Assay stack with separation membrane containing soluble polymer coating to control flow rate

Abstract

This present disclosure provides devices for analyzing a fluid sample and contemplates an assay device that includes a separation membrane and a detection membrane. The separation membrane includes a first reagent and a soluble polymer. The first reagent can precipitate one or more fluid components other than the target analyte or can sequester the target analyte. A soluble polymer in the separation membrane ensures that there is sufficient time for the precipitation or sequestration to reach completion before the fluid sample containing the target analyte or other analyte being used to indirectly determine its concentration flows to the next membrane. The assay stack also includes a detection membrane with a second reagent that is configured to elicit a quantifiable response in the presence of the target analyte or other analyte being used to indirectly determine its concentration. Methods for manufacturing and performing an assay with the device are also provided.

Description

ASSAY STACK WITH SEPARATION MEMBRANE CONTAINING SOLUBLE POLYMER COATING TO CONTROL FLOW RATEFIELD

[0001] The present disclosure relates generally to a point-of-care (POC) testing system that includes an assay device. More particularly, the present disclosure relates to assay devices and methods for ensuring that a fluid sample containing a target analyte has sufficient time to react with a first reagent on a first membrane before flowing to a second membrane.BACKGROUND

[0002] Point-of-care (POC) testing refers to performing medical diagnostic tests at the time and place that the patient is being treated. POC testing is advantageous over traditional diagnostic testing where patient samples are sent out to a laboratory for further analysis, because the results of traditional diagnostic tests may not be available for hours, if not days or weeks, making it difficult for a caregiver to assess the proper course of treatment in the interim.

[0003] In particular, vertical flow assays are a commonly used POC platform for testing for the presence of analytes in a fluid sample. When the fluid sample is blood, for example, such assays are generally optimized to remove red blood cells from the fluid sample while the remaining plasma flows to a membrane in which the target analyte is catalyzed to produce a color. These steps should be as efficient as possible in order to limit the total testing time. Thus, it is undesirable to require lengthy incubation steps in such a testing platform. Vertical flow technologies have been developed to perform the same functions as traditional assays which can be performed by adding all reagents to a sample in a single step. However, it has been a challenge to adapt vertical flow technologies to assays which require incubation steps without adding significant cost to the overall price of the assay device.

[0004] Therefore, a need exists for a vertical flow assay that progresses at a controlled rate, allowing a reaction at one layer or membrane of the assay device to take place for an addressable amount of time to allow a reaction between a reagent on the layer or membrane to run to completion before the fluid being tested flows to the next layer or membrane.SUMMARY

[0005] Aspects and advantages of embodiments of the present disclosure will be set forth in part in the following description, or can be learned from the description, or can be learned through practice of the embodiments.

[0006] One example aspect of the present disclosure is directed to an assay device for determining a concentration of a target analyte in a fluid sample. The assay device includes a separation membrane that includes a first reagent and a soluble polymer, as well as a detection membrane located downstream from the separation membrane and comprising a second reagent. Further, the detection membrane is configured to elicit a quantifiable response in the presence of the target analyte, the quantifiable response corresponding to an amount of the target analyte present in the detection membrane.

[0007] In one aspect, the polymer can be carboxymethyl cellulose or its derivatives, dextran or its derivatives, polyvinyl alcohol or its derivatives, polyethylene glycol or its derivatives, or a combination thereof. Further, the soluble polymer can be present on the separation membrane at a concentration ranging from about 0. 1 milligrams / square centimeters to about 0.6 milligrams / square centimeters.

[0008] In one more aspect, the soluble polymer can be present on the separation membrane at a concentration ranging from about 0.05 wt.% to about 4.0 wt.% of a mass of an amount of liquid necessary to saturate the separation membrane.

[0009] In another aspect, the first reagent is configured to precipitate a component of the fluid sample to prevent the component from flowing to the detection membrane. For instance, the soluble polymer is configured to initially block passage of the fluid sample through pores in the separation membrane until precipitation of the component of the fluid sample is complete, after which the fluid sample is able to pass through the pores. Further, the target analyte can be a high density lipoprotein and the precipitated component can be a non-high density lipoprotein. In addition, the first reagent can be a poly anionic non-high density lipoprotein binding reagent that can include a sulfonated polysaccharide, heparin, or phosphotungstic acid. Moreover, in some instances, the first reagent can include group II cation selected from the group of Mg2+, Mn2+, or Ca2+. In one specific non-limiting embodiment, the first reagent can include phosphotungstic acid and Mg2+.

[0010] In still another aspect, the second reagent can include cholesterol esterase, cholesterol oxidase, horseradish peroxidase, a Trinder reagent, 4-amino antipyrine, or a combination thereof.

[0011] In yet another aspect, the target analyte can be sodium. Further, the first reagent can include a sodium sequestering agent that can include a complex comprising an ion and a cryptand having an affinity for the ion, and the second reagent can be an ion-dependent dye that is dependent on the ion from the complex. The ion can be a divalent ion that is Ca2+, Fe2+, Mg2+, or Mn2+or the ion can be K+, while the ion-dependent dye can be an azoic dye.

[0012] In one more aspect, the assay device can be used in-vitro for determining the concentration of the target analyte in the fluid sample.

[0013] Another example aspect of the present disclosure is directed to a method of forming an assay device for determining a concentration of a target analyte in a fluid sample. The method includes applying a first reagent and a soluble polymer to a separation membrane; allowing the first reagent and the soluble polymer to dry on the separation membrane; applying a second reagent to a detection membrane; allowing the second reagent to dry on the detection membrane; and positioning the detection membrane adj acent the separation membrane, the detection membrane configured to elicit a quantifiable response in the presence of the target analyte, the quantifiable response corresponding to an amount of the target analyte present in the detection membrane.

[0014] In one aspect, the soluble polymer can include carboxymethyl cellulose or its derivatives, dextran or its derivatives, polyvinyl alcohol or its derivatives, polyethylene glycol or its derivatives, or a combination thereof. Further, the soluble polymer can be present on the separation membrane after drying at a concentration ranging from about 0. 1 milligrams / square centimeters to about 0.6 milligrams / square centimeters.

[0015] In another aspect, the soluble polymer can be present on the separation membrane at a concentration ranging from about 0.05 wt.% to about 4.0 wt.% of a mass of an amount of liquid necessary to saturate the separation membrane.

[0016] In addition, the first reagent can be configured to precipitate a component of the fluid sample to prevent the component from flowing to the detection membrane, and the soluble polymer can be configured to initially block passage of the fluid sample through pores in the separation membrane until precipitation of the component of the fluid sample is complete, after which the fluid sample is able to pass through the pores.

[0017] In another aspect, the target analyte can include a high density lipoprotein and the precipitated component comprising a non-high density lipoprotein, and the first reagent can include a poly anionic non-high density lipoprotein binding reagent. In such an aspect, the first reagent can include a sulfonated polysaccharide, heparin, or phosphotungstic acid and can also include a group II cation selected from the group of Mg2+, Mn2+, or Ca2+. Forinstance, in one non-limiting example, the first reagent can include phosphotungstic acid and Mg2+. Further, the second reagent can include cholesterol esterase, cholesterol oxidase, horseradish peroxidase, a Tnnder reagent, 4-amino antipyrine, or a combination thereof.

[0018] In still another aspect, the target analyte can include sodium. In such an aspect, the first reagent can include a sodium sequestering agent such as a complex comprising an ion and a cryptand having an affinity for the ion, while the second reagent can include an iondependent dye that is dependent on the ion from the complex. Further, the ion can include a divalent ion that is Ca2+, Fe2+, Mg2+, or Mn2+or the ion comprising K+, while the iondependent dye can include an azoic dye.

[0019] These and other features, aspects, and advantages of various embodiments of the present disclosure will become better understood with reference to the following description and appended claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate example embodiments of the present disclosure and, together with the description, serve to explain the related principles.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Detailed discussion of embodiments directed to one of ordinary skill in the art is set forth in the specification, which makes reference to the appended figures, in which:

[0021] FIG. 1 provides a schematic drawing of a system comprising a cartridge and an assay reader according to one embodiment of the disclosure;

[0022] FIGs. 2A-2C illustrate an embodiment of the cartridge utilized in the system;FIG. 3 illustrates various layers of a metering stack contained within the cartridge;

[0023] FIG. 4 illustrates various layers of an assay stack contained within the cartridge;

[0024] FIG. 5 A shows a longitudinal cross-sectional view of an assay reader according to one embodiment of the disclosure;

[0025] FIG. 5B shows a longitudinal cross-sectional view of an assay reader with an inserted cartridge according to one embodiment of the disclosure;

[0026] FIG. 6A shows a transverse cross-sectional view' of an assay reader according to one embodiment of the disclosure;

[0027] FIG. 6B shows a transverse cross-sectional view of the assay reader with an inserted cartridge according to one embodiment of the disclosure;

[0028] FIG. 7 shows a block diagram of the sensor system of the assay reader, according to an exemplary implementation of the disclosure;

[0029] FIGs. 8A-8F illustrate a non-limiting embodiment of a cartridge that includes a metering stack and an assay stack during various stages of the assay process after a blood fluid sample has been introduced to the cartridge;

[0030] FIGs. 9A-9F illustrate another non-limiting embodiment of a cartridge that includes a metering stack and an assay stack during various stages of the assay process after a blood fluid sample has been introduced to the cartridge;

[0031] FIG. 10 shows a flow chart illustrating a method of using the assay system according to an exemplary implementation of the disclosure; and

[0032] FIG. 11 shows a flow chart illustrating a method of manufacturing a cartridge according to one exemplary implementation of the disclosure.

[0033] Reference numerals that are repeated across plural figures are intended to identify the same features in various implementations.DETAILED DESCRIPTION

[0034] Any of the features, components, or details of any of the arrangements or embodiments disclosed in this application, including without limitation any of the cartridge embodiments and any of the testing or assay embodiments disclosed below, are interchangeably combinable with any other features, components, or details of any of the arrangements or embodiments disclosed herein to form new arrangements and embodiments.

[0035] Generally, the present disclosure is related to an assay stack for determining a concentration of a target analyte in a fluid sample. The assay stack includes a separation membrane and a detection membrane located dow nstream from the separation membrane. The separation membrane includes a first reagent as well as a soluble polymer. In one embodiment, the first reagent can precipitate one or more fluid components other than the target analyte. In another embodiment, the first reagent can sequester the target analyte. In either embodiment, a soluble polymer in the separation membrane ensures that there is sufficient time for the precipitation or sequestration to reach completion before the fluid sample containing the target analyte or other analyte being used to indirectly determine the concentration of the target analyte flows to the next membrane. In addition, the assay stack includes a detection membrane that includes a second reagent that is configured to elicit a quantifiable response in the presence of the target analyte or other analyte being used to indirectly determine the concentration of the target analyte. Additionally, the quantifiable response corresponds to an amount of target analyte or other analyte being used to indirectly determine the concentration of the target analyte present in the fluid sample. The presentdisclosure also provides methods for forming an assay device to quantify the amount of target analyte that is present in the fluid sample.

