Binding assays using magnetic beads without a washing step or the use of moving parts

Abstract

The present disclosure provides devices, systems, and methods for the point-of-care analysis of target analytes in biological fluids by binding assays. The present disclosure includes a cartridge for collecting target analytes contained in a fluid sample and performing an assay. The cartridge includes an assay stack having a first separation layer, a second separation layer, and a detection membrane. The cartridge also includes a plurality of first complexes containing capture molecules and magnetic beads and a plurality of second complexes containing detection molecules and detection labels. Further, the detection membrane contains a substrate that interacts with the detection labels to initiate a quantifiable reaction in the presence of the target analytes. The quantifiable reaction corresponds to the amount of detection antibodies present in the detection membrane, and the amount of detection antibodies present corresponds to the amount of target analytes present.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to point-of-care (POC) testing systems. More specifically, the present disclosure relates to systems and methods for performing binding assays without the use of any washing steps, incubation steps, or moving parts. BACKGROUND

[0002] Point-of-care (POC) testing refers to medical diagnostic testing performed at the time and place of patient treatment. POC testing has advantages over traditional diagnostic testing, where patient samples are sent to a laboratory for further analysis, because the results of traditional diagnostic testing can not be available for hours or even days or weeks, making it difficult for caregivers to assess the appropriate course of treatment during the interim.

[0003] Generally, when measuring certain chemical analytes in biological fluids, such as blood, binding assays, such as immunoassays, are the gold standard for detecting such chemical analytes. However, binding assays are rarely used in POC diagnostics because they conventionally require several washing steps and several incubation steps. Due to the complexity of performing binding assays properly and accurately in a POC environment, this makes binding assays difficult to incorporate into POC testing systems.

[0004] For example, designing a POC testing system for home use is particularly challenging because such systems are typically operated by personnel with limited or no training at all. Current systems often require the user to follow multiple operational steps of multiple separate components, where user-introduced errors can easily result in inaccurate or failed assays.

[0005] Further, in most POC testing systems for blood samples, certain sample preparation steps are required prior to the final chemical reaction that provides the test result. Depending on the assay, these sample preparation steps can include complex preparation steps such as plasma separation, cell lysis, incubation, washing steps, etc. The time required to complete such complex preparation steps can be comparable to the time required for blood to undergo unwanted clotting, which further introduces error into the assay result. Although many attempts have been made or implemented to address this issue, these solutions often use complex fluidics or moving parts to create the necessary incubation time and washing steps, and such mechanisms result in increased cost, failure rate, and complexity.

[0006] Accordingly, it would be desirable to have a POC system that can detect target analytes using binding assays that address the above issues. SUMMARY

[0007] Various aspects and advantages of the implementations of the present disclosure will be set forth in part in the following description, or can be learned from the description, and / or can be learned through practice of the implementations.

[0008] One exemplary aspect of the present disclosure relates to a cartridge for collecting a target analyte contained in a biological fluid sample and assaying the target analyte. The cartridge includes an assay stack having a first separation layer. The assay stack further includes a plurality of first complexes having a capture molecule and a magnetic bead; a plurality of second complexes having a detection molecule and a detection label; a second separation layer; and a detection membrane. The detection membrane includes a substrate that interacts with the detection label to initiate a quantifiable reaction in the presence of the target analyte. The quantifiable reaction corresponds to an amount of the detection molecule present in the detection membrane, and the amount of the detection molecule present in the detection membrane corresponds to an amount of the target analyte present in the fluid sample.

[0009] Another aspect of the present disclosure relates to a method of manufacturing a cartridge. The method includes, in no particular order, applying a plurality of first complexes containing a capture molecule and a magnetic bead and a plurality of second complexes containing a detection molecule and a detection label to a first separation layer; allowing the plurality of first complexes and the plurality of second complexes to dry on the first separation layer; applying a substrate to a detection membrane; allowing the substrate to dry on the detection membrane; and placing a second separation layer between the first separation layer and the detection membrane. Further, the substrate is configured to interact with the detection label to initiate a quantifiable reaction in the presence of a target analyte in a fluid sample introduced to the cartridge, the quantifiable reaction corresponding to an amount of the detection molecule present in the detection membrane, and the amount of the detection molecule present in the detection membrane corresponding to an amount of the target analyte present in the fluid sample.

[0010] Another aspect of the present disclosure relates to a system for collecting a target analyte contained in a fluid sample and assaying the target analyte. The system includes an assay stack, wherein the assay stack includes a first separation layer; a plurality of first complexes containing a capture molecule and a magnetic bead; a plurality of second complexes containing a detection molecule and a detection label; a second separation layer; and a detection membrane, wherein the detection membrane includes a substrate that interacts with the detection label to initiate a quantifiable reaction in the presence of the target analyte, wherein the quantifiable reaction corresponds to an amount of the detection molecule present in the detection membrane, and wherein the amount of the detection molecule present in the detection membrane corresponds to an amount of the target analyte present in the fluid sample; and an electromagnet for drawing a third complex containing the target analyte bound to one of the first complexes and one of the second complexes through the second separation layer to the detection membrane.

[0011] Another aspect of the present disclosure relates to the in vitro use of the proposed cartridge for performing an assay on a target analyte in an isolated fluid sample.

[0012] Another aspect of the present disclosure relates to the use of the cartridge in a diagnostic method for performing an assay on a target analyte in an isolated fluid sample.

[0013] These and other features, aspects, and advantages of various different 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 form a part of the specification, illustrate examples of the present disclosure and, together with the description, serve to explain the principles involved. BRIEF DESCRIPTION OF DRAWINGS

[0014] In this specification, a detailed discussion of the implementation is set forth with reference to the accompanying drawings, in which:

[0015] Figure 1 A schematic of a system comprising a cartridge and an assay reader according to one embodiment of the present disclosure is provided;

[0016] Figures 2A-2C An embodiment of a cartridge used in the system is shown;

[0017] Figure 3 The various different layers of a dosing stack comprised in the cartridge are shown;

[0018] Figure 4 The various different layers of an assay stack comprised in the cartridge are shown;

[0019] Figure 5A A longitudinal cross-sectional view of an assay reader according to one embodiment of the present disclosure is shown;

[0020] Figure 5B A longitudinal cross-sectional view of an assay reader with a cartridge inserted according to one embodiment of the present disclosure is shown;

[0021] Figure 6A A transversal cross-sectional view of an assay reader according to one embodiment of the present disclosure is shown;

[0022] Figure 6B A transversal cross-sectional view of an assay reader with a cartridge inserted according to one embodiment of the present disclosure is shown;

[0023] Figure 7 A block diagram of a sensor system of an assay reader according to an example embodiment of the present disclosure is shown;

[0024] Figures 8A-8FA cartridge containing a metering stack and an assay stack at various stages of an immunoassay process is shown after introduction of a fluid to be analyzed for the presence of a target analyte into the cartridge;

[0025] Figure 9 A flow chart illustrating a method of using an assay system according to an exemplary embodiment of the present disclosure is shown; and

[0026] Figure 10 A flow chart illustrating a method of manufacturing a cartridge according to one exemplary embodiment of the present disclosure is shown.

[0027] Reference numerals that repeat in multiple figures are intended to identify the same features in the various different embodiments. DETAILED DESCRIPTION

[0028] Any feature, component, or detail of any arrangement or embodiment disclosed in the present specification, including but not limited to any cartridge embodiment and any test or assay embodiment disclosed below, can be interchanged with any other feature, component, or detail of any arrangement or embodiment disclosed herein to form new arrangements and embodiments.

[0029] In general, the present disclosure relates to devices and systems for rapid POC detection of a target analyte contained in a biological fluid sample and subsequent analysis of the target analyte by immunoassay or other binding type of assay without any washing step and without any moving parts. In certain embodiments, the binding assay can also be performed without any incubation step. The present disclosure also provides methods and systems for analyzing the fluid sample by immunoassay or other binding type of assay to quantify the level of the target analyte present in the fluid sample using the devices.

[0030] The device can take the form of a cartridge containing an assay stack. The assay stack includes a first separation layer, a second separation layer, and a detection membrane containing a substrate that interacts with a detection label to initiate a quantifiable reaction. The second separation layer can be disposed between the first separation layer and the detection membrane. A plurality of first complexes each comprising a capture molecule and a magnetic bead and a plurality of second complexes each comprising a detection molecule and a detection label can be dried on the first separation layer, where it should be understood that the capture molecule and detection molecule are selected based on their ability to bind to the target analyte. Upon contact of a fluid sample with the first separation layer, any target analyte present in the fluid sample will couple with the first and second complexes to form one or more third complexes. In an exemplary embodiment, an electromagnet can be activated to draw any third complexes through the second separation layer to the detection membrane, while any unbound second complexes remain in the second separation layer. It should also be understood that any unbound first complexes will also be drawn through the second separation layer to the detection membrane. However, because such unbound first complexes are not coupled with a target analyte, a detection molecule, or a detection label, the presence of the unbound first complexes in the detection membrane does not affect the accuracy of the binding assay. Subsequently, in the presence of the target analyte, the substrate can interact with the detection label to initiate a quantifiable reaction (e.g., colorimetric, fluorescent, electrochemical, etc.). The quantifiable reaction can correspond to the amount of detection molecule present in the detection membrane, and the amount of detection molecule present in the detection membrane can correspond to the amount of target analyte present in the fluid sample. It should be understood that any binding assay known to one of ordinary skill in the art can be used in the systems and devices of the present disclosure, such as, but not limited to, a sandwich assay, a competitive assay, or a labeled antigen assay. Furthermore, while immunoassays are described in the embodiments below, the present disclosure also contemplates other detection and capture molecules other than antibodies.

