surface  308  arranged in the wash addition zone  409 . The hydrophilic surface  308  is useful with an aqueous wash fluid  301 . In an example, the substrate  9  includes, or is coated with or bonded to, a material with which the wash fluid  301  has a contact angle of less than 45°. As used herein, the term “hydrophilic surface” refers specifically to a surface that is wetted by the wash fluid  301 . In at least one example, the wash fluid  301  includes numerous surfactants that permit the wash fluid  301  to wet certain types of plastic that are hydrophobic to pure water. Hydrophilic surfaces such as the hydrophilic surface  308  can include such plastics, which are hydrophilic with respect to the wash fluid  301 . 
     The lateral-flow assay device  300  also includes one or more flow constriction(s)  310 . As used herein, a “flow constriction” is a structural feature that assists in containing the wash fluid  301  within the wash addition zone  409  or that assists in restricting the wash fluid  310  from spreading out of the wash addition zone  409 . Some exemplary flow constrictions narrow the cross-section of flow across the hydrophilic surface  308  or otherwise impede, resist, or arrest (even if only temporarily) the flow of the wash fluid  301  across the hydrophilic surface  308 . Examples of flow constrictions include a nozzle nearing the substrate  9 , and the substrate  9  turning a corner out of plane, e.g., at the edge of a groove in the substrate  9 . Such flow constrictions are discussed below. The flow constriction(s)  310  are spaced apart laterally from the fluid flow path  64 , as shown more clearly in  FIG. 4 . 
     The flow constriction(s)  310  are arranged to define, with the at least one hydrophilic surface  308 , a reservoir  535  ( FIG. 5 ) configured to retain the wash fluid  301  by formation of a meniscus between the hydrophilic surface  308  and the one or more flow constriction(s)  310 , as discussed below. The fluid flow path  64  is configured to draw the wash fluid from the reservoir by capillary pressure. 
     As discussed above with reference to  FIGS. 1 and 2 , the lateral-flow assay device  300  can include, e.g., in the fluid flow path  64 , a plurality of the microposts  7 ,  FIG. 1 . The microposts  7  can extend upwardly from the substrate  9  proximal to the wash addition zone  409  or other zones described herein. The microposts  7  have heights, diameters and reciprocal spacing between the microposts  7  that induce lateral capillary flow of the sample  101 , the wash fluid  301 , or both. Moreover, the lateral-flow assay device  300  can include at least one reagent zone  303 , disposed along the fluid flow path  64  downstream of the sample addition zone  2 . 
     Furthermore, the lateral-flow assay device  300  can include at least one detection zone  56  disposed along the fluid flow path downstream of the sample addition zone  2  and the wash addition zone  409 . The at least one detection zone  56  can include a detection material responsive to an analyte of the sample  101  to produce a detectable signal, as discussed below with reference to  FIG. 37 . 
     Referring to  FIG. 4 , there is shown a plan view of an exemplary lateral-flow assay device  400  according to various embodiments. In aspects such as that shown, groove(s) are used as the only flow constriction(s)  310 . For example, the lateral-flow assay device  300  can be an open-top lateral-flow assay device, i.e., a lateral-flow assay device with no cover. 
     In this example, the substrate  9  includes the at least one hydrophilic surface  308 . The one or more flow constriction(s)  310  include at least one groove  410  formed in the hydrophilic surface  308 , and laterally within the wash addition zone  409 . In various examples, the grooves  410  can be elongated, straight or curved, short, circular or elliptical, or other shapes (when viewed from above). In at least one example, the grooves  410  are elongated and have widths between 50 μm and 200 μm. In other examples, the widths of the grooves  410  can be between 5 μm and 1000 μm, or can be greater than 1000 μm. 
     In the example shown, the lateral-flow assay device  400  includes a plurality of the flow constriction(s)  310 , each of the flow constrictions  310  including groove(s)  410  formed in the hydrophilic surface  308 . The groove(s)  410  are arranged along respective arcuate paths  411  about the centerline  464  of the fluid flow path  64 . The respective arcuate paths  411  can be circular, elliptical, or another shape. Circular grooves advantageously provide greater stability, since capillary pressure operates to pull the wash fluid  301  into a circular configuration in the absence of flow constriction(s)  310 . Accordingly, in at least one example, the grooves  410  are circularly arcuate in shape to maintain a round fluid dome above the fluid flow path  64 . The geometric center of the arcuate path for each of the grooves  410  is preferably on the geometric centerline of the fluid flow path  64  if the fluid flow path  64  is straight, as in this example, so that the wash fluid  301  enters the fluid flow path  64  symmetrically along the centerline of the fluid flow path  64 . 
     As noted above with reference to  FIG. 3 , the grooves  410  are spaced apart laterally from the fluid flow path  64 . This spacing advantageously restricts or impedes the wash fluid  301  or the sample  101  in the fluid flow path  64  from flowing into the grooves  410  by capillary pressure. 
     In various examples, such as that shown, the flow constriction(s)  310 ,  FIG. 3 , include at least three spaced-apart grooves  410 , e.g., four spaced-apart grooves, formed in the hydrophilic surface  308 . In various examples, such as that shown, at least one of the grooves  410  is arranged along a substantially arcuate path  411  disposed substantially about a portion of the fluid flow path  64 . Aspects using a plurality of grooves advantageously are more robust to different wash fluid volumes (e.g., permitting a reduction in the precision with which volumes of the wash fluid  301  should be metered) or provide increased reliability of maintaining the dome shape of the reservoir  535 ,  FIG. 5 , in case the inner groove is covered by the wash fluid  301  during dispensing, or due to imperfections in the grooves  410  that permit the spread of the wash fluid  301  over the substrate  9 . 
       FIG. 5  is a front elevational section along the line V-V in  FIG. 4  and shows flow constrictions according to various aspects. The substrate  9  has the fluid flow path  64  recessed therein. Two of the flow constrictions  310 ,  FIG. 3 , are the grooves  410  recessed into the substrate  9  within the area covered by the hydrophilic surface  308 . The illustrated grooves  410  have substantially rectangular cross-sections. The wash fluid  301  wets the hydrophilic surface  308  to form a dome-shaped meniscus  520 ,  530  above the fluid flow path  64  due to capillary pressure and surface tension. The volume of a reservoir  535  bounded by the meniscus  520 ,  530  is variable, depending on the volume of the wash fluid  301  delivered by a wash-metering mechanism  3725 ,  FIG. 37 . The sizes of the meniscus  520 ,  530  and the reservoir  535  shrink as the wash fluid  301  is drawn from the reservoir  535  into the fluid flow path  64  to perform the wash. 
     The reservoir  535  provides a stable meniscus that advantageously accommodates a wide range of volumes of the delivered wash fluid (e.g., between 7 and 17 μL) while keeping substantially the same wash performance. Another advantage of a fluid meniscus  520 ,  530  is that it can buffer large variations in the delivery rate of the wash fluid  301 . The grooves  410  in the illustrated embodiment also advantageously assist in maintaining a round shape of the wash fluid  301  in the reservoir  535  at the hydrophilic surface  308 . 
     In a hypothetical example using a wash fluid  301  with a contact angle of 45° against the hydrophilic surface  308 , if the hydrophilic surface  308  were flat and did not have the grooves  410  (graphically represented by the dotted lines across the tops of the grooves  410 ), a meniscus  520  (shown stippled) would form. The contact angle of 45° in this hypothetical example is shown at an angle  521  with respect to the horizontal hydrophilic surface  308 . 
     In an example using the wash fluid  301  with the contact angle of 45° and with the grooves  410 , a meniscus  530  forms. The 45° contact angle is shown at an angle  531  with respect to the vertical edge of the grooves  410 . The volume under the meniscus  530  is the reservoir  535  defined by the groove(s)  410 , i.e., the flow constriction(s), and the at least one hydrophilic surface  308 . The reservoir  535  is configured to retain the wash fluid  301  by formation of the meniscus  530  between the hydrophilic surface  308  and the one or more flow constriction(s)  310 . 
     The wash fluid  301  is drawn around the corner  511  by surface tension and contact forces, and consequently the cross-sectional area of the flow is restricted. The grooves  410  and the resultant angle  531  raise the meniscus  530  compared to the meniscus  520 . This increases the radius of the reservoir  535 , increasing or substantially increasing the volume of the reservoir  535 . For example, hemispherical reservoirs have the volumes indicated in Table 1, below, for various radii. As can be seen, increasing radius rapidly increases volume. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 R (mm) 
                 1 
                 1.25 
                 1.5 
                 1.75 
                 2 
               