[0036] In particular, the assay stack allows for the concentration of a target analyte in a fluid sample to be determined efficiently and accurately in a point-of-care setting without the need for user intervention, carefully controlled environments, moving parts, or expensive equipment. In addition, the assay stack allows for vertical flow assays to progress at a controlled rate, allowing a required reaction between the fluid sample and the first reagent to take place in a first membrane, such as the separation membrane, before the target analyte flows to the second membrane, such as the detection membrane. For instance, the first membrane, which contains pores, is coated with the first reagent and the soluble polymer at concentration to fill in the pores of the membrane until the soluble polymer dissolves over a time period that allows the reaction between the first reagent and the one or more fluid components other than the target analyte to proceed to completion, after which the fluid sample still containing the target analyte can flow to the detection membrane via the now open pores.

[0037] The assay stack can be part of a cartridge that forms an assay device. In combination with an assay reader configured to be compatible with a proposed assay device so that the proposed assay device can be inserted into the assay ready for distributing a fluid sample to the separation membrane, the detection of the target analyte may be automated in an easy way, since any target analyte present in the blood fluid sample may flow through the assay stack from the separation membrane to the detection membrane, while any interfering or unwanted fluid components are left behind in the separation membrane. Based on the detection membrane allowing for qualitatively or quantitatively determining the amount of target analyte in the fluid sample fluid based on a resulting signal (e.g., a color change) in the presence of a reagent for the target analyte, an automated assessment of the presence of the target analyte in the fluid sample is possible.

[0038] In one aspect, the assay stack can include reagents used to determine cholesterol levels in a fluid sample to assess a patient's risk for coronary artery disease. Total cholesterol includes low density lipoproteins (LDL), intermediate density lipoproteins (IDL), and very low density lipoproteins (VLDL), hereinafter collectively referred to as non-HDL, and high density lipoproteins (HDL). It is generally known that there is a positive correlation between LDL, IDL, and VLDL values and coronary artery disease and a negative correlation between HDL values and coronary artery diseases. Thus, LDL, IDL, and VLDL are often referred to as “bad cholesterol” while HDL is often referred to as “good cholesterol.” Due to thisdistinction, there is a benefit to determining the amount of HDL in a blood fluid sample to determine a patient’s risk of heart disease. This requires separating the HDL from other certain proteins and components (e.g., red blood cells or erythrocytes) in a blood fluid sample in order to arrive at an accurate reading of the HDL present. The present disclosure allows for such separation in order to efficiently provide accurate detection of the target analyte. The assay stack can include a separation membrane containing a first reagent. The first reagent can be used for precipitating a component such as a first protein contained in, for example, a blood fluid sample containing a target analyte. Further, the separation layer can have a maximum pore size ranging from about 20 micrometers to about 100 micrometers on an upstream side of the membrane and can have a minimum pore size ranging from about 0. 1 micrometers to about 2 micrometers on a downstream side of the membrane. Further, a soluble polymer in the separation membrane ensures that there is sufficient time for the precipitation to reach completion before the fluid sample containing the target analyte flows to the next membrane, which can be a detection membrane. The detection membrane is configured to elicit a quantifiable response in the presence of the target analyte, which can be a second protein present in the blood fluid sample. Further, the quantifiable response corresponds to an amount of the second protein present in the detection membrane. In addition, the separation membrane prevents red blood cells and the precipitated component (i.e., the first protein) from entering the detection membrane. In one embodiment to determine cholesterol levels, the precipitated component or first protein can be a non-high density (non-HDL) lipoprotein and the target analyte or second protein can be a high density lipoprotein (HDL).

[0039] Unlike other assay devices, which separate red blood cells from the blood fluid sample in a first layer, followed by the separation of non-high density lipoproteins in a second layer, which requires large blood volumes, the present disclosure, in some aspects, contemplates concurrent separation of the red blood cells and the non-high density lipoproteins. This requires a reduced blood fluid sample volume compared to traditional commercially available assay devices. In such devices, a spreading mesh, a plasma separation layer, and a non-high density lipoprotein separation layer, and a high density lipoprotein detection layer or analysis layer are required. In contrast, the assay device and methods of the present disclosure can utilize just a separation membrane or layer and a detection membrane or layer, although it is to be understood that a spreading mesh, plasma separation layer, or other layers can also be utilized.

[0040] The proposed solution allows for providing a compact POC testing system with a proposed assay device capable of an in-vitro assay of an isolated blood fluid sample with concurrent separation of a first protein (e.g., non-HDL) and red blood cells without the need for additional layers or membranes in the POC system to carry out such separation from the blood fluid sample to be analyzed for a target analyte of interest (e.g., a second protein such as HDL). A cartridge constructed by an embodiment of a proposed assay device with an assay stack comprising only a separation membrane and a detection membrane in this context allows for a cost-efficient design and requires less blood fluid sample volume compared to conventional HDL assay systems.

[0041] In another aspect, the assay stack can include a separation membrane and a detection membrane located downstream with respect to a flowing direction of the blood fluid sample through the assay device from the separation membrane. The separation membrane can include a first reagent that is a complex comprising an ion and a cryptand having an affinity for the ion. In addition, the detection membrane can include an iondependent dye that is dependent on the ion from the complex. Further, the ion-dependent dye is configured to elicit a quantifiable response after the ion binds to the ion-dependent dye when the ion is released from the cryptand after the sodium from the blood fluid sample binds to the cryptand portion of the complex in the separation membrane, where such a reaction is carefully controlled due to the presence of a soluble polymer that initially blocks the pores and allows the reaction to occur at a controlled rate until all of the sodium has bound to the cryptand and the ion that was initially bound to the cryptand has been released. Additionally, the quantifiable response corresponds to an amount of the ion bound to the ion-dependent dye, and the amount of the ion bound to the ion-dependent dye corresponds to a concentration of sodium present in the blood fluid sample. The present disclosure also provides methods for using an assay device to quantify the amount of sodium that is present in the blood fluid sample.

[0042] In particular, the assay device allows for the concentration of sodium in a blood fluid sample to be determined in a point-of-care setting with high accuracy without the need for carefully controlled environments, titration steps, manual manipulation, or expensive equipment that are required by current laboratory methods. Such parameters have typically been required due to the high level of accuracy and sensitivity required for measuring sodium concentrations given that there is only about an 8% difference in concentration for dangerously low sodium concentrations and dangerously high sodium concentrations in the blood.

[0043] For instance, the present disclosure contemplates an ion that can activate a color change in the ion-dependent dye upon binding thereto, and a cryptand that has an affinity for the ion to measure the concentration of sodium in blood, where the cryptand and ion form a first reagent that is applied as a complex on a layer of the assay device, as well as a second reagent in the form of an ion-dependent dye,. The ion-dependent dye can be used in higher concentrations than would be typical such that the resulting color change happens over a slightly larger range of concentrations, thus allowing the assay to be quantitative over the entire range of physical sodium concentrations (about 135 millimolar to about 145 millimolar). Further, the cryptand that is used preferentially binds sodium, but can also bind the ion to which it is initially complexed on a layer as a first reagent, such as a separation membrane, of the assay device more strongly than the ion-dependent dye in the absence of sodium such that the ion is initially sequestered from the ion-dependent dye. Then, when sodium is introduced to the layer of the assay device, the ion that is part of the complex with the cryptand is eventually released, travels to the detection membrane after the reaction between the sodium and the cryptand has been completed and the soluble polymer has dissolved, and binds to the ion-dependent dye, producing a color change from which the concentration of the released ion can be determined. For example, a degree of color change, in particular a change to a particular color, could thus be indicative for a predefined range of ion concentration (a change in color and in particular to a certain color) may correlate with a released ion concentration. Thereafter, from this concentration, the concentration of sodium can be determined indirectly.

[0044] Further, it should be understood that an excess of cryptand is present compared to the ion that forms part of a complex with the cryptand (i.e., some of the cryptand is not bound to the ion). Initially, this allows for most of the sodium present in a blood fluid sample to bind to be bound to the free cryptand, creating no measurable color change. Then, once all of the free cryptand is bound, any remaining sodium from the blood fluid sample will displace the ions that are part of the ion-cryptand complex. These released ions (e.g., divalent ions) will thus be present in much lower concentrations than the sodium, making the measured range proportionally much larger. In the theoretical limit of perfect affinity balances, this would mean that the divalent ion concentration would be from 0 to 20 mM, corresponding to 130 to 150 mM sodium. To achieve this behavior, the following relationships control: (1) the affinity of the cryptand for sodium is greater than the affinity of the cryptand for the ion that is part of the ion (e.g., divalent ion) and cryptand complex; (2) the affinity of the cryptand for the ion that is part of the ion (e.g., divalent ion) and cryptand complex is greater than theaffinity of the ion-dependent dye for that ion; (3) the affinity of the ion-dependent dye for the ion is high relative to the concentration of the ion; and (4) the affinity of the ion-dependent dye for sodium is low relative to the concentration of sodium.

[0045] In the embodiments contemplated by the present disclosure, the assay device is configured to measure a concentration of sodium ranging from about 135 millimolar to about 145 millimolar with an error rate of less than about 3%.

[0046] Regardless of the particular target analyte and specific first reagent and second reagent configurations described above, the first reagent is applied to the separation membrane in conjunction with a soluble polymer. The soluble polymer “clogs” the pores in the separation membrane until the desired reaction between the first reagent and the target analyte or other fluid component takes place to completion to improve the accuracy of sequential assays. The soluble polymer can include carboxymethyl cellulose or its derivatives, dextran or its derivatives, polyvinyl alcohol or its derivatives, polyethylene glycol or its derivatives, or a combination thereof. Moreover, the present inventor has found that the concentration at which the soluble polymer is present on the separation membrane allows for it to dissolve in a controlled manner to ensure that any reactions on the separation membrane have completed while the fluid still flows to the next membrane efficiently. For instance, the soluble polymer can be present on the separation membrane at a concentration ranging from about 0.1 milligrams / square centimeter to about 0.6 milligrams per square centimeter, such as from about 0. 15 milligrams / square centimeter to about 0.55 milligrams per square centimeter, such as from about 0.2 milligrams / square centimeter to about 0.5 milligrams per square centimeter. In other aspects, the soluble polymer can be present on the separation membrane at a concentration ranging from about 0.05 wt.% to about 4.0 wt.%, such as from about 0.075 wt.% to about 3.5 wt.%, such as from about 0. 1 wt.% to about 3 wt.% of a mass of an amount of liquid necessary to saturate the separation membrane. Additionally, the soluble polymer can have a molecular weight ranging from about 40,000 Daltons to about 1,500,000 Daltons, such as from about 50,000 Daltons to about 1,000,000 Daltons, such as from about 60,000 Daltons to about 500,000 Daltons, such as from about 70,000 Daltons to about 200,000 Daltons.