[0031] The proposed solution allows to provide a compact POC testing system capable of performing in vitro assays of a separated (bio)fluid sample, which does not require washing and incubation steps, and therefore physical washing or complex moving parts in the POC system. The cartridge built according to the proposal has an assay stack comprising a second separation layer sandwiched between a first separation layer and a detection membrane, allowing in this case to detect target analytes in a fluid sample with high cost-effectiveness and lower complexity compared to conventional POC testing systems. In combination with the proposed assay reader, detection can be performed in an easy way automatically, as any target analyte present in the fluid sample can bind to the plurality of first complexes and the plurality of second complexes to produce a third complex, which can then be pulled through the assay stack at a specific point in time after activation of an electromagnet of the assay reader. Based on the detection membrane allowing to qualitatively or even quantitatively determine the amount of target analytes in the sample fluid on the basis of the resulting signal (e.g. color change), it is also possible to automatically assess the presence of target analytes in the fluid sample.

[0032] It is furthermore to be understood that the first and second complexes have time to interact with target analytes in the fluid sample when the electromagnet is not activated, and then move through the assay stack to the detection membrane after the electromagnet is activated to pull any third complex through to reach the detection membrane. This allows to precisely control the fluid sample incubation time, which is not possible in many other assay platforms, let alone assay platforms with physical washing steps.

[0033] Exemplary embodiments of the present disclosure will now be discussed in more detail with reference to the accompanying drawings. First the components of the cartridge and the assay reader will be discussed, followed by the components for performing the immunoassay envisaged by the present disclosure.

[0034] Figure 1A point-of-care (POC) testing system according to an exemplary embodiment of the present disclosure is illustrated. The POC testing system includes a cartridge 100 and a assay reader 110. As described herein, the cartridge 100 is used to collect a biological sample that may potentially contain a target analyte. The collection process also dispenses the target analyte within the cartridge 100. After the target analyte is collected in the cartridge 100, the user inserts the cartridge 100 into the assay reader 110. As described herein, the act of inserting the cartridge 100 into the assay reader 110 results in compression of the cartridge 100, thereby dispensing the target analyte onto multiple assay pads. In this way, the act of inserting the cartridge 100 into the assay reader 110 initiates one or more assay reactions that provide information about the contents of the target analyte. However, it should also be understood that other insertion methods without compression are contemplated. Furthermore, it should be understood that although various assays can be used to determine the contents of a target sample, each assay is typically specific to a particular target analyte. As described herein, the assay reader 110 is equipped with a detection system for detecting the result of one or more assay reactions occurring at one or more assay pads of the cartridge 100. The detection system is not particularly limited and can be any detection system that causes a measurable signal change as a result of the assay reaction. Non-limiting examples of suitable detection systems include the colorimetric, fluorescence, electrochemical, and optical detection systems described herein, and any other detection systems that will be understood by one of ordinary skill in the art.

[0035] Figure 2A A top perspective view of an embodiment of box 100 in the form of box 200 is shown. Figure 2A In this device, cartridge 200 includes a housing 201 attached to a handle 202. Typically, cartridge 200 is designed for easy user operation and provides a protective shell for the microfluidic dispensing system and assay components housed within cartridge 200. Suitable materials for housing 201 and handle 202 typically include polyolefin compounds such as polyethylene, polypropylene, and other polymeric resins or compounds known in the field of medical device manufacturing. During sample collection, cartridge 200 comes into contact with a target analyte in a fluid sample (e.g., blood). The target analyte is drawn into channel 203 via capillary action through channel opening 204. In some embodiments, channel 203 includes a plurality of receiving chambers 205 positioned along channel 203. In some embodiments, each receiving chamber is located between two vents, which facilitates the division of the target analyte in the channel into multiple equal aliquots flowing to the assay pad in the assay stack. It should be appreciated that channel opening 204 can function as a vent, and adjacent receiving chambers can share a common vent. The vent is combined with the porous or mesh material described herein to prevent unwanted bubble formation when the target analyte is drawn into the receiving chamber. Figure 2BA bottom view of an embodiment of the cartridge 200 is shown. In Figure 2B the bottom of the housing 201 contains a plurality of assay detection ports 206 aligned with the channel openings 204. The assay detection ports 206 allow for interrogation of assay results, for example, by the optical detection methods described herein. In addition, the bottom of the housing 201 can contain a plurality of holes 207 that are additional assay detection ports that can be used with assay components and microfluidic channels arranged in corresponding configurations.

[0036] Figure 2C An exploded view of the components of the cartridge 200 according to one embodiment of the disclosure is provided. In Figure 2C the housing of the cartridge 200 includes a handle 202, a bottom housing portion 227, and a lid 223 equipped with a slot 228. The bottom housing portion 227 can be a cuboid-shaped enclosure having one open side. The enclosure shape of the bottom housing portion 227 protects the components in the internal compartment and can prevent accidental actuation of the system. The lid 223 can fit to the open side of the bottom housing portion 227 and have a shape and size corresponding to the open side of the bottom housing portion 227. When the bottom housing portion 227 and the lid 223 of the housing are assembled together, an internal compartment can be formed for enclosing other components of the cartridge in the internal compartment. In other embodiments, the lid 223 and the bottom housing portion 227 do not form an enclosure with an internal compartment and can be rigid structures placed on top of the metrology stack and under the assay stack described herein.

[0037] In preferred embodiments, the bottom housing portion 227 and the lid 223 can be formed of a material that provides a rigid structure for the cartridge 200. For example, the bottom housing portion 227 and the lid 223 can be the plastic materials described herein. The bottom housing portion 227 and the lid 223 can be movable or immovable relative to each other. In certain embodiments, when the cartridge 200 is inserted into an assay reader, the components in the internal compartment are compressed, resulting in delivery of at least a portion of the collected target analyte to the plurality of assay components. The compression can be caused, for example, by a user closing a lid of the assay reader. However, it should also be understood that other methods for inserting the cartridge 200 into an assay reader that do not require compression are contemplated.

[0038] In certain embodiments, the cartridge does not include a lid and a bottom housing portion. In such embodiments, the cartridge does not include the housing 201 (see, e.g., Figure 2A ), and the metrology stack and the assay stack can be inserted into an assay reader without an enclosure surrounding them.

[0039] As Figure 2CAs shown, cartridge 200 may include a metering stack 224, a spacer material 225, and a assay stack 226. The metering stack 224 is used to collect samples of biological fluids (e.g., blood), and the assay stack 226 contains assay components for performing binding assays (e.g., immunoassays) as discussed in detail herein. When used herein, the term “metering” refers to collecting a liquid sample of a biological fluid and delivering at least a portion of one or more predetermined volumes of said fluid to the assay components for further analysis by means of the assay components contained in the assay stack. When assembled in the cartridge, the metering stack 224, spacer material 225, and assay stack 226 may be arranged in a stack.

[0040] Spacer material 225 is a compressible layer, which can be as follows: Figure 2C The spacer 225 is positioned between the metering stack 224 and the measurement stack 226, as shown in the diagram. In one embodiment, the spacer 225 may be a flexible material that can be compressed vertically when the cartridge is inserted into the measurement reader, moving the metering stack 224 into contact with or near the measurement stack 226. In some embodiments, the spacer 225 may be a flexible material such as foam, rubber, porous polymer, metal, cotton, or other bending, folding, or moving mechanism such as a clamp or spring. In some embodiments, the metering and measurement stacks are initially separated by an air gap maintained by the spacer 225. In some embodiments, the spacer 225 is physically attached to another layer, such as the metering stack 224 or the measurement stack 226, before the layers of the cartridge are placed together. Typically, the metering and measurement stacks remain separated throughout the sample collection process. In such embodiments, the separation between the metering and measurement stacks prevents the initiation of a chemical reaction during the target analyte collection step. When the spacer 225 is compressed, the metering stack 224 and the measurement stack 226 may come into contact with or be adjacent to each other.

[0041] In preferred embodiments, the cartridge is inserted into an assay reader when the metering stack is completely filled with biological fluid. Preferably, the material used for the top surface of channel 230 is sufficiently transparent so that a user can visually determine when channel 230 is filled and the cartridge is ready for insertion into an assay reader. The assay reader is configured to accept the cartridge and contains a mechanism to compress the spacer material, thereby pushing the metering stack and assay stack together when the cartridge is inserted into the assay reader. Compression of the spacer material results in the flow of a predetermined volume of at least a portion of the collected fluid to the assay components in the assay stack. In this way, the act of compressing the metering stack and assay stack together can provide a clear point in time that marks the beginning of an immunoassay or other binding-type assay by the components in the assay stack. However, it should also be understood that other insertion methods that do not require compression of the metering stack and assay stack together are contemplated, as will be understood by one of ordinary skill in the art.

[0042] In certain embodiments, the biological fluid containing the target analyte is blood, and the cartridge can be used to collect a blood sample from a skin prick and deliver the sample to the assay stack, consistent with minimal user intervention. A user using a conventional lancet can draw blood in an appropriate body part, such as a fingertip, palm, hand, forearm, stomach area, etc. Upon the appearance of a drop of blood of sufficient volume on the skin, the user can collect it by touching the tip of the cartridge to the blood drop. After the metering stack is completely filled with blood, the user can insert the cartridge into an assay reader, which triggers the delivery of the blood sample to the assay stack. In certain embodiments, this can be performed by the patient, a supervisor, or a healthcare provider. The blood collection and testing described herein need not be performed by a trained healthcare professional.

[0043] Furthermore, the design of the cartridge allows for the dispensing of different predetermined volumes of blood sample to multiple assay locations without the use of any moving parts, such as pumps or valves, in the cartridge or assay reader. This improves the accuracy and flexibility of multiplexed quantitative POC analysis while reducing the complexity and cost of the cartridge and assay reader.