               
                   
                   
               
             
            
               
                   
                 V (μL) 
                 2.1 
                 4.1 
                 7.1 
                 11.2 
                 16.7 
               
               
                   
                   
               
            
           
         
       
     
     Accordingly, using the grooves  410  or similar flow constrictions  310  surrounding the fluid flow path  64  can advantageously permit maintaining and controlling the shape of the meniscus  530  and of the reservoir  535  to increase the volume of the reservoir  535  above the fluid flow path  64 . 
       FIG. 6  is a side elevational section along the line VI-VI in  FIG. 4 , and shows ingress of the wash fluid  301  into the fluid flow path  64 . The reservoir  535  is shown arranged over the fluid flow path  64 , and holding the wash fluid  301 . In this example, the fluid flow path  64  includes the microposts  7  arranged over the substrate  9 . The sample  101  has filled at least a portion of the fluid flow path  64 . In various examples, the wash fluid  301  enters fluid flow path  64  from the reservoir  535  proximate the edge of the reservoir  535 , e.g., being drawn by capillary pressure between the microposts  7 . 
     In various exemplary configurations using the microposts  7 , the wash fluid  301  is dispensed into the fluid flow path  64  between the sample addition zone  2  and the detection zone  56  to interrupt or displace the fluid of the sample  101 . The wash fluid  301  forms a dome shaped meniscus above the fluid flow path  64  so that fresh wash fluid  301  enters the fluid flow path  64  from above the fluid flow path  64  while the flow of sample stops flowing toward the reaction zone. A dome shaped wash fluid meniscus above the fluid flow path  64  is advantageous since the flow resistance is the smallest from above the fluid flow path  64  as compared with sample fluid flowing through between the microposts  7 . This low flow resistance will stop sample flow while supplying fresh wash fluid  301  from the front edge of the reservoir  535 . Prior geometry designs using a shallow well in a wash addition area do not reliably maintain the dome shape of the dispensed wash fluid  301 . The wash fluid  301  can easily spread and result in a thin layer of the wash fluid  301  above the fluid flow path  64  instead of a dome, especially when the wash fluid  301  has a low contact angle for the hydrophilic surface  308 ,  FIG. 3  (e.g. if the contact angle is 45°). In this case, wash efficiency is poor since little of the wash fluid  301  above the fluid flow path  64  is available and sample  101  will continue to flow even after the addition of wash fluid. Configurations described herein advantageously maintain the reservoir  535  to effectively supply the wash fluid  301  to the fluid flow path  64 . 
     Specifically, in at least one example, the wash fluid  301  has a large amount or a relatively high concentration of surfactants. These surfactants are useful for washing, but increase the difficulty of drawing from a thin layer of the wash fluid  301  into the fluid flow path  64 . Accordingly, in this example it is preferable to maintain a bulk fluid in the reservoir  535  from which the fluid flow path  64  can draw. The grooves  410 ,  FIG. 5 , advantageously increase the size of the reservoir  535 , permitting more effective flow of the wash fluid  301  into the fluid flow path  64  than in prior schemes with no flow constrictions. 
     In the example of  FIG. 5 , the sample  101  has filled the fluid flow path  64 , the wash fluid  301  has been applied, and the wash fluid  301  has begun to displace the sample  101  in the fluid flow path  64 . The wash fluid  301  can flow both downstream (along the flow direction F) and upstream (opposite the flow direction F). In various aspects, the wash fluid flows faster downstream than upstream. In an example, there is an area  655  of the fluid flow path  64  at least partly under the reservoir  535  in which there is no flow, i.e., in which the contents of the fluid flow path  64  are stagnant. 
     Referring to  FIG. 7 , there is shown a plan view of an exemplary groove configuration of a lateral-flow assay device  700  according to various embodiments. The fluid flow path  64  in the exemplary lateral-flow assay device  700  has a 90° bend in the wash addition zone  409 . In this example, at least one of the grooves  410  is disposed substantially about a reference point  710  along a centerline  764  of the fluid flow path  64  leaving the wash addition zone  409 . This placement of the reference point  710 , i.e., the geometry center of the grooves  410 , advantageously maintains symmetry in the flow of the wash fluid  301  along the fluid flow path  64  departing the wash addition zone  409 . 
     Also as shown here, it is not required that each of the grooves  410  or other flow constriction(s)  310 ,  FIG. 3 , have the same width W or other dimensions. In this example, the grooves  410  are arranged along substantially arcuate paths (not shown) having respective radii, e.g., radii R1, R2, R3, with respect to the reference point  710 . 
     Referring to  FIG. 8 , there is shown a plan view of an exemplary groove configuration of a lateral-flow assay device  800  according to various embodiments. In this example, at least one of the groove(s)  410  is configured as a segment of a spiral. The segment can have any length and number of turns (for the avoidance of doubt, fractional turns and grooves  410  with less than one full turn can be used). As a result, one or more of the groove(s)  410  can be a spiral passing through more than 360° of rotation around a center point. However, this is not required. 
     In the example shown, the grooves  810  are arranged along a spiral path  869 . The spiral path  869  is arranged to laterally extend on either side of the fluid flow path  64 . Accordingly, each of the grooves  810  follows the spiral path  869  until blocked by the fluid flow path  64 . In this and other aspects, the fluid flow path  64  and the grooves  410  (or, in various aspects, others of the flow constrictions  310 ) are separated by a barrier or gap so that fluid in the fluid flow path  64  is restricted from flowing to the grooves  410  by capillary pressure. 
     Various aspects using grooves  410  around the fluid flow path  64  maintain and control the meniscus shape of the wash fluid  301  so that a higher dome will be formed above the fluid flow path  64  and the wash fluid  301  will be restricted from spreading beyond the grooves  410 . 
     Referring to  FIGS. 9 and 10 , there are shown elevational sections of an exemplary lateral-flow assay device  900  in accordance with at least one embodiment.  FIGS. 9 and 10  illustrate stages in which fluid fills an internal volume of the lateral-flow assay device  900 . The exemplary lateral-flow assay device  900  does not use grooves  410 ,  FIG. 8 , as its flow constriction(s)  310 ,  FIG. 3 . Instead, the lateral-flow assay device  900  includes a cover  990  arranged over the substrate  9 . The cover  990  includes the hydrophilic surface  908  facing the substrate  9 . The substrate can also have a hydrophilic surface  308 , but this is not required. The cover  990  also includes an aperture  920  of diameter d defining a wash port  930  at least partly aligned with the wash addition zone  409 . The aperture  920  is configured to receive the wash fluid  301 . 
     At least one of the flow constriction(s)  310  comprises a first cover flow constriction  910 , including a protrusion  911  (e.g., a nozzle or nub; examples are discussed below) extending from the cover  990  towards the substrate  9  proximate the aperture  920 . In the example shown, the first cover flow constriction  910 , and specifically the protrusion  911 , includes a lip of the aperture  920  protruding to a first predetermined distance h 1  from the substrate  9 . Also in the example shown, a second cover flow constriction  912  is arranged outside the aperture  920  and includes a protrusion  913  extending to a second predetermined distance h 2  from the substrate  9 . The second predetermined distance h 2  can be greater than the first predetermined distance h 1 , as shown. In other configurations, h 2 &gt;h 1 , h 2 ≈h 1 , or h 2 =h 1 . As used herein, “higher” or “deeper” cover protrusions are those that extend relatively farther from the cover; “shorter” or “shallower” cover protrusions are those that extend relatively less far from the cover  990 . 
     The example of  FIGS. 9 and 10  can represent a nozzle (the cover flow constriction  910  with the interior aperture  920 ) having an inside diameter d. The nozzle can convey the wash fluid  301  from a fluid supply (not shown; e.g., a pipette or blister) to the hydrophilic surface  908  or to the hydrophilic surface  308  (if present). The nozzle can be annular in plan, e.g., a ring structure. The second cover flow constriction  912  can be an outer ring. Outside the double outer ring structure (cover flow constrictions  910 ,  912 ), the gap distance between the hydrophilic surface  908  and the facing surface of the substrate  9  is h 3 , which is larger than h 2  in this example. The inner ring (the protrusion  911 ) advantageously retains the wash fluid  301  in the reservoir  535  when the delivered fluid volume is small, e.g., 5 to 7 The outer ring (the protrusion  913 ) is spaced farther from the surface of the substrate  9  (h 2 &gt;h 1 ) so that more fluid, e.g., 15 to 17 μL, can be retained within a limited spatial extent (e.g., a diameter of the wash addition zone  409  substantially equal to 5 mm). 
     Referring specifically to  FIG. 9 , in an example, d=2 mm, h 1 =0.35 mm, h 2 =0.8 mm, and h 3 =1 mm. The outside diameter of the protrusion  911  is 3 mm. The inner diameter of the protrusion  911  is 4 mm, and the outer diameter of the protrusion  913  is 5 mm. Under the protrusion  911 , the volume of the gap is about 2.5 μL. The gap volume between the protrusions  911 ,  913  is 5.5 μL. The gap volume under the protrusion  913  is 4.6 μL. The total volume under the three parts is 12.6 μL. Since meniscus shape is not exactly straight, experiments showed that the feature can maintain stability and provide normal wash for a wash volume of 5 to 20 μL. 
       FIG. 9  shows the reservoir  535  when a relatively smaller amount of the wash fluid  301  has been added compared to  FIG. 10 . In this example, the hydrophilic surface  308  is used. The rounded end of the protrusion  911  permits the wash fluid  301  to form a dome in the nozzle (the aperture  920 ) and more readily contact the hydrophilic surface  308 . Specifically, the rounded end reduces back pressure to permit easier dispensing into the wash addition area  409  through the aperture  920 . A round bottom reduces back pressure by providing an increased radius as the meniscus of the wash fluid  301  moves down the aperture  920  towards the substrate  9 . This permits the wash fluid  301  to contact the substrate  9  or the hydrophilic surface  308  thereof without a large allied pressure. This can be particularly useful, e.g., with uncoated plastic nozzles with which the wash fluid  301  has a contact angle of, e.g., 100°. 
     Upon contact, the wash fluid  301  wets the hydrophilic surface  308  and thus spreads laterally. The lateral spreading causes the wash fluid  301  to also wet the hydrophilic surface  908 . Capillary pressure forms menisci, e.g., a meniscus  935 , that define the reservoir  535  as shown. 
     Referring specifically to  FIG. 10 , there is shown the reservoir  535  when a relatively larger amount of the wash fluid  301  has been added compared to  FIG. 9 , e.g., 15 μL. In this example, the meniscus  1035  is stabilized by the protrusion  913  (the outer ring). The reservoir  535  is defined by the meniscus  1035  and a meniscus (shown) inside the aperture  920 . 