[0047] With reference now to the figures, example embodiments of the present disclosure will be discussed in further detail. First, the components of the cartridge and assay reader will be discussed, followed by the components used to perform an assay as contemplated by the present disclosure.

[0048] FIG. 1 shows a point-of-care (POC) testing system according to one exemplary embodiment of the present disclosure. The POC testing system includes a cartridge 100 and an assay reader 110. As described herein, cartridge 100 is used to collect the fluid sample that may potentially contain a target analyte. The collection process also distributes the fluid sample within cartridge 100. After the fluid sample is collected in cartridge 100, the user inserts cartridge 100 into assay reader 110. As described herein, the act of inserting cartridge 100 into assay reader 110 results in the compression of cartridge 100, thereby causing the fluid sample to be distributed to a separation membrane portion of an assay stack, the details of which are discussed in more detail below. In this way, the act of inserting cartridge 100 into assay reader 110 commences one or more assay reactions that provide information regarding the contents of the fluid sample. However, it is also to be understood that other insertion approaches are contemplated that do not require compression. Further, it is to be understood that while multiple assays can be utilized to determine the contents of a target analyte in the fluid sample, each assay is generally specific for one particular target analyte. As described herein, assay reader 110 is equipped with a detection system that is used to detect the results of the one or more assay reactions that occur at one or more membranes in an assay stack of cartridge 100. The detection system is not particularly limited and may be a detection system which causes a measurable signal change as the result of an assay reaction. Non-limiting examples of suitable detection systems include colorimetric, fluorescence, electrochemical, and optical detection systems as described herein and any other detection system that would be understood by one of ordinary skill in the art.

[0049] FIG. 2A illustrates a top, perspective view of an embodiment of cartridge 100 in the form of a cartridge 200. In FIG. 2A, cartridge 200 includes a housing 201 attached to a handle 202. In general, cartridge 200 is designed to be easy to handle by the user and to provide a protective shell for the microfluidic distribution system and assay components housed within cartridge 200. In general, suitable materials for housing 201 and handle 202 include polyolefinic compounds, such as polyethylene, polypropylene, and other polymeric resins or compounds known in the medical device manufacturing art. During sample collection, cartridge 200 is brought into contact with a fluid sample (e.g., the blood fluid sample). The fluid sample is drawn into channel 203 and via channel opening 204 by capillary action. In some embodiments, channel 203 includes a plurality of receiving chambers 205 located along channel 203. FIG. 2B illustrates a bottom view of an embodiment of the cartridge 200. In FIG. 2B, the bottom portion of housing 201 includes a plurality of assay detection ports 206 aligned with channel opening 204. The assay detectionports 206 permit the assay results to be interrogated, for example, by optical detection methods as described herein. In addition, the bottom portion of housing 201 may comprise plurality of holes 207, which are additional assay detection ports that may be used with assay components and microfluidic channels that are arranged in a corresponding configuration.

[0050] FIG. 2C provides an exploded view of the components of the cartridge 200, according to one embodiment of the present disclosure. In FIG. 2C, the outer shell of cartridge 200 includes the handle 202, bottom housing portion 227, and a cap 223 that is equipped with a slot 228. The cap 223 can fit to the open side of the bottom housing portion 227 and have a shape and size that corresponds to the open side of the bottom housing portion 227. When the bottom housing portion 227 and cap 223 of the housing are assembled together, an interior chamber can be formed for enclosing other components of the cartridge within the interior chamber. In other embodiments, the cap 223 and bottom housing portion 227 do not form an enclosure with an interior chamber and can be rigid structures positioned on the top of a metering stack and bottom of an assay stack, which are described herein.

[0051] In some embodiments, when cartridge 200 is inserted into an assay reader, the components within the interior chamber are compressed to cause at least one portion of the collected blood fluid sample to be delivered to a plurality of assay components. The compression can be caused by the user closing a lid of the assay reader, for example. However, it is also to be understood that other approaches for insertion of the cartridge 200 into an assay reader are contemplated that do not require compression.

[0052] As shown in FIG 2C, cartridge 200 can include a metering stack 224, a spacer material 225, and an assay stack 226. The metering stack 224 can be used to collect a sample of a biological fluid (e.g., a blood fluid sample) and the assay stack 226 includes assay components necessary for a protein concentration assay to be carried out through one or more of the receiving chambers 205 as discussed in detail herein. However, it should also be understood that other assays (e.g., immunoassays or enzymatic assays) may also be carried out in additional receiving chambers 205 and the assay stacks 226 associated with each receiving chamber 205 can each be unique based on the particular assay associated with each receiving chamber 205. As used herein, the term "metering" refers to collecting a liquid sample of a biological fluid and delivering one or more predetermined volumes of at least a portion of the fluid to the assay components for further analysis via the assay components contained in the assay stack. When assembled into a cartridge, the metering stack 224, a spacer material 225, and an assay stack 226 can be arranged in a stack.

[0053] The spacer material 225 is a compressible layer that may be positioned between the metering stack 224 and assay stack 226 as shown in FIG. 2C. In an embodiment, the spacer material 225 may be a flexible material that can be compressed in the vertical direction when the cartridge is inserted into the assay reader and the metering stack 224 is moved into contact with or close proximity to the assay stack 226. In some embodiments, the spacer material 225 can be a flexible material, such as foam, rubber, porous polymer, metal, cotton, or other bending, folding, or moving mechanisms such as a clamp or spring. In some embodiments, the metering and assay stacks are initially separated by an air gap maintained by the spacer material 225. In certain embodiments, spacer material 225 is physically affixed to another layer, such as metering stack 224 or assay stack 226 before the layers of the cartridge are brought together. Typically, the metering and assay stacks remain separated throughout the sample collection process. In such embodiments, the separation between the metering stack and the assay stack can prevent a chemical reaction from starting during the blood fluid sample collection step. When the spacer material 225 is compressed, the metering stack 224 and assay stack 226 can come into contact with or brought into close proximity to each other.

[0054] In preferred embodiments, when the metering stack is fully filled with a fluid, the cartridge is inserted into an assay reader. Preferably, the material that is used for the top surface of channel 230 is sufficiently transparent so that a user can determine by visual inspection when the channel 230 is filled and the cartridge is ready for insertion into the assay reader. The assay reader is configured to accept the cartridge and includes a mechanism that compresses the spacer material, thereby pushing the metering stack and assay stack together when the cartridge is inserted into the assay reader. The compression of the spacer material causes a predetermined volume of at least a portion of the collected fluid to flow to assay components in the assay stack. In this way, the act of compressing the metering stack and assay stack together can, in certain embodiments, provide a well-defined point in time that marks the start of the assay through the components in the assay stack. However, it is also to be understood that other insertion approaches are contemplated that do not require compression of the metering stack and assay stack together as would be understood by one of ordinary skill in the art.

[0055] In some embodiments, the fluid sample containing the target analyte can be blood, and the cartridge can be used to collect a sample of blood from a skin prick and deliver the sample to the assay stack consistently with minimal user intervention. The user, with a regular pricking lancet, can elicit bleeding in a suitable body site such as a fingertip, palm,hand, forearm, stomach area, etc. Once a drop of blood of sufficient volume is on the skin, the user can collect it by touching the tip of the cartridge to the blood drop. Once the metering stack is fully filled with blood, the user can insert the cartridge into the assay reader, which triggers the delivery of the blood sample to the assay stack. In some embodiments, this can be performed by a patient, administrator, or healthcare provider. The blood collection and testing as described herein does not have to be performed by a trained heath care professional.

[0056] In addition, the cartridge design can allow for dispensing different pre-defined volumes of blood sample to multiple assay locations, without using any moving parts such as pumps or valves in the cartridge or in the assay reader. This increases the accuracy and flexibility of a multiplexed quantitative POC analysis, while reducing the complexity and cost of the cartridge and the assay reader.

[0057] Typically, as illustrated in FIG. 2C, the metering stack 224 includes a channel 230 to contain the biological fluid (e.g., a blood fluid sample containing a target analyte). In certain embodiments, the channel 230 can hold a volume of biological fluid containing a target analyte in the range of about 0.5 pl to about 100 pl, such as about 5 pl to about 80 pl, such as about 10 to about 60 pl, such as about 15 pl to about 40 pl, such about 20 pl to about 30 pl.

[0058] Preferably, the metering stack 224 is designed to direct the biological fluid to flow into the channel 230 and into any receiving chamber(s) that may be present. In some embodiments, the channel 230 can be formed of or coated with a hydrophilic material, nonlimiting examples of which include 93210 hydrophilic PET (Adhesives Research, Glen Rock PA) or 9984 Diagnostic Microfluidic Surfactant Free Fluid Transport Film, 9960 Diagnostic Microfluidic Hydrophilic Film, or 9962 Diagnostic Microfluidic Hydrophilic Film (3M Oakdale, MN).

[0059] FIG. 3 illustrates an exploded view of a metering stack 304 according to one exemplary embodiment of the present disclosure, where such metering stack 304 can be used as metering stack 224 in the embodiment of FIGs. 2A to 2C. In FIG. 3, the metering stack 304 is formed by assembling multiple layers. The first layer 341 can be a plastic sheet with a first side 342, which is in communication with the surrounding environment when the cartridge is located outside the assay reader, and a second side 343 that faces the assay stack. In some embodiments, the first layer 341 may be a cover layer or top layer of the metering stack. In preferred embodiments, first layer 341 may have a hydrophilic surface or coating on second side 343. Non-limiting examples of suitable hydrophilic surfaces coatings includepolyvinylpyrrolidone-polyurethane interpolymer, poly(meth)acrylamide, maleic anhydride polymers, cellulosic polymers, polyethylene oxide polymers, and water-soluble nylons or derivatives thereof, to name just a few. The presence of the hydrophilic surface or coating on second side 343 helps to draw the biological fluid (e.g., blood fluid sample) into the channel. The first layer 341 may include venting holes 311 positioned to align with the channel 310 defined by the layers below. In FIG. 3, for example, the venting holes 311 are aligned with the receiving chambers of channel 310 to allow air that otherwise would be trapped as an air bubble in the receiving chamber during channel filling to escape efficiently into the surrounding environment. It should be noted that the channel opening can also serve as a vent hole, if desired. In certain preferred embodiments, the first layer 341 includes polyethylene terephthalate (PET) with a hydrophilic coating on the second side 343 and venting holes 311.