[0044] Generally, as Figure 2CAs shown in the middle, the metering stack 224 includes a channel 230 to contain the target analyte (e.g., an analyte of interest contained in a blood sample). In certain embodiments, the channel 230 can hold a volume of a biological fluid containing a target analyte in a range of about 0.5 to about 100 μl, about 5 μl to about 90 μl, about 10 to about 80 μl, about 20 μl to about 60 μl, or about 30 μl to about 50 μl. The volume of the target analyte can be controlled by the dimensions of the channel, including the shape, width, length, and depth of the channels described herein. In certain embodiments, the depth of the channel can be in a range of about 5 μm to about 3 mm, about 10 μm to about 2 mm, or about 250 μm to about 1 mm. In certain embodiments, the width of the channel can be in a range of about 100 μm to about 10 mm, about 250 μm to about 5 mm, about 500 μm to about 3 mm, or about 750 μm to about 1 mm. In certain preferred embodiments, the dimensions of the channel are selected such that the target analyte is drawn into the channel by capillary action.

[0045] Preferably, the metering stack 224 is designed to direct the flow of the target analyte-containing fluid into the channel 230 and into any receiving compartments that can be present. In certain embodiments, the channel 230 can be formed from or coated with a hydrophilic material, non-limiting 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). The channel 230 can also have one or more porous or mesh materials along at least some portions of the channel 230, allowing at least a portion of the target analyte-containing biological fluid to be dispensed from the channel 230 of the metering stack 224 to contact assay components in the assay stack. In one non-limiting embodiment, the metering stack includes a porous or mesh material that can be positioned such that the porous or mesh material is aligned with the channel portions on the top surface of the metering stack and the assay dispensing ports and assay components on the bottom surface of the metering stack. In certain embodiments, the porous or mesh material is selected such that the pores in such material separate the target analyte into a portion that is to be delivered to an assay component and a portion that is not to be delivered to an assay component. For example, when the target analyte-containing biological fluid is blood, the pores of the porous or mesh material can have a size that is suitable to separate red blood cells from other blood components, such as plasma. In this way, only the plasma is delivered to the assay components for analysis when the cartridge is inserted into an assay reader for the assay. Of course, combinations of porous or mesh materials can be used such that all of the biological fluid is delivered to certain assay components, while only a portion of the biological fluid can be delivered to other assay components. For example, a combination of porous or mesh materials can allow only plasma to reach certain assay components, but allow all blood components to be delivered to other assay components. In certain embodiments, the channel can include a porous or mesh material at the bottom of the channel. The porous or mesh material at the bottom of the channel can be a hydrophilic material or a material coated or treated with a hydrophilic coating. In certain embodiments, the porous or mesh material can have a pore size of about 1 pm to about 500 pm. Advantageously, when the target analyte-containing biological fluid is blood, the pores of the porous or mesh material can have a size that allows the porous or mesh material to hold a blood sample within the channel during blood collection without dripping and to be absorbed by the assay stack during a blood dispensing step that occurs after the cartridge is inserted into an assay reader. In certain embodiments, the porous or mesh material can also be used to release air and prevent bubble formation when the channel is filled with the biological fluid.

[0046] Figure 3An exploded view of a metering stack 304 according to one exemplary embodiment of the present disclosure is shown, where such metering stack 304 can be used as Figures 2A to 2C the metering stack 224 in embodiments of the present disclosure. In Figure 3 the metering stack 304 is formed from a plurality of layers that are assembled. The first layer 341 can be a plastic sheet having a first side 342 that is in communication with the ambient environment when the cartridge is outside of the assay reader and a second side 343 that faces the assay stack. In certain embodiments, the first layer 341 can be a cover or top layer of the metering stack. In preferred embodiments, the first layer 341 can have a hydrophilic surface or coating on the second side 343. Non-limiting examples of suitable hydrophilic surface coatings include polyvinylpyrrolidone-polyurethane interpolymer, poly(meth)acrylamide, maleic anhydride polymer, cellulose polymer, polyethylene oxide polymer, and water-soluble nylon or derivatives thereof, to name a few. The presence of a hydrophilic surface or coating on the second side 343 facilitates the wicking of the target analyte into the channel, as the target analyte, if not all, is a majority of an aqueous mixture such as blood. The first layer 341 can include a vent hole 311 that is placed in alignment with a channel 310 defined by the underlying layers. For example, in Figure 3 the vent hole 311 is aligned with a receiving compartment of the channel 310, allowing air that would otherwise be trapped as an air bubble in the receiving compartment during channel priming to efficiently escape to the ambient environment. It should be noted that the channel opening can function as a vent if desired. In certain preferred embodiments, the first layer 341 comprises polyethylene terephthalate (PET) with a hydrophilic coating on the second side 343 and a vent hole 311.

[0047] 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 can itself be a combination of one or more layers as shown in Figure 3 Regardless of whether the second layer comprises one layer or more than one layer, the second layer substantially defines the shape and dimensions of the channel in the metering stack, including any receiving compartments that can be part of the channel. For example, the second layer 344 can be formed from one or more layers of polymeric material that are cut to define the volume and shape of the channel 310 that can contain the target analyte. Other non-limiting methods of forming the channel 310 include injection molding, stamping, machining, casting, lamination, and 3-D printing. Combinations of such manufacturing techniques are also expressly contemplated by the present disclosure. In Figure 3In the embodiment shown, the second layer 344 has a first side 347 facing the first layer 341 and a second side 348 facing the opposite side of the measuring stack. Furthermore, the second layer 344 includes an adhesive layer 345 and a plastic layer 346. The adhesive layer 345 secures the first layer 341 to the plastic layer 346. In some embodiments, the second layer 344 may be a combination of one or more plastic layers 346 and adhesive layers 345. Preferably, the adhesive layer 345 or the plastic layer 346, or both, are made of a material that presents a hydrophilic surface to the inner surface of the channel 310 to facilitate the distribution of the target analyte in the channel 310. In some embodiments, the hydrophilic plastic sheet may comprise a PET material in which the channel 310 is cut. If desired, the channel 310 may be as follows: Figure 3 The diagram shows one or more receiving chambers. Therefore, the thickness and geometry of channel 310 can control the volume of the sample to be collected. The hydrophilic inner surface of channel 310 allows the metering stack to collect blood samples by capillary forces. In some embodiments, the first layer 341 and the second layer 344 may be an integrated layer used in the metering stack 304.

[0048] exist Figure 3 In this embodiment, the third layer 349 may be formed of a hydrophobic adhesive layer. Non-limiting examples of suitable materials for manufacturing the third layer 349 include 3M 200MP adhesive or 3M 300MP adhesive (3M, Oakdale, MN). In a preferred embodiment, the same channel geometry as channel 310 is cut out in the third layer to match the channel 310 cut out in the second layer. In some embodiments, the third layer 349 may have a first side 351 and a second side 352 facing the second layer 344. In some embodiments, the third layer 349 may define a hydrophilic region in a fourth layer 350 placed below or above the second side 352 of the third layer.

[0049] In some embodiments, the fourth layer 350 may be a hydrophilic mesh or porous material. In some embodiments, substantially all of the fourth layers 350 may be as follows: Figure 3 The diagram includes the aforementioned mesh or porous material. In other embodiments, the hydrophilic mesh or porous material may be part of the fourth layer 350. In some embodiments, for example... Figure 3 In the example shown, the fourth layer 350 may have a first side 353 facing the third layer 349 and a second side 354 facing the measuring stack. The hydrophobic third layer 349 may be located above the fourth layer 350. The hydrophobic third layer 349 may be a hydrophobic adhesive layer to define the wettable regions of the mesh or porous material of the fourth layer 350.

[0050] The method used to manufacture the metering stack is not particularly limited, so long as it is compatible with the overall manufacturing requirements of the medical device. In certain embodiments, the layers that make up the metering stack are first fastened together as a large multi-layer sheet or strip, which is then subjected to a punching or cutting process to form the metering stack, including the channels and any receiving compartments that can be present. In certain embodiments, the first layer 341 and the second layer 344 can be combined in a sheet of plastic material, which has a hydrophilic surface that forms the channels. In certain embodiments, the third layer 349 and the fourth layer 350 can be combined in a sheet of molding mesh made by printing or other methods to define the hydrophilic porous area. In certain embodiments, the third layer is not used in the metering stack. Various different embodiments contemplate various different other combinations of two or more layers and other layers.

[0051] In the assay assay or POC system of the present disclosure, the assay reaction takes place in an assay stack. Generally, the assay stack contains one or more "assay components". As used herein, the term "assay component" refers to one or more active components and inactive support elements or masks, including but not limited to multiplexed assay pads. The number of assay pads in a particular assay component is not particularly limited, and can be scaled to meet the assay requirements needed for the diagnosis of the patient condition for which the assay stack is designed. In preferred embodiments, the top layer of the assay pads of a given assay component are vertically aligned with the appropriate area of the channels in the overlying metering stack to ensure that a predetermined volume of biofluid sufficient to perform the assay related to the particular target analyte of interest is delivered to the assay pads. The assay pads can act as wicking cores, drawing the sample through the mesh of the metering stack into the assay stack, for example by capillary action, gravity, etc. Thus, once the metering stack and assay stack are in contact or in close proximity to one another, the biofluid to be analyzed is directed to move into the assay pads, where it can encounter one or more chemical reagents needed to perform the assay related to the particular assay component. If needed, the assay stack can contain additional layers containing the chemicals needed to complete the assay. The number of layers needed can depend on the number of chemical reactions that need to be performed to complete the assay. In various different embodiments, the layers of the assay stack can be made from pads of various different shapes and various different sizes of different porous membrane materials, non-limiting examples of which include nylon, polyether sulfone (PES), nitrocellulose, cellulose filter paper, and glass fiber.