     Also in the example of  FIG. 10 , a first one of the flow constriction(s), e.g., the cover flow constriction  912  including the protrusion  913 , includes a proximal edge  1018  and a distal edge  1019  defined with respect to the fluid flow path  64 . The distal edge  1019  is more sharply curved than the proximal edge  1018 . This advantageously increases the dome height at the distal edge  1019 , e.g., as discussed above with reference to the angle  531 ,  FIG. 5 . In other aspects, the proximal edge  1018  is more sharply curved than the distal edge  1019 , or the edges  1018 ,  1019  are equally sharply curved. The curvature of the edges  1018 ,  1019  can be selected to determine the volume that can be held the reservoir  535  when the menisci  1035  are retained at the respective one of the edges  1018 ,  1019 . Increasing the sharpness of curvature of the edges  1018 ,  1019  increases the effectiveness with which the edges  1018 ,  1019  “pin” (retain) menisci. 
       FIG. 11A  is a sectioned perspective of a lateral-flow assay device  1100  according to various aspects, and  FIG. 11B  is an elevational section along the line XIB-XIB in  FIG. 9A . In the section shown in  FIG. 11B , dimensions are given in millimeters. As shown, the lip of the aperture  920  is substantially annular in shape. Since capillary force naturally tries to maintain circular configurations, using an annular nozzle can advantageously improves stability of menisci such as that shown in  FIG. 9 . Also as shown, in this example, the aperture  920  and the lip of the aperture (the protrusion  911 ) are coaxial to one another. 
     In this example, the cover  990  of the lateral-flow assay device  1100  is arranged over the substrate  9 . At least one of the flow constriction(s)  310  includes the nozzle  1120  extending from the cover  990  towards the substrate  9  and spaced apart from the substrate  9 . The nozzle defines a wash port  930 ,  FIG. 9 , at least partly aligned with the wash addition zone  409 ,  FIG. 9 , and configured to receive the wash fluid  301 ,  FIG. 9 . At least one said flow constriction  310  can include an annulus (the cover flow constriction  912 ) arranged around the nozzle  1120  and extending a smaller distance from the cover  990  than does the nozzle  1120 . The aperture  920  in the nozzle  1120  can be conical, as shown. This can provide humans dispensing the wash fluid  301  through the nozzle  1120  a larger target to hit, reducing the probability of spilling the wash fluid  301 . This can also assist in drawing the wash fluid  301  towards the substrate  9 , since the reduction in diameter of the aperture  920  causes the capillary pressure pulling the wash fluid  301  down near the bottom of the aperture  920  to exceed the capillary pressure pulling the wash fluid  301  up near the wider top of the aperture  920 . Alternatively, the nozzle  1120  can have a cylindrical or rectilinear aperture  920 , or an aperture  920  of another shape. 
     In various aspects such as that shown in  FIGS. 11A and 11B , nozzle(s)  1120  and groove(s)  420  are used together. The nozzles(s)  1120  and the groove(s)  420  both assist in maintaining meniscus stability and restricting the metered wash fluid  301  from spreading across the hydrophilic surface  308  of the substrate  9 . Various such aspects are discussed below with reference to  FIGS. 12, 13, and 15-19 . Moreover, various exemplary configurations of flow constrictions are described below. Unless otherwise specified, flow constrictions shown on substrates or on covers can be used independently or can be used together in any combination. 
     Referring to  FIG. 12 , there is shown an elevational section of an exemplary lateral-flow assay device  1200  and an illustration of effects of contact angle. In this example, the lateral-flow assay device  1200  includes the substrate  9  having the hydrophilic surface  308  facing the cover  990 . The one or more flow constriction(s)  310  include one or more recessed substrate flow constriction(s), in this example the grooves  1210 . For example, groove(s)  1210  and nozzle(s)  1120  can be used together when the contact angle of the wash fluid  301  on the hydrophilic surface  308  is less than 40°. At least one of the substrate flow constriction(s) can be arranged along a substantially arcuate path  411 ,  FIG. 4 , disposed substantially about a portion of the fluid flow path  64 , e.g., as shown in  FIG. 11A . 
     As the wash fluid  301  is added to the lateral-flow assay device  1200  through the aperture  920 , it wets the hydrophilic surface  908  of the cover  990  and the facing hydrophilic surface  308  of the substrate  9  and forms menisci. In an example, the wash fluid  301  creeps along the hydrophilic surface  908  on the underside of the cover  990 . The shape of the meniscus and thus the lateral extent of the reservoir  535  for a given volume of the wash fluid  301  can be controlled by selecting materials having desired contact angles. In an example in which the wash fluid  301  has a contact angle of less than 45° with the hydrophilic surfaces  308 ,  908 , one or more menisci  1235  form. In an example in which the wash fluid  301  has a contact angle of greater than 45° with the hydrophilic surfaces  308 ,  908 , one or more menisci  1237  form. As shown, the menisci  1237  extend farther from the aperture  920  than do the menisci  1235 . Accordingly, in various aspects, the compositions of the wash fluid  301  and the hydrophilic surfaces  308 ,  908  are selected to provide a desired lateral extent of the reservoir  535 . 
     Moreover, the sizes and positions of the substrate flow constriction(s), e.g., the groove(s)  1210 , can be selected to cooperate with the nozzle  1120 . In this example, four grooves  1210 ,  1211 ,  1212 ,  1213  are visible (referred to collectively with reference number  1210 ). The grooves  1210  are configured so that the grooves  1210  farther from the aperture  920  will participate in forming reservoirs with larger volumes than the grooves  1210  closer to the aperture. For example, the groove  1213  can retain a meniscus behind which more of the wash fluid  301  is held than can the groove  1211 . In this non-limiting example, meniscus  1235  is held by the proximal edge (for clarity, not labeled) of the groove  1212 , and the meniscus  1237  is held by the distal edge of the groove  1213 . 
     In various aspects, the wash fluid  301  can form a stabilized meniscus at a location at which the gap size, i.e., the distance between the hydrophilic surfaces  308 ,  908 , is smaller at that location than at adjacent locations. The grooves  1210  cause this to be the case for the raised areas between the grooves, and the cover flow constrictions cause this to be true between the cover flow constrictions and the hydrophilic surface  308 . 
       FIG. 13  is an elevational section of an exemplary lateral-flow assay device  1300  illustrating stages in which the wash fluid  301  fills an internal volume of the lateral-flow assay device  1300 . For clarity, the stages are indicated with circled numbers, and menisci are indicated with dotted curves. Each of stages  2 ,  3 , and  4  includes the wash fluid  301  in the areas marked indicated by earlier stages, starting from stage  2 . 
     In stage  1 , the wash fluid  301  is retained within the nozzle  1120  and forms a dome, as described above. 
     In stage  2 , the wash fluid  301  is retained between the protrusion  911  (the lip of the nozzle  1120 ) and the hydrophilic surface  308 . The menisci are concave. In an example, the reservoir  535  holds about 5 μL in stage  2 . 
     In stage  3 , more of the wash fluid  301  has been added. The volume of the reservoir  535  has expanded, so the menisci between the hydrophilic surface  308  and the protrusion  311  are convex rather than concave. As a result, the reservoir  535  holds, e.g., about 7 μL in stage  3 . 
     In stage  4 , more of the wash fluid  301  has been added, and the reservoir  535  has expanded to the menisci  1335 . In an example, the reservoir  535  holds about 20 μL in stage  4 . 
       FIG. 13  shows one example of a configuration of flow constrictions  310 ,  FIG. 3 , that provides a reservoir  535  with a selected capacity in each of a selected number of steps. The number and arrangement of the flow constrictions  310 , e.g., the nozzle  1120  or other cover flow constrictions, or the grooves  1210  or other substrate flow constrictions, can be selected to effectively retain the wash fluid  301  in the reservoir  535  above the fluid flow path  64 . For example, the flow constrictions  310  can be configured to effectively retain volumes of the wash fluid  301  in 2 μL increments. Each set of flow constrictions, e.g., each ring protruding from the hydrophilic surface  908 , provides a range of stable volumes of the reservoir  535 . In this example, the protrusion  911  provides stable ones of the reservoirs  535  between volumes of 5 μL (stage 2) and 7 μL (stage  3 ). These ranges, and configurations using multiple flow constrictions, increase the range of possible uses of a single design of the lateral-flow assay device  1300 . 
       FIG. 14  is an elevational section of another exemplary lateral-flow assay device  1400 . The lateral-flow assay device  1400  includes the nozzle  1120  having a lip  1411  (a cover flow constriction  910 ,  FIG. 9 ). The lip  1411  has a distal surface  1420  with respect to the aperture  920 . The distal surface  1420  is sloped and does not have a sharply-curved edge. Capillary pressure will tend to retain the wash fluid  301 ,  FIG. 13 , in the reservoir  535  as long as the menisci  1435 ,  1436  contact the sloped distal surface  1420 . Since capillary pressure is stronger in narrower apertures, if the reservoir  535  moves, e.g., right, the capillary pressure pulling the meniscus  1435  to the left will increase and the capillary pressure pulling the meniscus  1436  to the right will decrease, returning the reservoir  535  to a more central position. 
       FIGS. 15-27  are perspectives of components of lateral-flow assay devices according to various aspects.  FIGS. 15-18  show examples similar to those discussed above with reference to  FIGS. 11A-11B .  FIG. 15  shows a configuration with a single ring (the lip of the nozzle) spaced apart from the surface of the substrate  9  by 0.75 mm.  FIG. 16  shows a configuration similar to that of  FIG. 15 , but with the ring spaced apart from the substrate by 0.35 mm. This configuration can be useful, e.g., for lateral-flow assay devices designed for only a single volume of the wash fluid  301 .  FIG. 17  shows a configuration having two rings, each spaced apart by 0.35 mm.  FIG. 18  shows a configuration having two rings, the inner (the lip of the nozzle) spaced apart by 0.75 mm and the outer spaced apart by 0.35 mm. Exemplary devices were constructed according to configurations shown in  FIGS. 15-18  and were tested. The results are given in Table 2, below. 
     Table 2 shows the wash performance of the four wash feature designs shown in  FIGS. 15-18  at different wash volumes. The wash fluid used in this test was POC wash having properties listed below in Table 3. In Table 2, “overflow” signifies that wash fluid flowed above the fluid flow path  64  (this is undesirable since the wash efficiency will be poor). “Meniscus out” signifies that the fluid meniscus extends laterally at least partly beyond the third wash groove  410 ,  FIG. 4 , in the tested lateral-flow assay device. “Off-center” signifies that the fluid meniscus is not centered in the tested arcuate grooves  410 . “Good” signifies that the wash fluid  301  is stable in the reservoir  535 ,  FIG. 5 , and the meniscus is substantially a desired size. Cells in Table 2 marked “*” represent preferred embodiments. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 experimental results 
               