[0060] The second layer 344 is positioned below the first layer 341 on the second side or assay facing side of the first layer 341. The second layer 344 itself can be a combination of one or more layers as illustrated in FIG. 3. Regardless of whether the second layer is comprised of one layer or more than one layer, the second layer essentially defines the shape and size of channel in the metering stack, including any receiving chambers that may be part of the channel. For example, the second layer 344 can be formed from one or more layers of polymeric material cut to define the volume and shape of the channel 310 that can contain the biological fluid (e.g., blood fluid sample). Other non-limiting methods of forming the channel 310 include injection-molding, stamping, machining, casting, laminating, and 3-D printing. Combinations of such fabrication techniques are also expressly contemplated by the present disclosure. In the embodiment shown in FIG. 3, second layer 344 has a first side 347 facing the first layer 341 and an opposite, second side 348 that faces the assay stack. Furthermore, second layer 344 includes adhesive layer 345 and plastic layer 346. Adhesive layer 345 fastens the first layer 341 to plastic layer 346. In some embodiments, the second layer 344 can be a combination of one or more plastic layer(s) 346 and adhesive layers 345. Preferably, adhesive layer 345 or plastic layer 346 or both are fabricated from materials which present a hydrophilic surface to the interior surfaces of the channel 310 in order to facilitate the distribution of the biological fluid within channel 310. In some embodiments, the hydrophilic plastic sheet(s) can include a PET material with a channel 310 cut into it. If desired, channel 310 may include one or more receiving chambers as shown in FIG. 3. Thus, the thickness and geometry of channel 310 can control the volume of sample to be collected. The hydrophilic interior surfaces of the channel 310 allow the metering stack to collect bloodsample by capillary force. In some embodiments, the first layer 341 and the second layer 344 can be one integrated layer used in the metering stack 304.

[0061] In FIG. 3, third layer 349 can be formed from a hydrophobic adhesive layer. Nonlimiting examples of suitable materials for fabricating third layer 349 include 3M 200MP adhesive or 3M 300MP adhesive (3M, Oakdale, MN). In preferred embodiments, the same channel geometry as channel 310 is cut into the third layer to match channel 310 cut in the second layer. In some embodiments, the third layer 349 can have a first side 351 facing the second layer 344 and a second side 352. In some embodiments, the third layer 349 can define the hydrophilic region in a fourth layer 350 positioned below or on the second side 352 of the third layer.

[0062] In some embodiments, the fourth layer 350, which can be positioned beneath only a portion of the receiving chambers of the channel 310, can be a hydrophilic mesh or porous material. In some embodiments, substantially all of the fourth layer 350 can include the mesh or porous material as shown in FIG. 3. In other embodiments, the hydrophilic mesh or porous material can be a portion of the fourth layer 350. In some embodiments, such as the example shown in FIG. 3, the fourth layer 350 can have a first side 353 facing the third layer 349 and an opposite assay stack-facing second side 354. The hydrophobic third layer 349 can be positioned above the fourth layer 350. The hydrophobic third layer 349 can be a hydrophobic adhesive layer to define a wettable region of the mesh or porous material of the fourth layer 350.

[0063] In the assay device or POC systems of the present disclosure, the assay reactions occur in the assay stack of the assay device. In general, an assay stack includes one or more “assay components.” As used herein, the term “assay component” refers to one or more of the active component and a passive supporting element or mask, including but not limited to the multiplexed assay pads. The number of assay pads (e g., separation membranes, detection membranes, etc.) in a particular assay component is not particularly limited but is based on the particular assay requirements needed to diagnose the condition or analyze the fluid sample of the patients for whom the assay stack is designed. In preferred embodiments, the layers of the assay pads of a given assay component align vertically with the appropriate regions of the channel in the metering stack above to ensure that a predetermined volume of a biological fluid, sufficient to perform the assay associated with the particular target analyte of interest, is delivered to the detection membrane. The assay pads can act as a wick that draws the sample through the metering stack into the assay stack, for example through cap i 11 ary action, gravity, etc. Therefore, once the metering stack and the assay stack are in contactwith or within close proximity to each other, the biological fluid to be analyzed is directed to move into the detection membrane, where it may encounter one or more reagents required to perform the assay associated with the particular assay component. If desired, the assay stack may comprise additional layers that contain reagents required for the completion of the assay. The number of layers required can depend on the number of chemical reactions that need to take place in order to complete the assay. In various embodiments, layers of the assay stack can be made of variously shaped and variously-sized pads of different porous membrane materials, non-limiting examples of which include a sulfone polymer (e.g., a polysulfone, a polyethersulfone, a polyarylsulfone, etc.), nylon, cellulose (e.g., nitrocellulose, cellulose filter paper, etc.) and glass fiber.

[0064] The type of assays that may be performed using the assay systems of the present disclosure are not particularly limited and can be any assay for which the required reagents can be stably incorporated into one or more separation membranes and / or detection membranes and which can cause a change that can be detected by the assay reader. In some embodiments, the assay reactions cause a color change, which may be detected using the colonmetric detection methods as descnbed herein. Still other assay reactions may result in another optical change, a fluorescence change, an electrochemical change, or any other detectable change that may occur in a detection membrane of the assay stack. In certain embodiments, the assays may be porous material-based lateral flow assays, vertical flow assays, and / or a combination of lateral and vertical flow assays. Further, the assays may be sequential assays. In general, the target analyte is contained within a biological fluid, nonlimiting examples of which include blood, plasma, serum, saliva, sweat, urine, lymph, tears, synovial fluid, breast milk, and bile, or a component thereof, to name just a few. In certain preferred embodiments, the biological fluid is blood. For example, in one embodiment, the assay systems of the present disclosure are useful for providing patients with POC information regarding target analytes in their blood composition. In particular, the assay systems of the present disclosure can be used to determine the concentration of HDL, LDL, VLDL, total cholesterol, or combination thereof in a blood fluid sample. Other analytes that can be measured in blood via other receiving chambers that may be present as part of the assay system include, sodium, thyroid markers (e g., T3, free T4, thyroid stimulating hormone, etc.), inflammatory markers (e.g., C-reactive protein, etc.), vitamins (detected via a competitive assay structure), metabolic syndrome markers, glucose, glycated hemoglobin, glycated albumin, and serological levels of antibodies against a disease (detected by a labeledantigen architecture). Non-limiting examples of analyte that can be measured in urine include total protein, leukocyte esterase and myoglobin.

[0065] FIG. 4 illustrates an exemplary assay stack 406 of an assay device according to one embodiment of the present disclosure, where such assay stack 406 can in particular be used as assay stack 226 in the embodiment of FIGs. 2A to 2C. In FIG. 4, the assay stack 406 is formed of multiple layers, including one or more of the layers with active components and a passive supporting element or mask. More specifically, in FIG. 4, assay stack 406 includes assay stack cover layer 410 that features a cut-out portion 411 that is aligned with the channel in the overlying metering stack. Generally, assay stack cover layer 410 is fabricated from a polymeric material that provides rigidity to the assay stack and provides ease of handling during manufacturing of the cartridge. Furthermore, the cut-out portion 411 allows the biological fluid to flow past the assay stack cover layer 410 towards the underlying assay components when the cartridge is inserted into the assay reader, as described herein. As shown, the assay stack 406 includes a separation membrane 461 which can be the top-most layer facing the metering stack. The separation membrane 461 is used to separate components of the biological fluid (e.g., blood fluid sample) to prevent undesirable components from reaching the underlying assay components. For example, when the biological fluid is a blood fluid sample and the assay is focused on determining an HDL concentration in the blood fluid sample, the separation membrane 461 is designed specifically to prevent red blood cells and non-HDL proteins (e.g., LDL, VLDL, etc.) from reaching the detection membrane portion of the assay stack 406 after the cartridge has been inserted into the assay reader. This is advantageous because the strong spectral absorption by the hemoglobin present in red blood cells may overwhelm the color changes that occur in the detection membrane 463 of the assay stack 406 during performance of the assay. Further, the presence of non-HDL proteins in the detection membrane 463 of the assay stack 406 may also lead to interference and reduced accuracy in determining the HDL concentration in the blood fluid sample, so the separation membrane 461 is specifically designed to trap such non- HDL proteins and prevent their passage to the detection membrane 463. As another example, when the biological fluid is a blood fluid sample and the assay is focused on determining an sodium concentration in the blood fluid sample, the separation membrane 461 is designed specifically to sequester the sodium and release an ion that binds to a reagent in the detection membrane to elicit a colorimetric response that is then correlated with the amount of sodium that was sequestered. Such a separation membrane 461 can be made of a variety of materials, non-limiting examples of which include a sulfone polymer (e.g., a polysulfone, apolyethersulfone, a polyarylsulfone, etc.), nylon, cellulose (e.g., nitrocellulose, cellulose filter paper, etc.) and glass fiber. In some embodiments, the fabrication of the separation membrane can include surface treatments for improved wettability and / or other properties. Other additional components and features of the separation membranes of the assay devices contemplated by the present disclosure are discussed in more detail with respect to FIGs. BASF and FIGs. 9A-9F.

[0066] As shown in FIG. 4, in some embodiments, the assay stack 406 can include an assay component 462 positioned below the separation membrane 4 1. The assay component 462 includes a mask support layer 450 with a plurality of cut-outs 451 that are configured to receive and immobilize detection membranes 463 when the assay stack 406 is assembled. Preferably, cut-outs 451 are positioned to align detection membranes 463 with separation membranes 461 such that the biological fluid containing the target analyte will flow from separation membranes 461 into detection membranes 463. Detection membranes 463 (e.g., detection membranes such as but not limited to color generation membranes) may comprise reagents that are necessary to complete the assay reactions that are initiated once the target analyte (e.g., HDL or other protein of interest) flows through the separation membranes 461. In some embodiments, detection membranes 463 serve as a detection indicator layer that provides information corresponding to the results of the assay performed. For example, detection membranes 463 (e.g., color generation membranes) can include a visual indicator, such as a color change, to indicate the results of the assays, although it is to be understood that the detection membranes contemplated by the present disclosure also contemplate fluorescent and electrochemical changes or responses. Furthermore, while assay stack 406 in FIG. 4 contains only assay component 462, it should be understood that the assay stack 406 may contain additional assay components with assay pads that are impregnated with the reagents required to complete and / or report the results of a particular assay. For instance, the assay stack 406 can include any number of assay components necessary to perform the analysis of the blood sample. In particular, the assay stack 406 can have an architecture that can be serialized to perform multiple time-controlled steps in a single assay.

[0067] Assay stack 406 in FIG. 4 also includes an assay bottom layer 470, which is ty pically fabricated from a polymeric material to provide mechanical strength and ease of handling of assay stack 406 during the manufacturing process. In addition, assay bottom layer 470 typically includes a plurality of detection ports 471 which are aligned with the detection membranes of the assay stack and sized to permit interrogation of the assay results by the assay reader.