[0052] There are no particular limitations on the types of assays that can be formed using the assay system of this disclosure, and any assay can be performed where the desired reagents can be stably incorporated into one or more assay pads and can cause changes detectable by an assay reader. In some embodiments, the assay reaction causes a color change, which can be detected using the colorimetric detection method described herein. Other assay reactions may cause another optical change, fluorescence change, electrochemical change, or any other detectable change that may occur in the detection membrane of the assay stack. In some embodiments, the assay may be a transverse flow assay, a vertical flow assay, and / or a combination of transverse and vertical flow assays based on porous materials. Typically, the target analyte is contained in a biological fluid, non-limiting examples of which include blood, plasma, serum, saliva, sweat, urine, lymph, tears, synovial fluid, breast milk, and bile, or components thereof, to name just a few. In some preferred embodiments, the biological fluid is blood or a component thereof (e.g., plasma). For example, in one embodiment, the assay system of this disclosure can be used to provide patients with POC information about the target analyte in their blood composition. Non-limiting examples of analytes that can be measured in blood include thyroid markers (e.g., T3, free T4, thyroid-stimulating hormone, etc.), inflammatory markers (e.g., C-reactive protein, etc.), vitamins (detected by competitive assay structure), metabolic syndrome markers, glucose, glycated hemoglobin, glycated albumin, and serological levels of antibodies against the disease (detected by labeled antigen structure). Non-limiting examples of analytes that can be measured in urine include total protein, leukocyte esterase, and myoglobin.

[0053] Figure 4 An exemplary measurement stack 406 according to one embodiment of the present disclosure is shown, wherein such a measurement stack 406 can be specifically used as Figures 2A to 2C The measurement of the stack 226 in the implementation method. In Figure 4 In this context, the stack 406 is formed of multiple layers, including one or more layers having active components and inactive support elements or masks. More specifically, in... Figure 4In some embodiments, assay stack 406 includes an assay stack cover 410, characterized by a cutout portion 411 that is aligned with the channel in the overlying assay stack. Typically, assay stack cover 410 is made of a polymeric material that provides rigidity and ease of handling for the assay stack during cartridge manufacture. In addition, cutout portion 411 allows the flow of biological fluid through assay stack cover 410 to the underlying assay components when the cartridge is inserted into an assay reader as described herein. As shown, assay stack 406 includes a first separation layer 461 (e.g., a plasma separation membrane), which can be the topmost layer facing the metering stack. First separation layer 461 can be used to separate components of the biological fluid to prevent unwanted components from reaching the underlying assay components. For example, when the biological fluid is blood, first separation layer 461 can be a plasma separation membrane that prevents red blood cells from reaching the assay components upon insertion of the cartridge into an assay reader. This is advantageous because the strong spectral absorption of hemoglobin present in red blood cells can overwhelm the color change that occurs at the assay pads after the assay is performed. Such plasma separation membranes can be made of a variety of different materials, non-limiting examples of which include asymmetric polysulfone membranes, glass fibers, or cellulose. In certain embodiments, the manufacture of the plasma separation membrane can include a surface treatment for improved wettability and / or other properties. The plasma separation membrane can be a continuous sheet of membrane material for all of the assay components, or can be multiple discrete sheets of membrane material, which are specific to Figure 4 The first separation layer 461 can be the same or different (or some combination thereof) for each of the assay pads in the assay components in the assay stack. When the first separation layer 461 is discrete, cross-talk between adjacent assays can be prevented. In certain embodiments, certain assay pads of the assay components have a corresponding plasma separation membrane, while other assay pads do not have such a layer. Other additional components used in the immunoassay system contemplated by the present disclosure are discussed in more detail with respect to Figures 8A-8F more detail.

[0054] In Figure 4 In some embodiments, assay stack 406 includes an assay stack cover 410, characterized by a cutout portion 411 that is aligned with the channel in the overlying assay stack. Typically, assay stack cover 410 is made of a polymeric material that provides rigidity and ease of handling for the assay stack during cartridge manufacture. In addition, cutout portion 411 allows the flow of biological fluid through assay stack cover 410 to the underlying assay components when the cartridge is inserted into an assay reader as described herein. As shown, assay stack 406 includes a first separation layer 461 (e.g., a plasma separation membrane), which can be the topmost layer facing the metering stack. First separation layer 461 can be used to separate components of the biological fluid to prevent unwanted components from reaching the underlying assay components. For example, when the biological fluid is blood, first separation layer 461 can be a plasma separation membrane that prevents red blood cells from reaching the assay components upon insertion of the cartridge into an assay reader. This is advantageous because the strong spectral absorption of hemoglobin present in red blood cells can overwhelm the color change that occurs at the assay pads after the assay is performed. Such plasma separation membranes can be made of a variety of different materials, non-limiting examples of which include asymmetric polysulfone membranes, glass fibers, or cellulose. In certain embodiments, the manufacture of the plasma separation membrane can include a surface treatment for improved wettability and / or other properties. The plasma separation membrane can be a continuous sheet of membrane material for all of the assay components, or can be multiple discrete sheets of membrane material, which are specific to Figure 4As shown in FIG. 4, in certain embodiments, the assay stack 406 can include a second assay component 462 positioned below the first separation layer 461 (e.g., a plasma separation membrane) and the first assay component 420. The second assay component 462 includes a mask support layer 450 having a plurality of cutouts 451 configured to receive and secure an assay pad 463 when the assay stack 406 is assembled. Preferably, the cutouts 451 are positioned to align the assay pad 463 with the assay pad 440 (e.g., a hydrophobic membrane) such that a biological fluid containing a target analyte will flow from the assay pad 440 into the assay pad 463. The assay pad 463 (e.g., a detection membrane such as, but not limited to, a color generating membrane) can contain the chemical reagents necessary to complete an assay reaction that begins after the target analyte flows through the assay pad 440 (e.g., a hydrophobic membrane) of the assay component 420. In certain embodiments, the assay pad 463 acts as a detection indicating layer providing information corresponding to the results of the assay being performed. For example, the assay pad 463 (e.g., a color generating membrane) can include a visual indication such as a color change to indicate the results of the assay, although it should be understood that detection membranes contemplated by the present disclosure also contemplate fluorescent and electrochemical changes or reactions. Furthermore, although the assay pad 463 is shown as a single layer, it should be understood that the assay pad 463 can include multiple layers of different materials, each layer having a different function in the assay being performed. Figure 4 Although the assay stack 406 in FIG. 4 contains only two assay components 420 and 462, it should be understood that the assay stack 406 can contain additional assay components having assay pads impregnated with chemical reagents necessary to complete a particular assay and / or report the results thereof. For example, the assay stack 406 can include any number of assay components necessary to perform an analysis of a blood sample. Since certain assays require more chemical steps than others, the assay components can contain more non-functional assay pads that are only used to draw the completed assay products to the bottom of the assay stack where the results can be detected by an assay reader as described herein.

[0055] Figure 4 The assay stack 406 in FIG. 4 also includes an assay base layer 470, which is typically made of a polymeric material to provide mechanical strength and ease of handling to the assay stack 406 during the manufacturing process. Furthermore, the assay base layer 470 typically contains a plurality of detection ports 471 that are aligned with the assay pads of the assay stack and are sized to allow the assay results to be interrogated by an assay reader.

[0056] Figure 5A A longitudinal cross-sectional schematic view of an assay reader according to one non-limiting embodiment of the present disclosure is shown. In FIG. 5, the assay reader 500 includes a cartridge receiving chamber 510 that houses a cartridge when the cartridge is inserted as indicated by arrow 505. A flap 515 is disposed longitudinally along the assay reader 500 and extends into the cartridge receiving chamber 510. The flap 515 is configured to be inserted into a slot at the top of the cartridge when the cartridge is inserted into the assay reader, such as the slot 520 shown in FIG. 6. The flap 515 is configured to be secured to the cartridge when the cartridge is inserted into the assay reader. In certain embodiments, the flap 515 is secured to the cartridge by a friction fit, although it should be understood that other securement methods are contemplated by the present disclosure. Figure 5A In FIG. 5, the assay reader 500 includes a cartridge receiving chamber 510 that houses a cartridge when the cartridge is inserted as indicated by arrow 505. A flap 515 is disposed longitudinally along the assay reader 500 and extends into the cartridge receiving chamber 510. The flap 515 is configured to be inserted into a slot at the top of the cartridge when the cartridge is inserted into the assay reader, such as the slot 520 shown in FIG. 6. The flap 515 is configured to be secured to the cartridge when the cartridge is inserted into the assay reader. In certain embodiments, the flap 515 is secured to the cartridge by a friction fit, although it should be understood that other securement methods are contemplated by the present disclosure. Figure 2CThe cartridge is inserted into slot 228. Furthermore, the gap 525 between the bottom edge of the baffle 515 and the support surface 520 is configured such that when the cartridge is inserted, the baffle 515 compresses the metering stack and the measurement stack together, thereby allowing the target analyte to flow from the metering stack into the measurement stack and initiate the measurement reaction. In some embodiments, the measurement reader may include a slide-in engagement mechanism that locks the cartridge in place after it has been fully inserted into the measurement reader. This is advantageous because it prevents the user from accidentally removing the cartridge from the measurement reader before the measurement is complete, which could adversely affect the accuracy of the measurement results. In some embodiments, the measurement reader 500 also includes sensors 542a and 542b that detect and time the insertion of the cartridge. For example, when the cartridge is inserted into the cartridge receiving chamber 510 and begins to engage with the baffle 515, the bottom surface of the cartridge may pass over sensor 542a, which is detected by suitable electronics as the start of cartridge insertion. The second sensor 542b is located further inside the measurement reader 500 and detects the presence of the cartridge and the time of full insertion when the cartridge is fully inserted. The measurement reader 500 then compares the total insertion time of the cartridge to determine whether the insertion was timely and appropriate. In this way, the measurement reader will not perform any measurement readings if (1) the cartridge is only partially inserted or (2) the cartridge is partially inserted, removed, and reinserted. In either case, inaccurate measurement readings may be given because incomplete compression of the metrology stack and the measurement stack results in incomplete delivery of the required amount of target analyte to the measurement pad in the measurement stack.