            
           
           
               
               
               
            
               
                   
                 Gaps 
                 Metering volume and fluid types 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Inner 
                 Outer 
                 *5 μL 
                 *10 μL 
                 *15 μL 
                 20 μL 
                 25 μL 
               
               
                 FIG. 
                 ring 
                 ring 
                 POC 
                 POC 
                 POC 
                 POC 
                 POC 
               
               
                   
               
               
                 15 
                 0.75 
                 N/A 
                 overflow 
                 *good 
                 good 
                 stable, brief 
                 stable, brief 
               
               
                   
                   
                   
                   
                   
                   
                 overflow 
                 overflow, 
               
               
                   
                   
                   
                   
                   
                   
                   
                 meniscus out 
               
               
                 16 
                 0.35 
                 N/A 
                 *stable, fluid 
                 *good 
                 *good 
                 stable, long 
                 stable, long 
               
               
                   
                   
                   
                 short 
                   
                   
                 overflow 
                 overflow, 
               
               
                   
                   
                   
                   
                   
                   
                   
                 meniscus out 
               
               
                 17 
                 0.35 
                 0.35 
                 *stable, fluid 
                 *good 
                 *good 
                 *stable, 
                 stable, long 
               
               
                   
                   
                   
                 short 
                   
                   
                 brief 
                 overflow, 
               
               
                   
                   
                   
                   
                   
                   
                 overflow 
                 meniscus out 
               
               
                 18 
                 0.75 
                 0.35 
                 stable, off- 
                 *stable, 
                 *stable, 
                 *stable, 
                 stable, brief 
               
               
                   
                   
                   
                 center, brief 
                 brief 
                 brief 
                 brief 
                 overflow, 
               
               
                   
                   
                   
                 overflow 
                 overflow 
                 overflow 
                 overflow 
                 meniscus out 
               
               
                   
               
            
           
         
       
     