[0068] FIG. 5A shows a schematic drawing of an assay reader, in longitudinal crosssection, according to one non-limiting embodiment of the present disclosure. In FIG. 5 A, assay reader 500 includes cartridge receiving chamber 510 which houses the cartridge when it is inserted as indicated by arrow 505. Tab 515 runs longitudinally along assay reader 500 and extends into cartridge receiving chamber 510. Tab 515 is configured to insert into a slot at the top of the cartridge, such as slot 228 in FIG. 2C, when the cartridge is inserted into the assay reader. In addition, the spacing 525 between the bottom edge of tab 515 and support surface 520 is set such that when the cartridge is inserted, tab 515 compresses the metering stack and the assay stack together, thereby causing the biological fluid containing the target analyte to flow from the metering stack into the assay stack and initiating the assay reactions. In certain embodiments, the assay reader may comprise a snap-fit mechanism that locks the cartridge in place once it has been fully inserted into the assay reader. This is advantageous because it prevents the user from accidentally removing the cartridge from the assay reader before the assays are complete, which could adversely affect the accuracy of the assay results. In some embodiments, assay reader 500 also includes sensors 542a and 542b, which detect and time the insertion of the cartridge. For example, as the cartridge is inserted into cartridge receiving chamber 510 and begins to engage with tab 515, the bottom surface of the cartridge may pass over sensor 542a, which is detected by appropriate electronics as the beginning of the insertion of the cartridge. The second sensor 542b is located further inside the assay reader 500 and detects the presence of the cartridge when the cartridge is fully inserted as well as the time at which full insertion occurred Assay reader 500 may then compare the overall time for insertion of the cartridge to determine if the insertion of the cartridge was timely and proper. In this way, the assay reader will not perform any assay readings in situations where (1) the cartridge was only partially inserted, or (2) the cartridge was partially inserted, removed, and inserted again. Either case could give inaccurate assay readings, due to the incomplete compression of the metering stack and assay stack, resulting in incomplete delivery of the required amount of target analyte to the detection membranes in the assay stack.

[0069] In the exemplary embodiment shown in FIG. 5A, assay reader 500 detects the results of the assay by detecting the color change of the detection membrane caused by the assay reactions. To achieve this, assay reader 500 includes a plurality of light sources (not shown in this cross-sectional drawing) and light detection elements 550 arrayed within assay reader 500 such that they align with the detection membranes of the cartridge when the cartridge is fully inserted. In order for light detection elements 550 to be able to detect thecolor of the detection membranes, support surface 520 may be equipped with one or more apertures or be fabricated from a transparent material that allows light to penetrate therethrough. However, it is also to be understood that the assay reader 500 can alternatively include components to detect electrochemical or fluorescent changes in a detection membrane portion of the assay stack. FIG. 5B shows a schematic illustration of a longitudinal cross-section of assay reader 500 with cartridge 502 fully inserted. Cartridge 502, which may correspond to cartridge 100 or 200 of FIG. I or FIGs. 2A to 2C, includes metering stack 504 and assay stack 506, which are compressed together by tab 515 such that the target analyte (e.g., HDL) or another component (e.g., an ion) is delivered from the metering stack 504 to the detection membranes 530. Detection membranes 530 are aligned with light detection elements 550. Note, however, that assay reader 500 may include an additional light detection element 550a without a corresponding detection membrane 530. The presence of additional light detection elements, such as tight detection element 550a, allow the assay reader to be used with different types of cartridges for different assays, particularly cartridges that may be designed to perform more assays, as well as to identify the different types of cartridges for the different assays.

[0070] FIG. A shows a schematic drawing of a transverse cross-section of the assay reader shown in FIG. 5 in the form of an assay reader 600 that may be used to detect color changes. In FIG. 6A, the assay reader 600 includes a tab 615 that extends into cartridge receiving chamber 610 to engage with a slot on the cartridge. Such engagement then compresses the metering stack and the assay stack against support surface 620, initiating the assay reactions. Light sources 660a and 660b provide light for detecting the assay results and are positioned near tight detection device 650. Specifically, as illustrated in FIG. 6A, light sources 660a and 660b provide light to analyze the detection membrane corresponding to light detection device 650. In general, it is advantageous to dedicate one or more light sources to each light detection element in order to ensure that the photon flux onto the light detection element is sufficient to obtain an accurate reading. In some embodiments, the light sources dedicated to a particular light detection element have the same output spectrum. In other embodiments, however, the light sources corresponding to a given light detection element produce different output spectra. For instance, the light sources may be light emitting diodes (LEDs) that produce different colors of light. In general, it is advantageous to include optical elements to direct the light and / or reduce the amount of light scattering in the assay reader. In some embodiments, the optical elements are apertures that only allow light emanating from the light source that is line-of-sight to the respective detectionmembrane to reach the detection membrane. For example, in FIG. 6A, light source 660a is limited by aperture defining members 670a and 671a such that only the light from light source 660a that passes through aperture 673a will reach the detection membrane and subsequently be detected by light detection device 650. Similarly, light source 660b is limited by aperture defining members 670b and 671b, such that only the light from light source 660b that passes through aperture 673b will reach the detection membrane and subsequently be detected by light detection device 650. In preferred embodiments, aperture defining members 670a, 670b, 671a, and 671b are fabricated from a black matte material to reduce the amount of undesirable scattering when light sources 660a and 660b are turned on. Furthermore, in this embodiment, light detection device 650 located in a housing that is comprised of aperture defining members 671a and 671b that only permit light that passes through aperture 672 to reach light detection device 650. If desired, the aperture 672 may be fitted with a filter to admit only light of a predetermined wavelength or wavelength range for detection by light detection device 650. This may be useful, for example, when the light sources are equipped to provide only white light for colorimetric analysis. In addition, the light from light sources 660a and 660b and the light to be detected by light detection device 650 may be directed or manipulated using optical elements such as lenses, filters, shutters, fiber optics, light guides, and the like without departing from the spirit and the scope of the present disclosure.

[0071] FIG. 6B shows a schematic illustration of the operation of the assay reader described in FIG. 6A. In FIG. 6B, a cartridge comprising metering stack 604 and assay stack 606 are inserted into cartridge receiving chamber 610 of assay reader 600. Tab 615 compresses metering stack 604 and assay stack 606 against support surface 620 to cause the fluid sample (e.g., blood fluid sample) to flow from the channel 612 into detection membrane 630. As noted previously, assay reader 600 may be fitted with sensors to confirm that the cartridge has been inserted correctly and in a timely manner. Assay reader 600 may also be pre-programmed before sample collection, either by the user or during the manufacturing process, to illuminate the detection membranes at the appropriate time based on the type of cartridge being used. In this way, assay reader 600 collects assay data from detection membrane 630 only when the assay is completed. Alternatively, if desired, assay reader 600 may be configured to collect assay data from detection membrane 630 during the entire assay reaction after the cartridge has been inserted. As shown in FIG. 6B, light source 660a provides light beam 680a, which impinges on the bottom face of detection membrane 630 to produce reflected light beam 661. Similarly, light source 660b produces light beam 680b,which may impinge on the botom of the detection membrane 630 to produce reflected light beam 661 at the same time as light source 660a or a different time, depending on the requirements of the assays being detected.

[0072] FIG. 7 shows a block diagram 700 of a sensor configuration inside an assay reader according to one exemplary embodiment of the present disclosure. In FIG. 7, four detection membranes (identified by reference numerals 741, 742, 743, and 744) have completed their assay reactions with the target analyte, undergone the respective color changes, and are ready for colorimetric analysis. Note that, if desired, this configuration can also be used to collect data from the four detection membranes to monitor the progress of the assay reactions. Input signal 701 from a first microcontroller serial-peripheral interface bus (MCU SPI Bus) enters digital-to-analog converter unit 710, which includes individual digital-to-analog converters 711, 712, 713, and 714 that independently control current sources 721, 722, 723, and 724. These current sources, in turn, power light sources 731, 732, 733, and 734, respectively. In some embodiments, input signal 701 may be sent by a timing circuit at a predetermined time after the insertion of the cartridge into the assay reader. In such embodiments, the predetermined time corresponds to the known time or times for the assay reactions in the detection membranes to reach completion. In some preferred embodiments, the light sources 731, 732, 733, and 734 are activated at the same time to measure the assay-induced color change of detection membranes 741, 742, 743, and 744 simultaneously in a multiplexed mode. However, this present disclosure also contemplates operating all of the light sources separately and sequentially, or some simultaneously and some separately, depending on the timing requirements of the assays in the cartridge.

[0073] In this non-limiting example, each of light sources 731, 732, 733, and 734 includes individual three light emiting diodes (LEDs) which may be the same or different colors, depending on the requirements of the assay and any optical elements that may be present in the assay reader. Generally, the light sources that are useful in the assay readers of the present disclosure are not particularly limited, so long as they provide light of suitable wavelength(s) and brightness for the light detection element to make an accurate reading of the colored light reflected from the detection membrane.

[0074] In FIG. 7, element 740 is a schematic representation of optical elements that optionally may be present in the optical path between the light sources 731, 732, 733, and 734 and detection membranes 741, 742, 743, and 744. When desired, one or more optical elements may be located between the light source and its corresponding detection membrane to direct the light, focus the light, reduce undesirable scatering, select one or morewavelengths for assay detection, or some combination thereof. Non-limiting examples of such optical elements include apertures, lenses, light guides, bandpass filters, optical fibers, shutters, and the like. Similarly, element 745 represents optical elements that optionally may be present in the optical path between detection membranes 741, 742, 743, and 744 and corresponding light detection devices 751, 752, 753, and 754. These optical elements may be used to manipulate the light upstream of the light detector devices in a manner similar to that described for element 740. Light detecting devices 751, 752, 753, and 754 detect the light from the detection membranes 741, 742, 743, and 744. In this non-limiting example, the light detecting devices are photodiodes. More generally, the type of light detection device is not particularly limited, provided that it is capable of detecting the light that is reflected from the detection membranes used for colorimetric measurement of the assay results. Other examples of suitable light detection elements include photodiode arrays, CCD chips, and CMOS chips. The outputs from light detection devices (e.g., photodiodes) 751, 752, 753, and 754 are sent to transimpedance amplifier / low pass filter elements 761, 762, 763, and 764, which convert the current signal from the photodiodes to a voltage output, while filtering unwanted signal components. The output from transimpedance amphfier / low pass filter elements 761, 762, 763, and 764 are sent to analog-to-digital converter unit 770, which includes multiplexer unit 771, gain 772, and analog-to-digital converter 773. The output of analog-to-digital converter unit 770 may be sent to a component 780, which may be a second MCU SPI bus, a transmitter, or a processor. In certain embodiments, the transmitter allows for hardwired or wireless connectivity (e g., Bluetooth or Wi-Fi) with a personal computer, mobile device, or computer network. In addition, the assay results may be transmitted from the user’s mobile device or computer to a computer network, such as one belonging to the user’s physician. In this way, the assay systems of the present disclosure can allow a user’s physician to monitor a patient closely, by providing up-to-date medical information from the assay results obtained by the assay reader.