[0057] exist Figure 5A In the exemplary embodiment shown, the assay reader 500 detects the assay result by detecting a color change in the assay pad caused by the assay reaction. To achieve this, the assay reader 500 includes a plurality of light sources (not shown in this cross-sectional view) and light detection elements 550 arranged within the assay reader 500 such that they are aligned with the assay pad of the cartridge when the cartridge is fully inserted. To enable the light detection elements 550 to detect the color of the assay pad, the support surface 520 may be equipped with one or more apertures or made of a transparent material that allows light to pass through. However, it should also be understood that the assay reader 500 may also include components for detecting electrochemical or fluorescent changes in the detection membrane portion of the assay stack. Regardless of what changes in the detection membrane can be measured, the assay reader 500 also includes one or more electromagnets 552, which, when activated, react when a portion of the complex (e.g., an immune complex) includes, in particular, a reference... Figures 8A-8F When discussing magnetic beads in more detail, it is helpful to facilitate the transport of target analytes through the measurement of different layers of the stack. Figure 5B A schematic diagram of a longitudinal section of a measuring reader 500 with its cartridge 502 fully inserted is shown. This can correspond to... Figure 1 or Figures 2A to 2CThe cartridge 502 of the cartridge 100 or 200 includes a metering stack 504 and an assay stack 506 that are compressed together by a shutter 515 such that a target analyte is delivered from the metering stack 504 to an assay pad 530. The assay pad 530 is aligned with a light detection element 550. It should be noted, however, that the assay reader 500 can include additional light detection elements 550a that do not have a corresponding assay pad 530. The presence of additional light detection elements, such as light detection element 550a, allows the assay reader to be used with different types of cartridges for different assays, particularly cartridges that can be designed to perform more assays, and allows the different types of cartridges for different assays to be identified.

[0058] Figure 6A A transverse cross-sectional view of the assay reader shown in FIG. 5 is shown in the form of an assay reader 600 that can be used to detect color changes. In Figure 6A The assay reader 600 includes a shutter 615 that extends into the cartridge receiving chamber 610 to engage a slot on a cartridge. This engagement then compresses the metering stack and the assay stack against a support surface 620, initiating the assay reaction. Light sources 660a and 660b provide light for detecting assay results and are positioned proximate to light detection devices 650. In particular, as shown in Figure 6A The light sources 660a and 660b provide light for analysis of the assay pad corresponding to the light detection device 650, as shown in Figure 6AIn this embodiment, light source 660a is restricted by aperture limiting elements 670a and 671a, such that only light from light source 660a passing through aperture 673a can reach the measuring pad and subsequently be detected by the light detection device 650. Similarly, light source 660b is restricted by aperture limiting elements 670b and 671b, such that only light from light source 660b passing through aperture 673b can reach the measuring pad and subsequently be detected by the light detection device 650. In a preferred embodiment, aperture limiting elements 670a, 670b, 671a, and 671b are made of a black material to reduce unwanted scattering when light sources 660a and 660b are turned on. Furthermore, in this embodiment, the light detection device 650 is located within a housing containing aperture limiting elements 671a and 671b, which only allow light passing through aperture 672 to reach the light detection device 650. If needed, aperture 672 can be fitted with a filter to allow only light of a predetermined wavelength or wavelength range to be detected by the photodetector 650. This may be useful, for example, when the equipped light source only provides white light for colorimetric analysis. Furthermore, optical elements such as lenses, filters, shutters, optical fibers, and light guides can be used to guide or manipulate the light from light sources 660a and 660b and the light to be detected by the photodetector 650, without departing from the spirit and scope of this disclosure. The measurement reader 600 also includes one or more electromagnets 652, which, when activated, when a portion of a complex (e.g., an immune complex) includes, in particular, a reference... Figures 8A-8F When discussing magnetic beads in more detail, it is helpful to facilitate the transport of target analytes through the measurement of different layers of the stack.

[0059] Figure 6B It shows Figure 6A A schematic diagram illustrating the operation of the measurement reader as described in the diagram. Figure 6B In this process, a cartridge containing a metrology stack 604 and a measurement stack 606 is inserted into the cartridge receiving chamber 610 of the measurement reader 600. A baffle 615 presses the metrology stack 604 and the measurement stack 606 against a support surface 620 to allow the target analyte to flow from the channel 612 into the measurement pad 630. As previously mentioned, the measurement reader 600 may be equipped with a sensor to confirm that the cartridge has been inserted correctly and in a timely manner. The measurement reader 600 may also be pre-programmed by the user or during manufacturing before sample collection to irradiate the measurement pad at appropriate times depending on the type of cartridge used. In this way, the measurement reader 600 collects measurement data from the measurement pad 630 only when the measurement is complete. Alternatively, if desired, the measurement reader 600 may be configured to collect measurement data from the measurement pad 630 throughout the entire measurement reaction after the cartridge has been inserted. Figure 6BAs shown in FIG. 6B, light source 660a provides a light beam 680a that impinges on the bottom of assay pad 630 to produce a reflected light beam 661. Likewise, depending on the requirements of the assay to be detected, light source 660b produces a light beam 680b, which can impinge on the bottom of assay pad 630 to produce a reflected light beam 661, at the same time or at a different time than light source 660a.

[0060] Figure 7 A block diagram 700 showing the sensor configuration inside the assay reader according to one exemplary embodiment of the present disclosure is shown. In this example, four assay pads (identified by reference numerals 741, 742, 743, and 744) have completed their assay reactions with the target analyte, undergone the corresponding color change, and are ready for colorimetric analysis. Notably, this configuration can also be used to collect data from the four assay pads to monitor the progress of the assay reactions, if desired. Figure 7 In this example, four assay pads (identified by reference numerals 741, 742, 743, and 744) have completed their assay reactions with the target analyte, undergone the corresponding color change, and are ready for colorimetric analysis. Notably, this configuration can also be used to collect data from the four assay pads to monitor the progress of the assay reactions, if desired. Input signal 701 from the first microcontroller serial peripheral interface bus (MCU SPI bus) enters digital-to-analog conversion device 710, which contains separate 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 certain embodiments, input signal 701 can be sent by a timing circuit at a predetermined time after the cartridge is inserted into the assay reader. In such embodiments, the predetermined time corresponds to one or more known times at which the assay reactions in the assay pads reach completion. In certain preferred embodiments, light sources 731, 732, 733, and 734 are activated at the same time to measure the color change in assay pads 741, 742, 743, and 744 caused by the assay in a multiplexed manner simultaneously. However, the present disclosure also contemplates operating all of the light sources separately or sequentially or some simultaneously and some separately, depending on the time requirements of the assays in the cartridge.

[0061] In this non-limiting example, each light source 731, 732, 733, and 734 includes three individual light emitting diodes (LEDs), which can be of the same or different colors depending on the requirements of the assay and any optical elements that can be present in the assay reader. For example, in certain embodiments, the three LEDs in a particular light source (e.g., 731) can be red, green, and blue (RGB LEDs), such that when all three LEDs are activated the light impinging on the assay pad is white light. Of course, the light sources are not limited to any particular number or type of LEDs or other light-emitting devices. More generally, the light sources useful in the assay readers of the present disclosure are not particularly limited, so long as they provide light of the appropriate wavelength and brightness for the light detection elements to make accurate readings of colored light reflected from the assay pad. In certain non-limiting embodiments, the light sources are light emitting diodes (LEDs), organic light emitting diodes (OLEDs), active-matrix organic light-emitting diodes (AMOLEDs), or lasers. For example, the light source can be just one LED, which has sufficient brightness and the appropriate wavelength to allow colorimetric analysis of the assay reactions in a given assay pad. In certain embodiments, the light source can produce light of a particular wavelength. As one non-limiting example, when the biological fluid containing the target analyte is blood (with red blood cells removed), a dual-color light source producing light at 570 nm and 600 nm can be used to detect the presence of hemoglobin on a non-functional (i.e., not containing assay reagents) assay pad, which is indicative of unwanted hemolysis in the patient. Alternatively, the light source can be a broadband light source, paired with one or more narrow bandpass filters to select certain desired wavelengths of light. Typically, the light source produces light in the visible region of the electromagnetic spectrum (i.e., wavelengths between 400-700 nm), although the present disclosure also contemplates light sources that produce electromagnetic radiation in the infrared (700 nm to 10 6 nm) or ultraviolet region (10 nm - 400 nm) of the electromagnetic spectrum, so long as they are paired with appropriate light detection devices. Combinations of different light sources are also expressly contemplated by the present disclosure.

[0062] In Figure 7In the middle, element 740 is a schematic representation of an optical element that can optionally be present in the optical path between light sources 731, 732, 733, and 734 and assay pads 741, 742, 743, and 744. One or more optical elements can be placed between the light source and its corresponding assay pad to direct the light, focus the light, reduce unwanted scattering, select one or more wavelengths for assay detection, or some combination thereof, as needed. Non-limiting examples of such optical elements include apertures, lenses, light guides, bandpass filters, optical fibers, shutters, and the like. Likewise, element 745 represents an optical element that can optionally be present in the optical path between assay pads 741, 742, 743, and 744 and corresponding light detection devices 751, 752, 753, and 754. These optical elements can be used to manipulate light upstream of the light detection device in a similar manner as described for element 740. It should be understood that different types and numbers of optical elements can be used for each combination of light source, assay pad, and light detection device. Light detection devices 751, 752, 753, and 754 detect light from assay pads 741, 742, 743, and 744. In this non-limiting example, the light detection devices are photodiodes. More generally, the type of light detection device is not particularly limited, so long as it is capable of detecting light reflected from the assay pad that is used for colorimetric measurement of the assay result. Other examples of suitable light detection elements include photodiode arrays, CCD chips, and CMOS chips. The output from light detection devices (e.g., photodiodes) 751, 752, 753, and 754 is sent to transimpedance amplifier / low pass filter elements 761, 762, 763, and 764, which convert the current signal from the photodiode into a voltage output while filtering out unwanted signal components. The output from transimpedance amplifier / low pass filter elements 761, 762, 763, and 764 is sent to analog-to-digital conversion device 770, which includes multiplexing converter device 771, a gain 772, and an analog-to-digital converter 773. The output of analog-to-digital conversion device 770 can be sent to component 780, which can be a second MCU SPI bus, a transmitter, or a processor. In certain embodiments, the transmitter allows for hardwired or wireless connection (e.g., Bluetooth or Wi-Fi) to a personal computer, mobile device, or computer network. In one particularly useful embodiment, the assay results are transmitted to a user's mobile device or personal computer, where they are displayed in a graphical user interface (GUI). If desired, the GUI can display previous assay results in addition to the current result, in order to provide the user with information about the overall trend of the assay results. For example, if the user is a diabetic, the GUI can plot the glucose levels measured by the assay reader as a function of time, to allow the user to determine whether the blood glucose levels are being adequately controlled.Further, the assay results can be transmitted from the user's mobile device or computer to a computer network, such as a computer network belonging to the user's physician. In this way, the assay system of the present disclosure can allow the user's physician to closely monitor the patient by providing up-to-date medical information from the assay results obtained by the assay reader.