       FIG. 17  illustrates a configuration in which at least one of the flow constriction(s)  310  includes an annulus  1710  arranged around the nozzle  1120  and extending substantially the same distance from the cover  990  as does the nozzle  1120 . 
     In an example (not shown), the annulus  1710  can be interrupted periodically, e.g., every 90° around the annulus  1710 , thus forming a plurality of independent arcuate protrusions. 
       FIG. 18  illustrates a configuration in which at least one of the flow constriction(s)  310  includes an annulus  1810  arranged around the nozzle  1120  and extending a larger distance from the cover  990  than does the nozzle  1120 . 
       FIG. 19  is a top perspective view of components of a lateral-flow assay device according to various aspects. In the illustrated configuration, the aperture  1920  of the nozzle  1120  and the lip  1911  of the aperture  1920  are axially offset from one another. The wash fluid  301  is shown filling the aperture  1920  and being dispensed onto the substrate  9 . For clarity of explanation, the axes of the lip  1911  and of the aperture  1920  are shown, as is the offset  1995  between them in this example. Axial offset provides increased flexibility in the design of the lateral-flow assay device  1900 , since the location at which the wash fluid  301  is received (the aperture  1920 ) can be offset from the location at which the wash fluid  301  is dispensed onto the substrate  9 . In an aspect, the lowest tip of the aperture  920  is disposed above the fluid flow path  64  to be washed. Also as shown, the aperture  1920  can have a partly-conical, partly-cylindrical shape. 
       FIG. 20  is a perspective of components of the lateral-flow assay device  1900 ,  FIG. 19 , according to various aspects.  FIG. 20  shows a bottom perspective view of the nozzle  1120 . As shown, the nozzle  1120  has a conical portion, as indicated.  FIG. 20  also shows a portion of the hydrophilic surface  908 . Accordingly, in various embodiments, a first one of the flow constriction(s)  310  is shaped substantially as a convex closed figure such as a cone. Convex closed figures can include nubs, e.g., circular, elliptical, or polygonal in planwise cross-section. 
       FIG. 21  is a bottom perspective view of components of a lateral-flow assay device according to various aspects. In the illustrated configuration, at least one of the flow constrictions  310  includes a protrusion  2112  spaced apart from the nozzle  1120 . The protrusion  2112  permits menisci to form to differentially attract to a known location any excess wash fluid beyond the amount that can be held in a reservoir  535 ,  FIG. 5 , formed by the nozzle  1120  alone. This can advantageously permit, e.g., drawing excess amounts of the wash fluid  301  away from the fluid flow path  64  or a portion thereof. 
       FIG. 22  is a bottom perspective view of components of a lateral-flow assay device according to various aspects. In the illustrated configuration, a nozzle  2220  has a stepped surface  2225  facing the substrate  9 ,  FIG. 19 . This advantageously provides defined locations at which menisci will preferentially form, e.g., the edges of the steps. 
       FIG. 23  is a bottom perspective view of components of a lateral-flow assay device according to various aspects. In the illustrated configuration, at least one of the flow constrictions  310  is a protrusion  2312  is spaced apart from the nozzle  2220 . The protrusion  2312  can attract excess volumes of the wash fluid  301 , e.g., as described above with reference to  FIG. 21 . 
       FIGS. 24-27  are bottom perspective views of respective covers  990  of various exemplary lateral-flow assay devices. Each of the covers  990  includes the respective hydrophilic surface  908 . 
       FIG. 24  is a bottom perspective view of components of a lateral-flow assay device according to various aspects. In the illustrated configuration, the nozzle  2420  has a relatively broad plateau  2425  surrounding a relatively narrow aperture  920 . The aperture  920  can be broader where the wash fluid  301  is added to the aperture  920 , e.g., as shown in  FIG. 19 . The plateau  2425  is one of the flow constriction(s)  310  in this example. The plateau  2425  can be, e.g., 1.78 mm in diameter. 
       FIG. 25  is a bottom perspective view of components of a lateral-flow assay device according to various aspects. In the illustrated configuration, similar to the configuration shown in  FIG. 18 , at least one of the flow constriction(s)  320  includes an annulus  2520  arranged around the nozzle  2420  and extending a larger distance from the cover  990  than does the nozzle  2420 . In this example, the outside diameter of the annulus  2520  is 3 mm. The annulus  2520  can be concentric with the nozzle  2420 , or can be axially offset therefrom. The relative positions of the annulus  2520  and the nozzle  2420  can be selected to provide desired shapes of the menisci that form when the wash fluid  301  is added to the lateral-flow assay device. 
       FIG. 26  is a bottom perspective view of components of a lateral-flow assay device according to various aspects. In the illustrated configuration, at least one of the flow constriction(s)  310  includes a plurality of protrusions  2630  arranged substantially symmetrically about the annulus  2520  and spaced apart from the annulus  2520 . In this example, four of the protrusions  2630  are present, spaced at 90° intervals around the annulus  2520 . The annulus  2520 , the nozzle  2420 , and the protrusions  2630  can have any desired relationship of relative height off the cover  990 . As described above with reference to  FIG. 21 , the protrusions  2630  provide increased control of where excess volumes of the wash fluid  301  are stored. In various aspects, the protrusions  2630  can all have the same shape or can have any number of different shapes; any number of the protrusions  2630  can be used; and the protrusions  2630  can be spaced at any angles, evenly or unevenly. 
       FIG. 27  is a bottom perspective view of components of a lateral-flow assay device according to various aspects. In the illustrated configuration, at least one of the flow constriction(s)  310  includes a plurality of protrusions  2730 ,  2731  arranged substantially symmetrically about the nozzle  2420  and spaced apart from the nozzle  2420 . In this example, four of the protrusions  2730  are arranged alternating with four of the protrusions  2731  around the nozzle  2420  at 45° intervals. Solid and dotted lead lines are used for clarity only and without limitation. In various aspects, the protrusions  2730 ,  2731  can all have the same shape or can have any number of different shapes; any number of the protrusions  2730 ,  2731  can be used; and the protrusions  2730 ,  2731  can be spaced at any angles, evenly or unevenly. 
     The configurations shown in  FIGS. 26 and 27  can be useful for lateral-flow assay devices using high volumes of the wash fluid  301  compared to, e.g., the configurations shown in  FIGS. 24 and 25 . 
       FIGS. 28-36  are graphical representations of photographs of stages in experimental tests of an exemplary lateral-flow assay device according to various aspects. The tested exemplary lateral-flow assay device was configured as shown in  FIGS. 11A-11B . 
     Experiment 1 ( FIGS. 28-30 ), experiment 2 ( FIGS. 31-33 ), and experiment 3 ( FIGS. 34-36 ) illustrate that various flow constriction(s)  310  can, together with the hydrophilic surfaces  308 ,  908 , effectively deliver different wash fluids (water, POC wash and NDSB Wash) to accomplish wash effectively and maintain the stability of wash fluid menisci within the wash addition area  409  in the lateral-flow assay device during an assay fluid flow process. The properties of the tested wash fluids  301  are listed in Table 3: 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Wash Fluid 
                 Viscosity (cP) 
                 Surface Tension (dynes/cm) 
               
               
                   
               
             
            
               
                 DI Water 
                 0.88 
                 72.7 
               
               
                 POC Wash 1.0 
                 0.93 
                 32.8 
               
               
                 NDSB (FlumAb) 
                 0.92 
                 31.9 
               
               
                   
               
            
           
         
       
     