[0075] It should be noted that the optical detection systems described in the foregoing correspond to some exemplary embodiments of the system, but that the present disclosure expressly contemplates other types of detection systems as well. In general, any detection system which corresponds to a signal change caused by an assay reaction may be used in connection with the assay reader of the present disclosure. Thus, for example, in certain embodiments, the detection system is an optical detection system that is based on chemiluminescence. In such embodiments, light sources such as LEDS and OLEDS are not required to detect a color change caused by the assay reaction in the detection membranes.Rather, the signal change may be caused by the reaction of an oxidative enzyme, such as luciferase, with a substrate which results in light being generated by a bioluminescent reaction. In another exemplary embodiment, the signal change caused by the assay reaction may be detected by electrochemical reaction.

[0076] FIGs. 8A-8F illustrate a further embodiment of cartridges 100, 200, or 502 in the form of a cartridge 800 that includes a metering stack 802 and an assay stack 804 during various stages of performing an assay after a blood fluid sample 814 containing a first protein 824, a target analyte 816 (second protein), and one or more other blood components 820, which can include red blood cells, a first protein, etc., has been introduced to the cartridge 800. The metering stack 802 is configured to receive and distribute the blood fluid sample 814 along a channel from an upstream location U to a downstream location D, where the channel has one or more venting holes in communication with the channel, as described in detail above. As also described above, a spacer material may be disposed between the metering stack and the assay stack, wherein the spacer material provides a gap between the metering stack and the assay stack that prevents the target analyte from flowing from the metering stack into the assay stack when the cartridge is in an uncompressed state. Additionally, the blood fluid sample 814 can flow from the metering stack 802 to the assay stack 804 upon compression of the cartridge 800.

[0077] Initially, as shown in FIG. 8A, the fluid sample 814 is located outside of the metering stack 802. Then, once the fluid sample 814 comes into contact with or is introduced to the metering stack 802 in FIG. 8B, the blood fluid sample 814 passes to the separation membrane 806 as shown in FIG. 8C, and ultimately, the target analyte reaches a detection membrane 812 (e.g., a color generation membrane) of the assay stack 804 as shown in FIGs. 8D-8F and as discussed in more detail below. It should be understood the separation membrane 806 can be referred to as the plasma separation membrane, where the separation membrane 806 allows for the separation of blood components 820 such as a first protein (e.g., non-HDL) as well as other blood components such as red blood cells from the blood fluid sample while allowing for a target analyte 816 such as second protein (e.g., HDL) to pass through the separation membrane 806 to the detection membrane 812. Further, the separation membrane 806 can include pores 840 that have a pore size large enough to allow the target analyte 816 (second protein) to pass through to the detection membrane 812 but that also have a pore size small enough to prevent the passage of other blood components 820 such as the first protein that is bound to a first reagent 818 (precipitating reagent) specific for the other blood components 820 present in the separation membrane 806, as discussed in moredetail below, to the detection membrane 812, which could affect the accuracy of the assay results. This is because there is a strong spectral absorption by the hemoglobin present in red blood cells that may, for example, overwhelm the color changes that occur after the assay is performed, and the presence of other proteins besides the target analyte (second protein) in the detection membrane 812 would also affect the accuracy of the detection of the amount of the target analyte 816, such as the second protein, via the detection membrane 812.

[0078] Additionally, the separation membrane can include a soluble polymer 942 as referenced above, which serves to block the passage of the fluid being tested to the detection membrane 812 until polymer has dissolved, where the concentration of the polymer on the separation membrane 806 is controlled as described above to ensure the fluid does not pass to the detection membrane 812 until a reaction between a blood component 820 and the first reagent 818 has completed.

[0079] Further, as shown in FIGs. 8A and 8B and as mentioned above, the separation membrane 806 includes a first reagent 818 which can be a precipitating reagent that is specific for the first protein (e.g., non-HDL) 820. It should be understood that for ease of manufacturing and for efficiency purposes, the precipitating reagent 818 is not immobilized on the separation membrane 806. In some embodiments, the precipitating reagent 818 includes a poly anionic non-high density lipoprotein binding reagent. For instance, the precipitating reagent 818 can include a sulfonated polysaccharide, heparin, or phosphotungstic acid. In addition, the precipitating reagent 818 can also include a group II cation. The weight ratio of the sulfonated polysaccharide, heparin, or phosphotungstic acid to the group II cation can range from about 9: 1 to about 2:1, such as from about 7: 1 to about 2.5:1, such as from about 5: 1 to 3:1. In some embodiments, the group II cation can include Mg2+, Mn2+, or Ca2+. In one particular embodiment, the precipitating reagent 818 can include phosphotungstic acid and Mg2+. For example, the precipitating reagent 818 can include phosphotungstic acid and magnesium chloride hexahydrate (MgCh (H2O)6). In other embodiments, the precipitating reagent 818 can include heparin and manganese (II) chloride, dextran sulfate, or magnesium chloride.

[0080] Further, the precipitating reagent 818 can be present in separation membrane 806 at a concentration ranging from about 35 milligrams / milliliter to about 250 milligrams / milliliter, such as from about 45 milligrams / milliliter to about 245 milligrams / milliliter, such as from about 55 milligrams / milliliter to about 240 milligrams / milliliter based on the wet weight of the precipitating reagent 818 present on the detection membrane 812. In addition, because about 20 microliters to about 40 microliters ofthe precipitating reagent 818 can be applied to the separation membrane per square centimeter of the separation membrane 806, the precipitating reagent 818 can be present in the separation membrane 806 at a concentration ranging from about 0.7 milligrams / square centimeter to about 10 milligrams / square centimeter, such as from about 0.9 milligrams / square centimeter to about 9.8 milligrams / square centimeter, such as from about 1.1 milligrams / square centimeter to about 9.6 milligrams / square centimeter based on the dry weight of the precipitating reagent 818. In still other embodiments, the precipitating reagent 818 can be present in the separation membrane at concentration ranging from about 1 milligrams / square centimeter to about 7.5 milligrams / square centimeter, such as from about 1.5 milligrams / square centimeter to about 7 milligrams / square centimeter, such as from about 2 milligrams / milliliter to about 6.5 milligrams / square centimeter. Additionally, the separation membrane 806 can have a surface area of about 0.25 square centimeters in some embodiments. In such embodiments, the precipitating reagent 818 can be present in the separation membrane 806 an amount ranging from about 175 micrograms to about 2500 micrograms, such as from about 225 micrograms to about 2450 micrograms, such as from about 275 micrograms to about 2400 micrograms. In still other embodiments, the precipitating reagent 818 can be present in the separation membrane 806 in an amount ranging from about 250 micrograms to about 1875 micrograms, such as from about 325 micrograms to about 1750 micrograms, such as from about 500 micrograms to about 1625 micrograms.

[0081] Ultimately, as shown in FIGs. 8C and 8D, any blood components 820 such as a first protein (non-HDL) present in the blood fluid sample 814 can be precipitated by the first reagent 818 present and immobilized in the separation membrane 806 to form a complex 826 while the soluble polymer 842 blocking the pores 840 dissolves in a controlled manner (compare the polymer 842 present in FIGs. 8C and 8D). Eventually, all of the blood components 820 will have bound to the first reagent 818 as the soluble polymer 842 dissolves fully, where the dissolution is controlled based on the concentration of the soluble polymer 842 initially present, where the timing needs are considered when determining the concentration of the soluble polymer, where the concentration and rate of dissolution of the polymer allow for a complete reaction between the blood components 820 and the first reagent 818 to finish to completion before the blood fluid sample 814 moves to the next membrane or layer in the assay stack 804. Further, it is to be understood that the complex 826 and red blood cells (not shown) present in the blood fluid sample 814 are trapped or slowed down by the size of the pores 840, even after dissolution of the polymer 842, suchthat only the target analyte 816, which is a second protein (HDL), which remains unbound to any reagent, can quickly pass through to the detection membrane 812 (see FIG. 8D) for a determination of the amount of the target analyte 816 present in the blood fluid sample 814 before any lingering blood components 820 and / or complexes 826 are able to reach the detection membrane 812.

[0082] Turning now to FIGs. 8E-8F, after the target analyte 816 (second protein, HDL) passes through the pores 840 in the separation membrane 806, the target analyte 816 can react with a second reagent 830 present in the detection membrane 812. The reaction between the second reagent 830 and the target analyte 816 present can elicit a quantifiable response 844 (e.g., a colorimetric response, a fluorescent response, an electrochemical response, etc.) in the presence of the target analyte 816 (second protein), wherein the quantifiable response 844 corresponds to an amount of the target analyte 816 (second protein) present in the detection membrane 812 as shown in FIGs. 8E and 8F. In one embodiment, the second reagent 830 can be specific for the target analyte 816 (second protein, HDL) and can include cholesterol esterase, cholesterol oxidase, horseradish peroxidase, a Trinder reagent, 4-amino antipyrine, or a combination thereof, although any suitable reagents for measuring HDL are contemplated by the present disclosure and known to those having ordinary' skill in the art. For example, in one embodiment, the second reagent 830 can include one or more reagents which are catalyzable by target analyte 816 (second protein) to provide a detectable signal within the detection membrane 812.

[0083] FIGs. 9A-9F illustrate a further embodiment of cartridges 100, 200, or 502 in the form of a cartridge 900 that includes a metering stack 902 and an assay stack 904 during various stages of performing an assay after a blood fluid sample 914 containing a target analyte 916 (sodium) has been introduced to the cartridge 900. The metering stack 902 is configured to receive and distribute the blood fluid sample 914 along a channel, where the channel has one or more venting holes in communication with the channel, as described in detail above. As also described above, a spacer material may be disposed between the metering stack and the assay stack, wherein the spacer material provides a gap between the metering stack and the assay stack that prevents the target analyte from flowing from the metering stack into the assay stack when the cartridge is in an uncompressed state. Additionally, the blood fluid sample 914 can flow from the metering stack 902 to the assay stack 804 upon compression of the cartridge 900.

[0084] Initially, as shown in FIG. 9A, the fluid sample 914 is located outside of the metering stack 902. Then, once the fluid sample 914 comes into contact with or is introducedto the metering stack 902 in FIG. 9B, the blood fluid sample 914 passes to the separation membrane 906 as shown in FIGs. 9C and 9D, and ultimately, the target analyte reaches a detection membrane 912 (e.g., a color generation membrane) of the assay stack 904 as shown in FIGs. 9E-9F and as discussed in more detail below. In particular, a fluid sample 914 is introduced to the metering stack 902 at an upstream location U of the cartridge 900, after which the blood fluid sample 914 passes to a separation membrane 906. Ultimately, the fluid sample 914 to be tested reaches a detection membrane 912 (e g., a color generation membrane) of the assay stack 904, where the detection membrane 912 is located at a downstream location D of the cartridge 900, as shown in FIG. 9A. It should be understood the separation membrane 906 can be referred to as the plasma separation membrane, where the separation membrane 906 allows for a reaction between the target analyte 916 in the blood fluid sample 914 and a first reagent 918 to occur such that a concentration of a reaction product can be measured in the detection membrane 912. The separation membrane 906 can also separate other components 920 such as red blood cells from the fluid sample 914 that ultimately passes to the detection membrane 912 for analysis. The separation membrane 906 can be formed from one or more hydrophobic polymers, such as a sulfone polymer, a mixed cellulose ester, or a combination thereof, although it should be understood that it is not required that the separation membrane 806 be hydrophobic. The sulfone polymer can be a polysulfone, a polyethersulfone, a poly arylsulfone, or a combination thereof. In some instances, the hydrophobic polymers described above can be reacted with or mixed with a hydrophilic polymer (e g., polyvinylpyrrolidone or PVP) for use in applications involving aqueous environments. For example, the separation membrane 906 can include a sulfone polymer cross linked with polyvinylpyrrolidone.