[0063] It should be noted that the optical detection system described above corresponds to certain exemplary embodiments of the system, but other types of detection systems are expressly contemplated by the present disclosure. In general, any detection system that corresponds to a signal change resulting from an assay reaction can be used in conjunction with the assay reader of the present disclosure. Thus, for example, in certain embodiments, the detection system is an optical detection system based on chemiluminescence. In such embodiments, no light source, such as an LED or an OLED, is needed to detect a color change resulting from an assay reaction in the assay pad. Instead, the signal change can result from a reaction of an oxidizing enzyme, such as luciferase, with a substrate that results in light produced by a bioluminescent reaction. In another exemplary embodiment, the signal change resulting from an assay reaction can be detected by an electrochemical reaction.

[0064] Figures 8A-8F Another embodiment of the cartridge 100, 200, or 502 in the form of a cartridge 800 comprising a metering stack 802 and an assay stack 804 is shown in various different stages of an immunoassay after a fluid sample 814 in the presence of a target analyte 816 to be analyzed has been introduced into the cartridge 800. The metering stack 802 is configured to receive the target analyte 816 and distribute it along a channel, where the channel has a bottom comprising a porous or mesh material and one or more vent holes in communication with the channel, as described in detail above. As also described above, a spacer material is configured between the metering stack and the assay stack, where 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. Further, the porous or mesh material allows the target analyte 816 to flow from the metering stack 802 to the assay stack 804 after the cartridge 800 is compressed.

[0065] After the introduction of the metrological stack 802, the fluid sample 814 containing the target analyte 816 passes through the first separation layer 806, and finally the target analyte 816 reaches the detection membrane 812 (e.g., a hydrophobic membrane, a low molecular weight cutoff membrane, or a combination thereof) via a second separation layer 808, discussed in more detail below. When the fluid sample 814 is blood, the first separation layer 806 may be referred to as a plasma separation membrane. Furthermore, when the fluid sample 814 is blood, the first separation layer 806 may include orifices 840 having an orifice size large enough to allow the target analyte 816 to be drawn through the other layers of the cartridge 800, but also having an orifice size small enough to prevent any red blood cells from passing through the other layers of the cartridge 800 (which would affect the accuracy of the measurement results) (e.g., less than about 2 micrometers). This is because hemoglobin present in red blood cells has strong spectral absorption, which may, for example, overwhelm the color change that occurs after the measurement.

[0066] Furthermore, as Figure 8A As shown, the first separation layer 806 may further include a plurality of first complexes 822 each comprising a coupled capture molecule 818 (e.g., a capture antibody in the case of an immunoassay) and magnetic beads 820, and a plurality of second complexes 828 each comprising a coupled detection molecule 824 (e.g., a detection antibody in the case of an immunoassay) and a detection marker 826. Ultimately, as... Figure 8B As shown, any target analyte 816 present in the fluid sample 814 can bind to multiple first complexes 822 and multiple second complexes 828 to generate a third complex 834 (e.g., an immune complex in the case of an immunoassay), which can activate the electromagnet 852 to cause the electromagnet 852 to emit an electromagnetic force or signal 853 (see [link to documentation]). Figure 8C and 8D The first composite 822 is then pulled through the measuring stack 804, where the specific structure and composition of the first separating layer 806 substantially replaces the washing and incubation steps typically used in standard assays, although it should be understood that in some embodiments, the composite may be located on different layers besides the first separating layer 806, where the washing and incubation steps are still eliminated. In any case, regardless of where the first composite 822 and the second composite 828 are initially configured on the measuring stack 804, the assay system contemplated in this disclosure eliminates the need for the use of fluid physical cleaning or complex moving parts. As previously mentioned... Figure 1 and 5A As discussed in section 7, the electromagnet 852 may be part of the measurement reader.

[0067] The capture molecules 818 can be capture molecules that specifically bind to the target analyte 816 (e.g., capture antibodies in the case of immunoassays). Capture molecules 818 for target analytes 816 are well known to those of ordinary skill in the art and can be produced by routine techniques or can be readily commercially available. Furthermore, the magnetic beads 820 to which the capture molecules 818 are coupled can be ferromagnetic particles that can be readily coupled to biomolecules such as the capture molecules 818. The magnetic beads 820 can have diameters ranging from about 10 nanometers to about 10 micrometers, for example, from about 20 nanometers to about 7.5 micrometers, for example, from about 30 nanometers to about 5 micrometers. Suitable magnetic beads are well known to those of ordinary skill in the art and are available from commercial suppliers. The magnetic beads 820 can comprise iron oxide particles such as magnetite (Fe304), although it should be understood that any other iron oxide particles can also be used, so long as the magnetic beads 820 have superparamagnetic properties, as the beads exhibit magnetic behavior only in the presence of an external magnetic field. This property is due to the small size of the particles in the magnetic beads 820 and enables the magnetic beads 820 to be isolated in suspension along with the capture molecules 818 coupled to the magnetic beads 820. Since the magnetic beads 820 do not attract each other outside of a magnetic field, the magnetic beads 820 can be used without any concern for unwanted clumping. The capture molecules 818 can be coupled to the magnetic beads 820 directly or indirectly through a linker molecule that can be covalently or non-covalently bound to the capture molecules 818 and the magnetic beads 820. In any case, suitable methods for forming the first complex 822 containing the capture molecules 818 (e.g., capture antibodies) and the magnetic beads 820 are well known to those of ordinary skill in the art.

[0068] In addition, like the capture molecules 818, the detection molecules 824 (e.g., detection antibodies in the case of immunoassays) are also molecules that specifically bind to the target analyte. Detection molecules 824 for the target analyte 816 are well known to those of ordinary skill in the art and can be produced by routine techniques or readily purchased commercially. The detection molecules 824 are linked to detection labels 826. As discussed in greater detail below, the detection labels 826 can initiate a chemical reaction with a reagent or substrate 830 located in the detection membrane 812 (e.g., a color generating membrane, a fluorescent generating membrane, an electrochemical signal generating membrane, etc.) to produce a detectable signal. For example, the detection labels 826 can catalyze the oxidation of the substrate 830. The oxidized form of the substrate 830 can then provide a detectable signal in the form of a color change, a fluorescent change, or an electrochemical change. Suitable detection labels 826 are well known in the art and can include peroxidase, glucose oxidase, and alkaline phosphatase. In one particular embodiment, the detection labels 826 can be peroxidase, such as horseradish peroxidase (HRP), or in another embodiment, the detection labels 826 can be β-galactosidase. The detection molecules 824 can be coupled to the detection labels 826 directly or indirectly through a linker molecule that can be covalently or non-covalently bound to the detection molecules 824 and the detection labels 826. In any case, suitable methods for forming a second complex 828 containing the detection molecules 824 (e.g., detection antibodies) and the detection labels 826 are well known to those of ordinary skill in the art. Further, it should be appreciated that when the assay is a sandwich assay, the capture molecules 818 and the detection molecules 824 can be selected to be specific for the target analyte 816 and to pair to ensure targeting of different epitopes of the target analyte 816, such that both molecules can bind to the target analyte 816 to produce a complex 834 including the capture molecules 818, the detection molecules 824, and the target analyte 816 (as well as the magnetic beads 820 and the detection labels 826). It should also be appreciated that other assay architectures also fall within the scope of the present disclosure, such as, but not limited to, competitive and labeled antigen architectures.

[0069] The assay stack 804 also includes a second separation layer 808 (e.g., a hydrophobic membrane, a low molecular weight cutoff membrane, or a combination thereof) positioned adjacent to the first separation layer 806. The second separation layer 808 can include pores 842 that are large enough in size to allow a third complex 834 containing the target analyte 816 bound to one of the first complexes 822 and one of the second complexes 828 to pass in the presence of the activated electromagnet 852 to reach the detection membrane 812. The second separation layer 808 can also include pores 842 that are small enough in size to prevent any unbound second complexes 836 from passing in the presence of the activated electromagnet 852 to reach the detection membrane 812 (see Figures 8B-8F), where passage of such unbound second complex 836 would reduce the accuracy of the assay because it would allow passage of excess detection label 826 that is not coupled to a target analyte 816 as part of a third complex 834 to reach the detection membrane 812 and potentially interact with the substrate 830, which would cause the assay to indicate a higher concentration of target analyte 816 in the fluid sample 814 than actually exists. It should also be appreciated that passage of unbound first complex 838 to reach the detection membrane 812 in the presence of the activated electromagnet 852 is acceptable because the unbound first complex 838 does not contain the detection label 826.