     Referring to  FIGS. 28-30 , there are shown stages in Experiment 1. The sample  101 ,  FIG. 1 , was 1% silwet surfactant in plasma and included red food dye for visibility. Eight microliters of the sample  101  were added to the sample addition zone  2 ,  FIG. 1 . Once the sample  101  filled about 40% of the volume of the wicking zone  5 ,  FIG. 1 , 17 □L of POC wash fluid (at room temperature) with blue food dye was added to the wash addition zone  409 . Red food dye is added to the sample, and blue dye is added to the wash fluid.  FIGS. 28-30  show fluid flow and wash patterns at three different stages of the tested assay process with wash addition. 
       FIG. 28  shows the sample  101  (red color) having filled about 40% of the volume of the wicking zone  5  prior to wash addition.  FIG. 28  shows the tested lateral-flow assay device immediately after adding the wash fluid  301  (blue color). The grooves  410 ,  FIG. 4 , are retaining the wash fluid  301 .  FIG. 30  shows the distribution of the wash fluid  301  distribution when fluid, in this test the sample  101 , reaches the end  3005  of the wicking zone  5 . In this experiment, the fluid of the sample  101  was completely displaced by the wash fluid in the detection zone channel  3064  of the fluid flow path  64 ,  FIG. 3 . Moreover, the wash fluid extended into the wicking zone  5  in a region  3001 . The fluid under the wash addition zone  409  is still pinned within the grooves  410  and is still stable after the wash flow is complete. 
     Referring to  FIGS. 31-33 , there are shown stages in Experiment 2. The sample  101  as in Experiment 1 was added to the sample addition zone  2 .  FIG. 31  shows the sample  101  (red color) having filled about 30% of the volume of the wicking zone  5  prior to wash addition. At that point, the wash fluid  301  was added.  FIG. 32  shows the tested lateral-flow assay device immediately after adding the wash fluid  301 , in this experiment 17 □L de-ionized water (at room temperature) plus green food dye (green color). The wash fluid  301  (green color) is retained within the grooves  410 .  FIG. 33  shows the lateral-flow assay device when fluid, in this instance the sample  101 , reached the end  3005  of the wicking zone  5 . The fluid of the sample  101  (red color) is completely displaced by the wash fluid  301  (green color) in the detection zone channel  3064 . Moreover, the wash fluid extended into the wicking zone  5  in a region  3301 . The wash fluid  301  in the wash addition zone  409  is still pinned within the grooves  410  and is still stable after the wash flow is complete. 
     Referring to  FIGS. 34-36 , there are shown stages in Experiment 3. The sample  101  as in Experiment 1 was added to the sample addition zone  2 .  FIG. 34  shows the sample  101  (red color) having filled about 60% of the volume of the wicking zone  5  prior to wash addition. At that point, the wash fluid  301  was added.  FIG. 35  shows the tested lateral-flow assay device immediately after adding the wash fluid  301 , in this experiment 17 □L NDSB Wash fluid (at room temperature) plus blue food dye (blue color). The wash fluid  301  (blue color) is retained within the grooves  410 ,  FIG. 4 .  FIG. 36  shows the lateral-flow assay device when fluid, in this instance the sample  101 , reached the end  3005  of the wicking zone  5 . The fluid of the sample  101  (red color) is completely displaced by the wash fluid  301  (blue color) in the detection zone channel  3064 . Moreover, the wash fluid extended into the wicking zone  5  in a region  3601 . The wash fluid  301  in the wash addition zone  409  is still pinned within the grooves  410  and is still stable after the wash flow is complete. 
     Various experiments were conducted for the configurations shown in  FIGS. 24-27  using POC wash fluid. For all four of those tested configurations, wash was performed effectively for all three tested dispense volumes (10, 15 and 20 μL) of the POC wash fluid  301 . The wash fluid  301  was clearly visible in the detection zone channel  3064  and the wicking zone  5 . In some configurations, the wash fluid  301  moved only downstream if the sample  101  was not touching the cover  990 . In some configurations, the wash fluid moved both upstream and downstream if the sample  101  touched the cover  990 . All tested configurations provided stable menisci for volumes of the wash fluid  301  of 10 μL and 15 μL. The wash fluid  301  was retained within the third (outermost) ring of the grooves  410  at those volumes. For a volume of 20 μL, the wash fluid  301  passed the third ring in some tests. In one test, non-stable meniscus behavior was observed. Accordingly, the flow constrictions  310  can be designed based on the volumes of the sample  101  and the wash fluid  301  to provide stable meniscus behavior. In various tested configurations using nubs (e.g., the protrusions  2630 ,  FIG. 26 ), the nubs did attract the dispensed wash fluid  301 . The menisci were not symmetric in every test. Accordingly, the flow constrictions  310  can be designed based on the volumes of the wash fluid  301  and the configuration of the fluid flow path  64  to provide menisci with a desired degree of symmetry. 
     Referring to  FIG. 37 , there is shown an apparatus  3700  for analyzing a fluidic sample  101  according to at least one exemplary embodiment. The apparatus  3700  includes a transport system  3710  for conveying the lateral-flow assay device  300  between components described below. For simplicity, the transport system  3710  is represented as a continuous conveyor belt. However, this is not limiting. The transport system  3710  can include conveyor(s), gripper(s), robotic arm(s), or other device(s) for moving the lateral-flow assay device  300  with respect to below-described components, or can include stage(s), conveyor(s), or other device(s) for moving below-described components with respect to the lateral-flow assay device  300 , in any combination. Various examples of the transport system  3710  are described in commonly-assigned U.S. Pat. No. 8,080,204 to Ryan et al. and U.S. Pat. No. 8,043,562 to Tomasso et al., each of which is incorporated herein by reference, and in U.S. Pat. No. 7,632,468 to Barski, et al, incorporated herein by reference. Positions of the lateral-flow assay device  300  at various stages of processing are shown in phantom. 
     In this example, the lateral-flow assay device  300  includes the sample addition zone  2 , the wash addition zone  409 , and the wicking zone  60  disposed in that order along the fluid flow path  64 , e.g., as discussed above with reference to  FIG. 3 . Any of the above-described embodiments of lateral-flow assay devices can be used in addition to or in place of the lateral-flow assay device  300 , e.g., the lateral-flow assay devices  300 ,  400 ,  700 ,  800 ,  900 ,  1100 ,  1200 ,  1300 ,  1400 ,  1900 , or other illustrated or described lateral-flow assay devices. 
     A sample-metering mechanism  3720  is configured to selectively apply the fluidic sample  101  to the sample addition zone  2  of the at least one lateral-flow assay device  300 . The illustrated sample-metering mechanism  3720  includes a disposable metering tip  3724  holding, e.g., 250 μL of the fluidic sample  101 . In various aspects, there is a one-to-one correspondence between a particular fluidic sample  101  and a particular disposable metering tip  3724 . In an example, each metering event meters between ˜5 μL and ˜10 μL of the fluidic sample  101 . 
     In the illustrated example, and for explanation only, the sample-metering mechanism  3720  includes a piston  3721  and a driving system  3722  operating the piston  3721  to dispense a selected volume of the fluidic sample  101  from the metering tip  3724 . Other structures for metering can also be used, e.g., air or fluid pressure sources or piezoelectric or thermal actuators. An exemplary metering tip  3724  is described in U.S. Publication No. 2004/0072367 by Ding, et al., the disclosure of which is incorporated herein by reference. Metering the sample  101  onto a lateral-flow assay device  100  is referred to herein as “spotting.” 
     The exemplary apparatus  3700  further includes the wash-metering mechanism  3725  configured to selectively apply the wash fluid  301  to the wash addition zone  409  of the lateral-flow assay device  300 . In an example, the wash-metering mechanism  3725  includes a metering nozzle  3726  and an actuator (not shown), e.g., a piston such as the piston  3721 . In another example, the wash-metering mechanism includes a blister. 
     The wash addition zone  409  includes one or more flow constriction(s)  310  spaced apart from the fluid flow path  64  to form a meniscus in the applied wash fluid. Examples of the wash addition zones  409  and the flow constrictions  310  are discussed above with reference to  FIGS. 3-27 . As discussed above, the fluid flow path  64  is configured to draw the applied wash fluid  301  out of a reservoir  535 ,  FIG. 5 , defined at least partly by the meniscus. 
     The exemplary apparatus  3700  includes at least one incubator  3730 . Various types of sample testing, including potentiometric, rate chemistry, and endpoint tests, may be required for any given patient sample, necessitating both different incubation intervals and different test apparatus within the incubator  3730 . Accordingly, more than one incubator, or a tandem or other multi-test-capable incubator can be used. For clarity, only one incubator  3730  is shown. Various examples of the incubators  3730  and related components are described in U.S. Pat. Nos. 4,287,155 and 7,312,084 to Jakubowicz, et al., entitled “Tandem Incubator for Clinical Analyzer,” each of which is hereby incorporated by reference in its entirety. 
     The incubator  3730  retains the lateral-flow assay device(s)  300 , e.g., at room temperature or under selected environmental conditions, until an accurate measurement can be taken. Some lateral-flow assay devices  300  require endpoint testing, which requires only a single read be performed following a predetermined incubation interval (e.g., approximately 5 minutes). Other lateral-flow assay devices  300 , such as those requiring rate chemistries, require a number of reads to be taken throughout the course of incubation. The incubator  3730  or the transport system  3710  can therefore include structures for transporting lateral-flow assay device(s)  300  between the incubator  3730  and a measurement device  3740 , discussed below. 
     The exemplary apparatus  3700  shown further includes at least one measurement device  3740 . The measurement device  3740  can include a potentiometric sensor, e.g., a voltmeter, ammeter, or charge meter, or a colorimetric or other photometric sensor. Exemplary photometric sensors include photodiodes and line-scan or area-scan reflectometers or imagers, e.g., charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) imagers. Colorimetric sensors can operate in reflective or transmissive modes. Reflective colorimetric sensors can be arranged to measure the front or back of the lateral-flow assay device  300 . 
     In an example, the measurement device  3740  includes a light source  3742  (represented graphically as a lamp). The light source  3742  can include a lamp, light-emitting diode (LED), laser, or other source of optical radiation. The exemplary measurement device  3740  also includes a photosensor  3744  that captures light of the light source  3742  reflected from the detection zone  56  of the lateral-flow assay device  300 . 
     The exemplary apparatus  3700  further includes a controller  3786  configured to operate each of the sample-metering mechanism  3720 , the wash-metering mechanism  3725 , and the at least one measurement device  3740  in accordance with a predetermined timing protocol in order to determine at least one characteristic of the applied fluidic sample  101 . The controller  3786  is configured to operate the wash-metering mechanism  3725  after operating the sample-metering mechanism  3720 . The controller  3786  can also be configured to operate the incubator  3730 . 
     For clarity only, communications connections between the controller  3786  and other components are shown dashed. Further and according to this exemplary embodiment, the controller  3786  is configured to operate the transport system  3710 . For example, the controller  3786  can sequence the motion of the lateral-flow assay device  300  through the sample-metering mechanism  3720 , the incubator  3730 , and the at least one measurement device  3740  to perform a potentiometric or colorimetric measurement of the fluidic sample  101 . The exemplary controller  3786  can be further configured to receive data from the photosensor  3744  and provide a graphical representation of the measured data via an electronic display. The controller  3786  can include various components discussed below with reference to  FIG. 39 , e.g., a processor  3986 . 