[0085] Meanwhile, the detection membrane 912 can be formed from one or more hydrophobic polymers, such as a sulfone polymer, a mixed cellulose ester, or a combination thereof. The sulfone polymer can be a polysulfone, a polyethersulfone, a polyarylsulfone, or a combination thereof. In some instances, the hydrophobic polymers described above can be reacted with or mixed with a hydrophilic polymer (e.g., polyvinylpyrrolidone or PVP) for use in applications involving aqueous environments. For example, the detection membrane 912 can include a sulfone polymer crosslinked with polyvinylpyrrolidone.

[0086] It should be understood that the separation membrane 906 and the detection membrane 912 should utilize materials that contain little to no residual sodium, as this can negatively impact the accuracy of some embodiments of the assay device of the present disclosure. As such, care must be taken to select membranes that are manufactured withoutthe use of materials and reagents containing sodium. In other words, the separation membrane 906 and the detection membrane 912 should contain essentially no sodium, such as less than 100 parts per million of residual sodium, such as less than 10 parts per million of residual sodium, such as less than 1 part per million of residual sodium.

[0087] As shown in FIG. 9A, the detection membrane 912 includes a first reagent 918 that can be a sodium sequestering agent. In particular, the first reagent 918 can include a cryptand 918a and an ion 918b, where the cryptand 918a has an affinity for the ion 918b that can be referred to as a cryptand-ion complex. The ion 918b can be a divalent ion in some embodiments, and can include Ca2+, Fe2+, Mg2+, or Mn2+. In another embodiment, it is not required that the ion be divalent, and in some instances, the ion 918b can be K+. Regardless of the particular ion 918b utilized, the ion 918b can be present on the separation membrane 906 at a molar quantity ranging from about 0. 1 micromoles / square centimeter to about 10 micromoles / square centimeter, such as from about 0.25 micromoles / square centimeter to about 5 micromoles / square centimeter, such as from about 0.5 micromoles / square centimeter to about 2.5 micromoles / square centimeter.

[0088] Further, the cryptand 918a, which has an affinity' for the ion 918b but a stronger affinity for the target analyte 916 (e.g., sodium), can be selected from bicyclic molecules produced from joining nitrogen bridgeheads with chains of ( — OCH2CH 2 — ) groups, for example [2. 1.1] cryptand, [2.2.1]cryptand, [2.2.2] cryptand, [3.2.2]cryptand, [3.3.2] cryptand, [3.3.3] cryptand, etc., where the numerals indicate the length of the ethylene oxide subunits in each of the three bridges between the nitrogens. However, it is to be understood that any suitable cryptand known by one of ordinary skill in the art can be used in the assay device of present disclosure. In addition, it should be understood that a crown ether can be used in place of the cryptand 918a. Further, the cryptand 918a (or crown ether) can be present on the separation membrane 906 at a molar quantity ranging from about 0.2 micromoles / square centimeter to about 5 micromoles / square centimeter, such as from about 0.5 micromoles / square centimeter to about 4 micromoles / square centimeter, such as from about 0.75 micromoles / square centimeter to about 3 micromoles / square centimeter.

[0089] Additionally, the separation membrane can include a pores 940 that are initially blocked by a soluble polymer 942 as referenced in detail above, which serves to prevent the passage of the fluid being tested to the detection membrane 912 until polymer has dissolved, where the concentration of the polymer on the separation membrane 906 is controlled as described above to ensure the fluid does not pass to the detection membrane 912 until areaction between the target analyte 916 and the first reagent 918 has completed. This reaction will be discussed in more detail below.

[0090] Meanwhile, the detection membrane 912 includes a second reagent 930 that can be an ion-dependent dye. In some embodiments, the ion-dependent dye 930 can be an azoic dye, in particular an azoic aromatic dye. The azoic dye can include, eriochrome black T, eriochrome blue black T, xylenol orange, or a combination thereof. Other dyes that can alternatively be used include calcium green-5N, calcium green 2, fluo-4, fluo-5N, fluozin-1, newport green DCF, phen green FL, calcein, curcumin, fast sulphon black, hematoxylin, and murexide. Further, the ion-dependent dye can be present on the detection membrane 912 at a molar quantity ranging from about 0.2 micromoles / square centimeter to about 5 micromoles / square centimeter, such as from about 0.5 micromoles / square centimeter to about 4 micromoles / square centimeter, such as from about 0.75 micromoles / square centimeter to about 3 micromoles / square centimeter.

[0091] Turning now to FIG. 9B, once the blood fluid sample 914 containing the target analyte 916 (sodium) is introduced to the cartridge 900, the blood fluid sample 914 enters the metering stack 902. Then, as shown in FIG. 9C, the blood fluid sample 914 enters the separation membrane 906 portion of the assay stack 904. Thereafter, and referring to FIG. 9D, the target analyte 916 (sodium) is separated from other components 920 (red blood cells) in the blood fluid sample 914 and preferentially binds to the cry ptand 918a of the first reagent 918 to form a complex 926 since the cryptand 918a has a higher affinity for the target analyte 916 than the ion 918b that is part of the first reagent 918 (original ion-cryptand complex). This releases the ion 918b from the cryptand 918a so that it is free in the separation membrane 906.

[0092] Ultimately, as shown in FIGs. 9C and 9D, any target analyte 916 in the blood fluid sample 914 can be sequestered by the cryptand portion 918a of the first reagent 918 and at least temporarily immobilized in the separation membrane 906 along with any blood components 920 such as red blood cells while the soluble polymer 942 blocking the pores 940 dissolves in a controlled manner (compare the polymer 942 present in FIGs. 9C and 9D). Eventually, all of the target analyte 916 (sodium) will have bound to the cryptand portion 918a of the first reagent 918 as the soluble polymer 942 dissolves fully, where the dissolution is controlled based on the concentration of the soluble polymer 942 initially present, where the concentration is governed based on the timing for a complete reaction between the target analyte 916 and the first reagent 918 to finish to completion. Ultimately, the resulting target analyte 916 and cryptand 918a complex 926 forms so that the target analyte 916 (sodium) issequestered by the cryptand 918a such that the released ion 918b can pass through to the detection membrane 912 (see FIG. 9E) for a determination of the amount of the ion 918b present, leading to an indirect determination of the amount of target analyte 916 present in the blood fluid sample 814 before any lingering blood components 920 and / or complexes 926 are able to reach the detection membrane 912. By breaking this process down into a two-step reaction, the target analyte 916 (sodium) can be sequestered by the cryptand 918a (and the ion 918b released by the cryptand 918a) in the separation membrane 906 prior to introduction of the ion-dependent dye 930 in the detection membrane 912, ensuring that the slow rate of dissociation between the ion-dependent dye 930 and the ion 918b and / or target analyte 916 (sodium) does not influence or decrease the accuracy of the assay.

[0093] Referring more specifically to FIGs. 9C and 9D, once the fluid sample 914 containing the target analyte 916 (sodium) reaches the separation membrane 906 portion of the assay stack 904, the target analyte 916 (sodium) binds with the cryptand 918a in the first reagent 918, which has a higher affinity for the target analyte 916 (sodium) than the ion 918b to which it is bound in forming the first reagent 918. As such, the ion 918b is released from the cryptand 918a as the target analyte 916 (sodium) binds to the cryptand 918a. Thereafter, as shown in FIG. 9E, the ion 918b, which had been sequestered from the ion-dependent dye 930, binds to the ion-dependent dye 930 after passing to the detection membrane 912 after dissolution of the soluble polymer 942. This initiates a quantifiable response 944 (e.g., a colorimetric response, a fluorescent response, an electrochemical response, etc.) as shown in FIG. 9F due to the activation of the ion-dependent dye 930 once the ion 918b that had once been sequestered from the ion-dependent dye 930 binds to it. The quantifiable response 944 can then be measured and corresponds to an amount of the ion 918b bound to the iondependent dye 930. Then, the amount of the ion 918b can be correlated to the concentration of the target analyte 916 (sodium) in the blood fluid sample 914, allowing for the concentration of the target analyte 916 (sodium) to be determined indirectly. Moreover, it should be understood that initially the detection membrane 912 includes an excess of cryptand 918a compared to the ion 918b, such that the first 130 millimolar or so of the target analyte 916 (sodium) present does not affect the release of the sequestered ion 918b to the ion-dependent dye 930. This ensures that the narrow concentration range of the target analyte 916 (sodium), which ranges from about 135 millimolar to about 145 millimolar in the nomial range for a blood fluid sample 914 (and thus has a range of just 7%), can be accurately measured with minimal error. This improvement is due to the range moving from7% overall (135-145 millimolar) to 200% (135 millimolar minus 130 millimolar to 145 millimolar minus 130 millimolar.)

[0094] In any event, the quantifiable response 844 or 944 (e.g., a colorimetric response, a fluorescent response, an electrochemical response, etc.) in the detection membrane 812 or 912 can be detected by one or more detection devices, which, for example, can include light detection devices 854 and 954 as shown in FIG. 8F / 9F when the quantifiable response is a color change. Meanwhile, one or more light sources 831 or 931 provide light for detecting the quantifiable response 844 or 944 (e g., color change) in the detection membrane 812 or 912 (e.g., color generation membrane) and can be positioned near light detection device 864 or 964. In the case of a colorimetric response, the one or more light sources 831 or 931 provide light to analyze the quantifiable response 844 or 944 (e.g., color change) in the detection membrane 812 or 912 (e.g., color generation membrane) corresponding to light detection device 854 or 954. As described above, it is advantageous to dedicate one or more light sources to each light detection element in order to ensure that the photon flux onto the light detection element is sufficient to obtain an accurate reading. In some embodiments, the light sources 831 or 931 dedicated to a particular light detection device 854 or 954 have the same output spectrum. In other embodiments, however, the light sources 831 or 931 corresponding to a given light detection devices 854 or 954 produce different output spectra. For instance, the light sources may be light emitting diodes (LEDs) that produce different colors of light. In general, it is advantageous to include optical elements to direct the light and / or reduce the amount of light scattering in the assay reader. If desired, the light detection device 854 or 954 may be fitted with a filter to admit only light of a predetermined wavelength or wavelength range for detection by light detection device 854 or 954. This may be useful, for example, when the light sources 831 or 931 are equipped to provide only white light for colorimetric analy sis. In addition, the light from light sources 831 or 931 and the light to be detected by light detection device 854 or 954 may be directed or manipulated using optical elements such as lenses, filters, shutters, fiber optics, light guides, and the like without departing from the spirit and the scope of the present disclosure. More generally, the light sources 831 or 931 that are useful in the assay readers of the present disclosure are not particularly limited, so long as they provide light of suitable wavelength(s) and brightness for the light detection devices 854 or 954 to make an accurate reading of the colored light reflected from the detection membrane 812 or 912.