[0070] In certain embodiments, the pore size of the pore 842 can be about 150,000 Daltons or less, such as about 125,000 Daltons or less, such as about 100,000 Daltons or less, to prevent passage of the unbound second complex 836 containing the detection molecule 824 and the detection label 826 through the pore 842 because the molecules (e.g., antibodies) can have a molecular weight of about 150,000 Daltons or more. It should also be appreciated that, although the first complex 822 also contains molecules (e.g., antibodies) that can have a molecular weight higher than about 150,000 Daltons or more (e.g., the capture molecule 818), the presence of the magnetic beads 820 in the first complex 822 provides sufficient force upon activation of the electromagnet 852 to allow passage of the second complex 828 through the second separation layer 808 to reach the detection membrane 812.

[0071] In addition to the pore size of the second separation layer 808, the second separation layer 808 can also include a hydrophilic treatment 810 (e.g., a coating) applied to modulate the second separation layer 808 according to the particular detection molecule 824 used in the second complex 828 such that it has a desired molecular weight cut-off. In one embodiment, the hydrophilic treatment 810 can include one or more surfactants. Any suitable surfactant known to one of ordinary skill in the art can be used to form the hydrophilic treatment 810, including but not limited to non-ionic surfactants (e.g., surfactants with hydrophilic polyoxyethylene chains and aromatic hydrocarbon lipophilic groups such as Triton X-100, surfactants such as polysorbate molecules containing a hydrophilic head group with oligo(ethylene glycol) chains and a hydrophobic tail consisting of a fatty acid ester moiety such as Tween 20, Tween 40, and Tween 80, etc.), anionic surfactants (e.g., sodium laureth sulfate, sodium dodecyl sulfate, etc.), and cationic surfactants (e.g., methyltriethanolammonium). In any case, it should be understood that one of ordinary skill in the art can optimize the combination of low molecular weight cut-off membrane material, hydrophobic membrane material, hydrophilic treatment or coating, etc. to form the second separation layer 808 according to the magnetic field strength of the electromagnet signal 853 of the electromagnet 852, the size of the magnetic beads 820, the molecular weight cut-off of the materials used.

[0072] After any formed third complexes 834 (which contain any target analyte 816 present and sandwiched between the first complex 822 and the second complex 828) and any unbound first complexes 838 (which contain the capture molecule 818 and the magnetic beads 820) respond to the magnetic force or signal 853 emitted by the one or more electromagnets 852 through the pores 842 in the second separation layer 808, the detection label 826 in each third complex 834 can react with the substrate 830 present in the detection membrane 812. See Figures 8D-8F In the presence of the target analyte 816, the reaction between the substrate 830 and any detection label 826 present can initiate a quantifiable reaction 844 (e.g., a colorimetric reaction, a fluorescent reaction, an electrochemical reaction, etc.) where the quantifiable reaction 844 corresponds to the amount of detection molecule 824 present in the detection membrane 812, as Figure 8E and 8FThe amount of detection molecules 824 present in the detection membrane 812 corresponds to the amount of target analyte 816 present in the fluid sample 814. In one embodiment, the substrate 830 can include one or more reagents for detecting the label 826. For example, in one embodiment, the substrate 830 can include one or more reagents that can be catalyzed by the detection label 826 attached to the detection molecule 824 to provide a detectable signal within the detection membrane 812. For example, the substrate 830 can include a first reagent and / or a second reagent, where the second reagent can be an oxidizing agent or a precursor thereof for the first reagent. Further, the reaction between the first reagent and the oxidizing agent can be catalyzed by the detection label 826 to provide a detectable signal in the detection membrane 812.

[0073] The selection of the first and second reagents can depend on the detection label 826 that is part of the second complex 828. The first reagent can be reactive with the second reagent in the presence of the detection label 826. Suitable first reagents can include tetramethylbenzidine (TMB), a-guaiacol, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), hydroquinone, phenylenediamine, o-anisidine, o-toluidine (dimethylbenzidine), 6-methoxyquinoline, and 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole, or combinations thereof. The second reagent can be an oxidizing agent or a precursor thereof and can be reactive with the first reagent in the presence of the detection label 826. Suitable second reagents for detecting a detection label 826 that includes a peroxidase can include hydrogen peroxide or a precursor thereof. For example, the second reagent can include urea hydrogen peroxide or sodium perborate. Thus, the first reagent can be a compound that is reactive with hydrogen peroxide in the presence of a peroxidase detection label 826. Further, suitable second reagents for detecting a glucose oxidase detection label 826 can include glucose or a precursor thereof. In certain preferred embodiments, the substrate 830 for detecting a peroxidase detection label 826 can include tetramethylbenzidine (TMB) and perborate (PER). In certain embodiments, the substrate 830 can include a single reagent (i.e., only a first reagent). The reaction between the reagent and the detection label 826 can provide a detectable signal without a second reagent. This can be useful, for example, in the detection of an alkaline phosphatase detection label 826. Suitable reagents for detecting alkaline phosphatase include 1-naphthyl-phosphate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), hydroquinone diphosphate, phenolphthalein phosphate, 4-aminophenyl phosphate, 3-indoxyl phosphate, and phenyl phosphate. It should be understood, however, that the substrate 830 can include other reagents known to those of ordinary skill in the art based on the particular detection label 826 used.

[0074] In any case, the quantifiable reaction 844 (e.g., colorimetric reaction, fluorescence reaction, electrochemical reaction, etc.) in the detection membrane 812 can be detected by one or more detection devices, which, for example, when the quantifiable reaction is a color change, may include... Figure 8F The light detection device 854 is shown. Simultaneously, one or more light sources 831 provide light for detecting a quantifiable reaction 844 (e.g., color change) in the detection film 812 (e.g., a color-generating film) and can be placed near the light detection device 864. In the case of a colorimetric reaction, one or more light sources 831 corresponding to the light detection device 854 provide light for analyzing the quantifiable reaction 844 (e.g., color change) in the detection film 812 (e.g., a color-generating film). As mentioned above, it is advantageous to dedicate one or more light sources to each light detection element to ensure that the photon flux on the light detection element is sufficient to obtain an accurate reading. In some embodiments, light sources 831 dedicated to a particular light detection device 854 have the same output spectrum. However, in other embodiments, light sources 831 corresponding to a given light detection device 854 produce different output spectra. For example, the light source may be a light-emitting diode (LED) that produces light of different colors. Generally, it is advantageous to include optical elements in the measuring reader to guide the light and / or reduce the amount of light scattering. If necessary, the light detection device 854 may be equipped with a filter to allow only light of a predetermined wavelength or wavelength range to be detected by the light detection device 854. This may be useful, for example, when the light source 831 is configured to provide only white light for colorimetric analysis. Furthermore, optical elements such as lenses, filters, shutters, optical fibers, and light guides can be used to guide or manipulate the light from the light source 831 and the light to be detected by the light detection device 854, without departing from the spirit and scope of this disclosure. More generally, there are no particular limitations on the light source 831 that can be used in the measurement reader of this disclosure, as long as they provide light of suitable wavelength and brightness to the light detection device 854 for accurate reading of colored light reflected from the detection film 812.

[0075] It should also be understood that, for example Figures 8A-8F As shown, the detection membrane 812 may include one or more stabilizers 832. Such stabilizers 832 may include new silk protein protectants, mannitol, trehalose or other sugars, polypropylene glycol-polyethylene glycol block copolymers or other hydrophilic-hydrophobic block copolymers or combinations thereof.

[0076] Figure 9The illustrated flow chart illustrates a method 900 of performing a plurality of assays using an assay system according to one embodiment of the disclosure. The method 900 includes a step 910 involving receiving a fluid sample that can contain a target analyte or analyte of interest in a channel in a cartridge. A step 920 involves inserting the cartridge into an assay reader, thereby compressing the cartridge to expose the target analyte stored in the channel to an assay stack in the cartridge to initiate one or more assay reactions. A step 930 involves detecting one or more signal changes associated with the plurality of assay reactions. The method 900 can include any other steps that would be understood by one of ordinary skill in the art to detect the one or more signal changes through the various different components of the meter stack and assay stack described in detail above.

[0077] Figure 10 The illustrated flow chart illustrates a method 1000 of manufacturing a cartridge according to one embodiment of the disclosure. The method includes the steps of obtaining a first separation layer, applying a plurality of first complexes comprising capture molecules and magnetic beads and a plurality of second complexes comprising detection molecules and detection labels to a plasma separation membrane, and allowing the plurality of first complexes and the plurality of second complexes to dry (step 1010), and obtaining a second separation layer (step 1020). The method 1000 further includes the step of obtaining a detection membrane, applying a substrate to the detection membrane, and allowing the substrate to dry (step 1030). In addition, the method 1000 further includes the step of placing the second separation layer between the first separation layer and the detection membrane. In this method, in the presence of a target analyte in a fluid sample introduced to the cartridge, the substrate interacts with the detection labels to initiate a quantifiable reaction. In addition, the quantifiable reaction corresponds to the amount of detection molecules present in the detection membrane, and the amount of detection molecules present in the color detection membrane corresponds to the amount of target analyte present in the fluid sample.

[0078] While various different 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 limitation. Likewise, the various figures can depict an example architectural or other configuration for the present disclosure in detail, but it will be understood that the various features and functions of the present disclosure can be combined or divided into separate embodiments with or without these specific details. Furthermore, although the present disclosure has been described in terms of various embodiments and implementations, a person of ordinary skill in the art will recognize that the various characteristics and features described herein can be applied to other embodiments and implementations without departing from the spirit and scope of the present disclosure. Therefore, the breadth and scope of the present disclosure should not be limited by any of the above described embodiments.