       FIG. 38  shows a flowchart illustrating an exemplary method for displacing a fluidic sample in a fluid flow path of an assay device. In at least one example, processing begins with step  3810 . For clarity of explanation, reference is herein made to various components shown in  FIGS. 1-27, 37  that can carry out or participate in the steps of the exemplary method. It should be noted, however, that other components can be used; that is, exemplary method(s) shown in  FIG. 38  are not limited to being carried out by the identified components. The method can include automatically carrying out the listed steps using a processor, e.g., the processor  3986 ,  FIG. 39 , or another processor in the controller  3786 ,  FIG. 37 . 
     In step  3810 , the fluidic sample  101  is dispensed from a sample supply, e.g., the sample-metering mechanism  3720 ,  FIG. 37 , onto a sample addition zone  2  of the lateral-flow assay device  300 . The dispensed fluidic sample  101  travels along the fluid flow path  64  of the lateral-flow assay device  300 . 
     In step  3820 , a wash fluid  301  is dispensed from a wash-fluid supply, e.g., the wash-metering mechanism  3725 ,  FIG. 37 , onto a wash addition zone  409  of the lateral-flow assay device  300  downstream of the sample addition zone  2  along the fluid flow path  64 . A meniscus is then formed in the dispensed wash fluid  301  by at least one flow constriction  310  of the lateral-flow assay device  300 . The fluid flow path  64  draws at least some of the dispensed wash fluid  301  out of the reservoir  535  defined at least partly by the meniscus. The drawn at least some of the dispensed wash fluid  301  displaces at least some of the fluidic sample  101  in the fluid flow path  64 . This is discussed above with reference to  FIG. 6 . Step  3820  permits performing assays that require washing with other than fluid of the sample  101  in order to provide accurate results. In various embodiments, step  3820  is followed by step  3830 . 
     In step  3830 , after said dispensing the wash fluid in step  3820 , the presence of a detectable signal corresponding to a characteristic of the dispensed fluid sample  101  is determined. This can be done using the incubator  3730 , the measurement device  3740 , or both. In embodiments using incubation, the incubation time can be selected as appropriate based on the fluidics and dimensions of the lateral-flow assay device  300  and the viscosities or surface tensions of the sample  101  or the wash fluid  301 . 
       FIG. 39  is a high-level diagram showing the components of an exemplary data-processing system  3901  for analyzing data, operating an apparatus  3700 ,  FIG. 37 , for analyzing samples  101  and performing other analyses described herein, and related components. The data-processing system  3901  includes a processor  3986 , a peripheral system  3920 , a user interface system  3930 , and a data storage system  3940 . The peripheral system  3920 , the user interface system  3930  and the data storage system  3940  are communicatively connected to the processor  3986 . The processor  3986  can be communicatively connected to a network (not shown). The following devices can each include one or more of the systems  3986 ,  3920 ,  3930 ,  3940 , and can each connect to one or more network(s): the controller  3786 , the sample-metering mechanism  3720 , the wash-metering mechanism  3725 , the incubator  3730 , the light source  3742 , and the photosensor  3744 , all  FIG. 37 . The processor  3986 , and other processing devices described herein, can each include one or more microprocessors, microcontrollers, field-programmable gate arrays (FPGAs), application-specific integrated circuits (ASICs), programmable logic devices (PLDs), programmable logic arrays (PLAs), programmable array logic devices (PALs), or digital signal processors (DSPs). 
     The processor  3986  can implement processes of various aspects described herein. The processor  3986  and related components can, e.g., carry out processes for performing assays or for displacing a fluidic sample  101  in a fluid flow path  64  of a lateral-flow assay device  300 . Examples of such processes are described above with reference to  FIGS. 37 and 38 . 
     The processor  3986  can be embodied in one or more device(s) for automatically operating on data, e.g., a central processing unit (CPU), microcontroller (MCU), desktop computer, laptop computer, mainframe computer, personal digital assistant, digital camera, cellular phone, smartphone, or any other device for processing data, managing data, or handling data, whether implemented with electrical, magnetic, optical, biological components, or otherwise. 
     The phrase “communicatively connected” includes any type of connection, wired or wireless, for communicating data between devices or processors. These devices or processors can be located in physical proximity or not. For example, subsystems such as the peripheral system  3920 , the user interface system  3930 , and the data storage system  3940  are shown separately from the processor  3986  but can be stored completely or partially within the processor  3986 . 
     The peripheral system  3920  can include one or more devices configured to provide digital content records to the processor  3986 . For example, the peripheral system  3920  can include or communicate with one or more measurement device(s)  3740 ,  FIG. 37 . The processor  3986 , upon receipt of digital content records from a device in the peripheral system  3920 , can store such digital content records in the data storage system  3940 . In various examples, the peripheral system  3920  is communicatively connected to one or more of the sample-metering mechanism  3720 , the wash-metering mechanism  3725 , the incubator  3730 , the light source  3742 , and the photosensor  3744 , all  FIG. 37 . 
     The user interface system  3930  can convey information in either direction, or in both directions, between a user  3938  and the processor  3986  or other components of the data-processing system  3901 . The user interface system  3930  can include a mouse, a keyboard, another computer (connected, e.g., via a network or a null-modem cable), or any device or combination of devices from which data is input to the processor  3986 . The user interface system  3930  also can include a display device, e.g., an electronic display  3935 , a processor-accessible memory, or any device or combination of devices to which data is output by the processor  3986 . The user interface system  3930  and the data storage system  3940  can share a processor-accessible memory. 
     The data storage system  3940  can include or be communicatively connected with one or more processor-accessible memories configured to store information. The memories can be, e.g., within a chassis or as parts of a distributed system. The phrase “processor-accessible memory” is intended to include any data storage device to or from which the processor  3986  can transfer data (using appropriate components of the peripheral system  3920 ), whether volatile or nonvolatile; removable or fixed; electronic, magnetic, optical, chemical, mechanical, or otherwise. Exemplary processor-accessible memories include but are not limited to: registers, floppy disks, hard disks, tapes, bar codes, Compact Discs, DVDs, read-only memories (ROM), erasable programmable read-only memories (EPROM, EEPROM, or Flash), and random-access memories (RAMs). One of the processor-accessible memories in the data storage system  3940  can be a tangible non-transitory computer-readable storage medium, i.e., a non-transitory device or article of manufacture that participates in storing instructions that can be provided to the processor  3986  for execution. 
     In an example, the data storage system  3940  includes a code memory  3941 , e.g., a RAM, and a disk  3943 , e.g., a tangible computer-readable storage device such as a hard drive or Flash drive. Computer program instructions are read into the code memory  3941  from the disk  3943 . The processor  3986  then executes one or more sequences of the computer program instructions loaded into the code memory  3941 , as a result performing process steps described herein. In this way, the processor  3986  carries out a computer implemented process. For example, steps of methods described herein, blocks of the flowchart illustrations or block diagrams herein (e.g.,  FIG. 38 ), and combinations of those, can be implemented by computer program instructions. The code memory  3941  can also store data, or can store only code. 
     Various aspects described herein may be embodied as systems or methods. Accordingly, various aspects herein may take the form of an entirely hardware aspect, an entirely software aspect (including firmware, resident software, micro-code, etc.), or an aspect combining software and hardware aspects These aspects can all generally be referred to herein as a “service,” “circuit,” “circuitry,” “module,” or “system.” 
     Furthermore, various aspects herein may be embodied as computer program products including computer readable program code stored on a tangible non-transitory computer readable medium. Such a medium can be manufactured as is conventional for such articles, e.g., by pressing a CD-ROM. The program code includes computer program instructions that can be loaded into the processor  3986  (and possibly also other processors), to cause functions, acts, or operational steps of various aspects herein to be performed by the processor  3986  (or other processor). Computer program code for carrying out operations for various aspects described herein may be written in any combination of one or more programming language(s), and can be loaded from the disk  3943  into the code memory  3941  for execution. 
     Various above-described embodiments advantageously use flow constriction(s)  310 ,  FIG. 3 , in the wash addition zone  409  to stabilize the dispensed wash fluid  301 , e.g., to pin the wash fluid  301  to selected locations in the wash addition zone  409 . The flow constriction(s)  310  advantageously encourage the formation of one or more partly-meniscus-delimited reservoir(s)  535  that can receive variable volumes of the wash fluid  301  with reduced sensitivity to the dispensing rate of the wash fluid  301 . Moreover, the pressure of such menisci is close to the ambient, reducing the probability of overflowing the fluid flow path  64 . 
     Various exemplary flow constriction(s) include nozzle(s) that connects a wash fluid supply to the fluid flow path  64  in the lateral-flow assay device in the wash addition zone  409 ; very low nozzle outlets to promote contact between the wash fluid  301  in the nozzle and the hydrophilic surface  308  on the substrate  9 ; and steps outside the nozzle (e.g., as in  FIG. 22 ) to permit variable fluid meniscus sizes (volumes of the reservoir  535 ) while maintaining meniscus stability. 
     Various aspects advantageously permit variable-rate, variable-amount delivery of the wash fluid  301 , and stabilize the received wash fluid  301  at a desired location. Various aspects reduce the probability of overflowing the fluid flow path  64 , which improves wash efficiency. Various aspects advantageously provide robust wash performance with respect to one or more of the following properties:
         Variation in the volume of the wash fluid  301  delivered to the wash addition zone  409  within the range, e.g., from 7 μL to 17 μL. This relaxed volume range can reduce the development cost of wash fluid delivery system (e.g., the blister).   Variation in the delivery rate of the wash fluid  301  within the range, e.g., 1 μL/sec to &gt;10 μL/sec. This relaxed range also facilitates more effective fluid delivery system design (e.g., a burst of wash fluid from a squeezed blister can be used).   Maintenance of a stable meniscus in the wash addition zone, independent of above-noted variations in the delivery volume and delivery rate of the wash fluid  301 .   Entry of the wash fluid  301  into the fluid flow path  64  at an appropriate location to effectively displace the fluid of the sample  101  in the fluid flow path  64  without “overflow,” i.e., the wash fluid  301  flowing over the sample  101  between the microposts  7  inside the fluid flow path  64 .   Termination of the fluid flow of the sample  101  when the wash fluid  301  is added. Various aspects restrict the sample  101  from flowing along the fluid flow path  64  downstream past the wash addition zone  409  once the wash fluid  301  is added.   Maintenance of meniscus stability in the wash addition zone  409  as the wash fluid  301  enters the fluid flow path  64  to perform the wash.   Variation in the amount of the wash fluid  301  to be delivered through the detection zone channel  3064  in the range from 1 μL to 4 □L, or in the range of &gt;4 μL.       