[0095] FIG. 10 shows a flowchart that illustrates a method 1000 of using the assay system according to one embodiment of the present disclosure to perform a plurality ofassays. The method 1000 includes step 1010, which involves receiving a blood fluid sample that contains the target analyte (e.g., HDL or sodium) into a channel in a cartridge. Step 1020 involves inserting the cartridge into an assay reader, thereby compressing the cartridge to the blood fluid sample stored in the channel to an assay stack in the cartridge to initiate one or more assay reactions. Step 1030 involves detecting one or more signal changes associated with the plurality of assay reactions. The method 1000 can include any additional steps that would be understood by one of ordinary skill in the art to detect the one or more signal changes via the various components of the metering stack and assay stack described in detail above.

[0096] FIG. 11 shows a flow chart that illustrates a method 1100 of fabricating a cartridge according to one embodiment of the present disclosure. The method includes the steps of obtaining a separation membrane, applying a first reagent (e.g., a precipitating reagent for a first protein such as non-HDL or a sodium sequestering agent) and a soluble polymer to the separation membrane, and allowing the first reagent to dry (step 1110). The method 1100 also includes applying a second reagent (e.g., a reagent specific to a second protein such as HDL or a dye specific to an ion released from the separation membrane) to a detection membrane and allowing the second reagent to dry on the detection membrane (step 1120). Further, the method 1100 also includes the step of positioning the detection membrane adjacent the separation membrane (step 1130). In utilizing an assay device fabricated by this method, in one embodiment, the second reagent interacts with the second protein (e.g., HDL) in the detection membrane without interference from the first protein (e g., non-HDL) or red blood cells to elicit a quantifiable response in when a blood fluid sample is introduced to the assay device. In another embodiment, the second reagent interacts with an ion released from a cryptand to the detection membrane as the sodium is sequestered in the separation membrane via the cryptand in order to elicit a quantifiable response in when a blood fluid sample is introduced to the assay device. In utilizing an assay device fabricated by this method, the concentration of sodium in a blood fluid sample can be determined indirectly by determining the concentration of the ion that binds to the ion-dependent dye after it is released from the cry ptand when sodium in the blood fluid sample reaches the detection membrane, at which point the ion is released from the cryptand to bind with the iondependent dye. Moreover, the timing of such reactions is carefully controlled via a soluble polymer that initially blocks the pores of the separation membrane and dissolves once the reactions in the separation membrane have taken place to improve the accuracy of the assay. Further, the quantifiable response corresponds to an amount of the analyte or ion present inthe detection membrane, which then directly or indirectly corresponds to an amount or concentration of the target analyte present in the fluid sample.

[0097] While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not by way of limitation. Likewise, the various diagrams may depict an example architectural or other configuration for the disclosure, which is done to aid in understanding the features and functionality that can be included in the disclosure. The disclosure is not restricted to the illustrated example architectures or configurations but can be implemented using a variety of alternative architectures and configurations. Additionally, although the disclosure is described above in terms of various exemplary embodiments and implementations, it should be understood that the various features and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described. They instead can be applied, alone or in some combination, to one or more of the other embodiments of the disclosure, whether or not such embodiments are described, and whether or not such features are presented as being a part of a described embodiment. Thus, the breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments.

[0098] Unless otherwise defined, all terms (including technical and scientific terms) are to be given their ordinary and customary meaning to a person of ordinary skill in the art, and are not to be limited to a special or customized meaning unless expressly so defined herein. It should be noted that the use of particular terminology when describing certain features or aspects of the disclosure should not be taken to imply that the terminology is being re-defined herein to be restricted to include any specific characteristics of the features or aspects of the disclosure with which that terminology is associated. Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the tenn ‘example’ is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; adjectives such as ‘known’, ‘normal’, ‘standard’, and terms of similar meaning should not be construed as limiting the itemdescribed to a given time period or to an item available as of a given time, but instead should be read to encompass known, normal, or standard technologies that may be available or known now or at any time in the future; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important to the structure or function of the present disclosure, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure. Likewise, a group of items linked with the conjunction ‘and’ should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as ‘and / or’ unless expressly stated otherwise. Similarly, a group of items linked with the conjunction ‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and / or’ unless expressly stated otherwise.

[0099] Where a range of values is provided, it is understood that the upper and lower limit, and each intervening value between the upper and lower limit of the range is encompassed within the embodiments.

[0100] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does not exclude a plurality. A single processor or other unit may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0101] It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should typically be interpreted to mean “at least one” or“one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc ). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to sy stems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0102] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term ‘about.’ Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0103] All of the features disclosed in this specification (including any accompanying exhibits, claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The disclosure is not restricted to the details of any foregoing embodiments. The disclosure extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanyingclaims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0104] While the present subject matter has been described in detail with respect to various specific example embodiments thereof, each example is provided by way of explanation, not limitation of the disclosure. Those skilled in the art, upon attaining an understanding of the foregoing, can readily produce alterations to, variations of, and equivalents to such embodiments. Accordingly, the subject disclosure does not preclude inclusion of such modifications, variations and / or additions to the present subject matter as would be readily apparent to one of ordinary skill in the art. For instance, features illustrated or described as part of one embodiment can be used with another embodiment to yield a still further embodiment. Thus, it is intended that the present disclosure cover such alterations, variations, and equivalents.

Claims

WHAT IS CLAIMED IS:

1. An assay device for determining a concentration of a target analyte in a fluid sample, the assay device comprising: a separation membrane comprising a first reagent and a soluble polymer; and a detection membrane located downstream from the separation membrane and comprising a second reagent, the detection membrane configured to elicit a quantifiable response in the presence of the target analyte, the quantifiable response corresponding to an amount of the target analyte present in the detection membrane.

2. The assay device according to claim 1, the soluble polymer comprising carboxymethyl cellulose or its derivatives, dextran or its derivatives, polyvinyl alcohol or its derivatives, polyethylene glycol or its derivatives, or a combination thereof.

3. The assay device according to claim 1 or 2, the soluble polymer being present on the separation membrane at a concentration ranging from about 0.1 milligrams / square centimeters to about 0.6 milligrams / square centimeters.

4. The assay device according to any one of the preceding claims, the first reagent configured to precipitate a component of the fluid sample to prevent the component from flowing to the detection membrane, and the soluble polymer configured to initially block passage of the fluid sample through pores in the separation membrane until precipitation of the component of the fluid sample is complete, after which the fluid sample is able to pass through the pores.

5. The assay device according to claim 4, the target analyte comprising a high density lipoprotein and the precipitated component comprising a non-high density lipoprotein.

6. The assay device according to claim 5, the first reagent comprising a polyanionic non-high density lipoprotein binding reagent.

7. The assay device according to claim 6, the first reagent comprising a sulfonated polysaccharide, heparin, or phosphotungstic acid.

8. The assay device according to claim 7, the first reagent further comprising a group II cation selected from the group of Mg2+, Mn2+, or Ca2+.

9. The assay device according to claim 8, the first reagent comprising phosphotungstic acid and Mg2+.

10. The assay device according to any one of the preceding claims, the second reagent comprising cholesterol esterase, cholesterol oxidase, horseradish peroxidase, a Trinder reagent, 4-amino antipyrine, or a combination thereof.

11. The assay device according to any one of claims 1 to 3, the target analyte comprising sodium.

12. The assay device according to claim 11, the first reagent comprising a sodium sequestering agent, the sodium sequestering agent comprising a complex comprising an ion and a cryptand having an affinity for the ion.

13. The assay device according to claim 12, the second reagent comprising an iondependent dye that is dependent on the ion from the complex.

14. The assay device according to claim 13, the ion comprising a divalent ion that is Ca2+, Fe2+, Mg2+, or Mn2+or the ion comprising K+.

15. The assay device according to claim 13 or 14, the ion-dependent dye comprising an azoic dye.

16. In-vitro use of the assay device according to any one of claims 1 to 15 for determining the concentration of the target analyte in the fluid sample.

17. A method of forming an assay device for determining a concentration of a target analyte in a fluid sample, the method comprising: applying a first reagent and a soluble polymer to a separation membrane; allowing the first reagent and the soluble polymer to dry on the separation membrane; applying a second reagent to a detection membrane; allowing the second reagent to dry on the detection membrane; and positioning the detection membrane adj acent the separation membrane, the detection membrane configured to elicit a quantifiable response in the presence of the target analyte, the quantifiable response corresponding to an amount of the target analyte present in the detection membrane.

18. The method of claim 17, the soluble polymer comprising carboxy methyl cellulose or its derivatives, dextran or its derivatives, polyvinyl alcohol or its derivatives, polyethylene glycol or its derivatives, or a combination thereof.

19. The method of claim 17 or 18, the soluble polymer being present on the separation membrane after drying at a concentration ranging from about 0. 1 milligrams / square centimeters to about 0.6 milligrams / square centimeters.

20. The method according to any one of claims 17 to 19, the first reagent configured to precipitate a component of the fluid sample to prevent the component fromflowing to the detection membrane, and the soluble polymer configured to initially block passage of the fluid sample through pores in the separation membrane until precipitation of the component of the fluid sample is complete, after which the fluid sample is able to pass through the pores.

21. The method according to claim 20, the target analyte comprising a high density lipoprotein and the precipitated component comprising a non-high density lipoprotein.

22. The method according to claim 21, the first reagent comprising a polyanionic non-high density lipoprotein binding reagent.

23. The method according to claim 22, the first reagent comprising a sulfonated polysaccharide, heparin, or phosphotungstic acid.

24. The method according to claim 23, the first reagent further comprising a group II cation selected from the group of Mg2+, Mn2+, or Ca2+.

25. The method according to claim 24, the first reagent comprising phosphotungstic acid and Mg2+.

26. The method according to any one of claims 17 to 25, the second reagent comprising cholesterol esterase, cholesterol oxidase, horseradish peroxidase, a Trinder reagent, 4-amino antipyrine, or a combination thereof.

27. The method according to any one of claims 17 to 19, the target analyte comprising sodium.

28. The method according to claim 27, the first reagent comprising a sodium sequestering agent, the sodium sequestering agent comprising a complex comprising an ion and a cryptand having an affinity for the ion.

29. The method according to claim 28, the second reagent comprising an iondependent dye that is dependent on the ion from the complex.

30. The method according to claim 29, the ion comprising a divalent ion that is Ca2+, Fe2+, Mg2+, or Mn2+or the ion comprising K+.

31. The method according to claim 29 or 30, the ion-dependent dye comprising an azoic dye.

Images