[0079] Unless otherwise defined, all terms (including technical and scientific terms) should be given their common and conventional meanings to those skilled in the art and should not be limited to specific or customary meanings unless expressly defined herein. It should be noted that the use of particular terms in describing certain features or aspects of this disclosure should not be construed as implying that the term is redefined herein to include any particular feature of the disclosure to which that term is associated. Unless expressly stated otherwise, the terms and phrases and their variations used in this application, particularly in the appended claims, should be interpreted as open-ended rather than restrictive. As examples of the above, the term "comprising" should be understood to mean "including but not limited to," etc.; when used herein, the term "comprising" is synonymous with "including," "containing," or "characterized as," and is inclusive or open-ended, not excluding other undescribed elements or method steps; the term "having" should be interpreted as "having at least"; the term "comprising" should be interpreted as "including but not limited to"; the term "example" is used to provide exemplary cases of the items discussed, not an exhaustive or limiting list; adjectives such as "known," "normal," "standard," and similar terms should not be interpreted as limiting the described items to items available for a given time period or for a given time, but should be understood to cover known, normal, or standard techniques that may be available or known at any time now or in the future; and the use of terms such as "preferred," "ideal," "required," or "ideal," and similar words should not be interpreted as implying that certain features are critical, necessary, or even important to the structure or function of this disclosure, but are merely intended to highlight optional or additional features that may or may not be used in a particular embodiment of this disclosure. Similarly, a group of entries connected by the conjunction "and" should not be understood as requiring each of these entries to appear in the group, but should be understood as "and / or" unless explicitly stated otherwise. Likewise, a group of entries connected by the conjunction "or" should not be understood as requiring mutual exclusivity within the group, but should be understood as "and / or" unless explicitly stated otherwise.

[0080] When a range of values ​​is provided, it should be understood that the upper and lower limits of the range, as well as each intermediate value between the upper and lower limits, are covered within the implementation.

[0081] For use of any plural and / or singular term herein, those skilled in the art can convert the plural to the singular and / or the singular to the plural, as appropriate, depending on the context and / or application in which the term is used. For clarity, various different arrangements of singular / plural permutations can be set forth herein. The indefinite article "a" or "an" does not exclude a plurality. A single processor or other unit can 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.

[0082] Those skilled in the art will further appreciate that, where a particular number of claim recitation items is intended to be introduced, such intent will be explicitly recited in the claims, and no such intent exists without such recitation. For example, to aid in understanding, the claims can contain the introductory phrases "at least one" and "one or more" to introduce claim recitation items. However, the use of such phrases should not be interpreted as implying that claim recitation items introduced by indefinite articles are limited to embodiments containing only one such recitation item, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles (e.g., the indefinite articles should generally be interpreted to mean "at least one" or "one or more"), unless such limitation is explicitly recited. The same holds true for the use of definite articles to introduce claim recitation items. In addition, even if a particular number of claim recitation items is explicitly recited, those skilled in the art will recognize that such recitation generally should be interpreted to mean at least the recited number (e.g., an unqualified recitation of "two items" generally means at least two items or two or more items) unless otherwise modified by further qualifiers. Further, where a phrase similar to "at least one of A, B, and C, etc." is used, such phrase is generally intended to mean that at least one of A, at least one of B, and at least one of C, etc. (e.g., a system having at least one of A, B, and C would include but would 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.). Where a phrase similar to "at least one of A, B, or C, etc." is used, such phrase is generally intended to mean that at least one of A, at least one of B, or at least one of C, etc. (e.g., a system having at least one of A, B, or C would include but would 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.). Those skilled in the art will further appreciate that virtually any disjunctive language presenting two or more

[0083] All numbers used herein to express quantities of ingredients, reaction conditions, and so forth should be understood as modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations. Where necessary, the numerical parameters have been indicated as approximations by the use of the terms "about" or "approximately." It will be further understood that the parameters and numeric identifiers used herein are merel y intended to illustrate the application, and that the application is not limited to the specific values recited. Each numerical parameter should at least susbstantially reflect actual values within the prior art measurements as divined from the literature and / or the actual values of the test measurements carried out.

[0084] All features disclosed in this specification, and / or of any method or process so disclosed, can be combined in any combination, provided that the resulting is not excluded by the law of the established prior art. The disclosure is not limited to any presently disclosed particular combination of features. The disclosure extends to any novel one, or any novel combination, of the features disclosed in this specification, to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0085] While the subject matter of the present disclosure has been described in detail with respect to various specific exemplary embodiments thereof, it should be understood that the words to the effect such as "for", "for example", "for instance", "e.g.", "in an embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in one embodiment", "in another embodiment", "in some embodiments", "in certain embodiments", "in

Claims

1. A cartridge for collecting and measuring a target analyte contained in a fluid sample, wherein the cartridge comprises a measurement stack, wherein the measurement stack comprises: First separation layer; Multiple first complexes containing trapping molecules and magnetic beads; Multiple second complexes containing detection molecules and detection markers; Second separation layer; and A detection membrane comprising a substrate that interacts with the detection marker to initiate a quantitatively reactive reaction in the presence of the target analyte, wherein the quantitatively reactive reaction corresponds to the amount of detection molecules present in the detection membrane, and wherein the amount of detection molecules present in the detection membrane corresponds to the amount of the target analyte present in the fluid sample. The second separation layer comprises a hydrophobic membrane, a low molecular weight cutoff membrane, or a combination thereof, the pores of the second separation layer being small enough to prevent any unbound second complex from passing through to the detection membrane in the presence of an activated electromagnet, and the second separation layer allowing a third complex containing a target analyte bound to one of (1) the first complex and (2) the second complex to pass through to the detection membrane in the presence of an activated electromagnet.

2. The box according to claim 1, wherein the second separation layer is subjected to a hydrophilic treatment.

3. The box according to claim 2, wherein the hydrophilic treatment is a coating comprising a surfactant.

4. The kit of claim 1, wherein the target analyte is contained in a fluid sample selected from blood, saliva, sweat, urine, lymph, tears, synovial fluid, milk, serum, plasma, bile, or components thereof.

5. The box according to claim 4, wherein the fluid sample is blood, and the first separation layer is a plasma separation membrane that prevents red blood cells from contacting the second separation layer.

6. The kit according to claim 1, wherein the detection marker comprises peroxidase.

7. The kit of claim 6, wherein the substrate comprises a reagent for the peroxidase.

8. The cartridge of claim 1, wherein the cartridge is configured to measure the target analyte without any cleaning steps or moving parts.

9. The box of claim 1, wherein at least one component of the box is compressible, thereby allowing the box to have an uncompressed state and a compressed state.

10. The cartridge of claim 1, further comprising a metering stack configured to receive a fluid sample containing a target analyte and to dispense the fluid sample along a channel of the cartridge, wherein the channel has a bottom comprising a porous or mesh material, and wherein the metering stack includes one or more vents communicating with the channel.

11. The cartridge of claim 10, wherein a spacer material is disposed between the metering stack and the measuring stack, wherein the spacer material provides a gap between the metering stack and the measuring stack to prevent the target analyte from flowing from the metering stack into the measuring stack when the cartridge is in an uncompressed state.

12. The cartridge of claim 10 or 11, wherein the porous or mesh material allows the target analyte to flow from the metering stack into the determination stack after at least one component of the cartridge is compressed.

13. A method of manufacturing a box, the method comprising: A first complex containing trapping molecules and magnetic beads and a second complex containing detection molecules and detection markers are applied to the first separation layer; Multiple first complexes and multiple second complexes are allowed to dry on the first separation layer; Apply substrate to the detection membrane; Allow the substrate to dry on the detection membrane; and The second separation layer is placed between the first separation layer and the detection membrane; The substrate is configured to interact with the detection marker to trigger a quantifiable reaction in the presence of the target analyte in a fluid sample introduced into the cartridge; The quantifiable reaction corresponds to the amount of the detection molecule present in the detection membrane; and The amount of detection molecules present in the detection membrane corresponds to the amount of target analyte present in the fluid sample. The second separation layer comprises a hydrophobic membrane, a low molecular weight cutoff membrane, or a combination thereof, the pores of the second separation layer being small enough to prevent any unbound second complex from passing through to the detection membrane in the presence of an activated electromagnet, and the second separation layer allowing a third complex containing a target analyte bound to one of (1) the first complex and (2) the second complex to pass through to the detection membrane in the presence of an activated electromagnet.

14. The method of claim 13, further comprising applying a hydrophilic treatment to the second separation layer.

15. The method according to claim 13 or 14, wherein the detection marker comprises peroxidase.

16. The method of claim 15, wherein the substrate comprises a reagent for the peroxidase.

17. A system for collecting a target analyte contained in a fluid sample and measuring the target analyte, the system comprising: An assay stack comprising: a first separation layer; a plurality of first complexes comprising trapping molecules and magnetic beads; a plurality of second complexes comprising detection molecules and detection labels; a second separation layer; and a detection membrane comprising a substrate that interacts with the detection label to initiate a quantitatively quantifiable reaction in the presence of the target analyte, wherein the quantitatively quantifiable reaction corresponds to the amount of detection molecules present in the detection membrane, and wherein the amount of detection molecules present in the detection membrane corresponds to the amount of the target analyte present in the fluid sample; and An electromagnet, used to draw a third complex containing a target analyte bound to one of the first complexes and one of the second complexes through the second separation layer to the detection membrane. The second separation layer comprises a hydrophobic membrane, a low molecular weight cutoff membrane, or a combination thereof, the pores of the second separation layer being small enough to prevent any unbound second complex from passing through to the detection membrane in the presence of an activated electromagnet, and the second separation layer allowing a third complex containing a target analyte bound to one of (1) the first complex and (2) the second complex to pass through to the detection membrane in the presence of an activated electromagnet.

18. The system of claim 17, wherein the system further comprises: A metering stack configured to receive the target analyte and dispense the target analyte along a channel having a bottom comprising a porous or mesh material, and wherein the metering stack includes one or more vents communicating with the channel.

19. The system of claim 17 or 18, wherein the system comprises a measurement reader including the electromagnet and a cartridge including the measurement stack.

20. The system of claim 19, wherein the box is the box of any one of claims 1 to 12.

21. The in vitro use of the cartridge according to any one of claims 1 to 12, for determining a target analyte in a separated fluid sample.

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