     PARTS LIST FOR FIGS.  1 - 39   
     
         
           1  lateral-flow assay device 
           2  sample addition zone 
           3  reagent zone 
           4  detection zone 
           5  wicking zone 
           7  microposts 
           9  substrate 
           20  lateral-flow assay device 
           40  substrate 
           44  top surface 
           48  sample addition zone 
           52  reagent zone 
           55  detection channel 
           56  detection zone 
           57  flow promoter 
           60  wicking zone 
           64  fluid flow path 
           70  hydrophilic layer 
           72  vents 
           100  lateral-flow assay device 
           101  sample 
           300  lateral-flow assay device 
           301  wash fluid 
           308  hydrophilic surface 
           310  flow constriction 
           311  protrusion 
           400  lateral-flow assay device 
           409  wash addition zone 
           410  groove 
           411  arcuate path 
           464  centerline 
           511  corner 
           520  meniscus 
           521  angle 
           530  meniscus 
           531  angle 
           535  reservoir 
           655  area 
           700  lateral-flow assay device 
           710  reference point 
           764  centerline 
           800  lateral-flow assay device 
           810  grooves 
           869  spiral path 
           900  lateral-flow assay device 
           908  hydrophilic surface 
           910  cover flow constriction 
           911  protrusion 
           912  cover flow constriction 
           913  protrusion 
           920  aperture 
           930  wash port 
           935  meniscus 
           990  cover 
           1018  proximal edge 
           1019  distal edge 
           1035  meniscus 
           1100  lateral-flow assay device 
           1120  nozzle 
           1200  lateral-flow assay device 
           1210 ,  1211 ,  1212 ,  1213  grooves 
           1235 ,  1237  menisci 
           1300  lateral-flow assay device 
           1335  meniscus 
           1400  lateral-flow assay device 
           1411  lip 
           1420  distal surface 
           1435 ,  1436  menisci 
           1710 ,  1810  annuli 
           1900  lateral-flow assay device 
           1911  lip 
           1920  aperture 
           1995  offset 
           2112  protrusion 
           2220  nozzle 
           2225  stepped surface 
           2312  protrusion 
           2420  nozzle 
           2425  plateau 
           2520  annulus 
           2630 ,  2730 ,  2731  protrusions 
           3001  region 
           3005  end 
           3064  detection zone channel 
           3301 ,  3601  regions 
           3700  apparatus 
           3710  transport system 
           3720  sample-metering mechanism 
           3721  piston 
           3722  driving system 
           3724  disposable metering tip 
           3725  wash-metering mechanism 
           3726  metering nozzle 
           3730  incubator 
           3740  measurement device 
           3742  light source 
           3744  photosensor 
           3786  controller 
           3810 ,  3820 ,  3830  steps 
           3901  data-processing system 
           3920  peripheral system 
           3930  user interface system 
           3935  electronic display 
           3938  user 
           3940  data storage system 
           3941  code memory 
           3943  disk 
           3986  processor 
         F flow direction 
       
    
     The invention is inclusive of combinations of the aspects described herein. References to “a particular embodiment” (or “aspect” or “version”) and the like refer to features that are present in at least one aspect of the invention. Separate references to “an embodiment” or “particular embodiments” or the like do not necessarily refer to the same embodiment or embodiments; however, such embodiments are not mutually exclusive, unless so indicated or as are readily apparent to one of skill in the art. The use of singular or plural in referring to “method” or “methods” and the like is not limiting. The word “or” is used in this disclosure in a non-exclusive sense, unless otherwise explicitly noted. The invention has been described in detail with particular reference to certain preferred aspects thereof, but it will be readily apparent that other modifications and variations are possible within the intended ambits of the concepts described herein and in accordance with the following claims.