METHODS AND SYSTEMS FOR CONCENTRATION OF SAMPLES FOR LATERAL FLOW ASSAYS

Methods and systems for capture concentration of analytes using lectins and other capture ligands are described. For example, stationary phase media functionalized with lectins are used for capture concentration and cleaning of TB lipoarabinomannan (TB LAM) prior to assay on a lateral flow assay (LFA) device, and filtration devices suitable for particulate or bulk capture media are described. Size-exclusion filtration is used to separate particles with captured analyte during washing and concentration steps. Captured analyte can be eluted from stationary phase media prior to application to a LFA or eluted directly onto a customized LFA device that includes a size-selective filter. In various aspects, a size-selective filter on a LFA is used to transfer particulate capture media on a LFA device.

CROSS-REFERENCE TO RELATED APPLICATIONS

PRIORITY APPLICATIONS

All subject matter of the Priority Applications and of any and all applications related to the Priority Applications by priority claims (directly or indirectly), including any priority claims made and subject matter incorporated by reference therein as of the filing date of the instant application, is incorporated herein by reference to the extent such subject matter is not inconsistent herewith.

BACKGROUND

Lateral flow assay (LFA) devices are commonly used to perform assays to detect presence or absence, or in some cases quantity, of an analyte in a sample. For example, lateral flow assays are used to detect hormones indicative of pregnancy or ovulation, infectious disease vectors, and drugs of abuse and various other analytes, e.g., as discussed in U.S. Pat. No. 4,703,017 to Campbell et al.; M. Sajid, A.-N. Kawde, and M. Daud, (2015) “Designs, formats and applications of lateral flow assay: A literature review,” J. Saudi Chem. Soc., Vol. 19, pp. 689-705; and “Rapid Lateral Flow Test Strips: Considerations for Product Development,” Lit. No. TB500EN00EM Rev. C 12/13, © 2013, EMD Millipore Corporation, Billerica, Mass., each of which is incorporated herein by reference.

A sample fluid applied to a lateral flow assay device flows through the assay flow path in response to capillary forces, solubilizing and interacting with dried reagents as it moves though the assay flow path. The analyte of interest binds to a capture reagent immobilized in the assay flow path, resulting in a detectable signal indicating the presence or absence of the analyte. The signal may be visually detectable by a human to provide qualitative information regarding presence or absence of the analyte. Some lateral flow assays are sufficiently sensitive that it is possible to obtain quantitative information regarding the amount of analyte in the sample. In some cases, lateral flow assay devices are used in combination with hand-held or table-top readers. Lateral flow assays frequently are used to perform immunochromatographic tests such as sandwich assays or competitive binding assays, e.g. as discussed in U.S. Pat. No. 4,376,110 to David et al. and U.S. Pat. No. 4,855,240 to Rosenstein et al., both of which are incorporated herein by reference. Other types of assays, using different components for capturing and visualizing analytes of interest may be used instead, for example competitive binding assays, inhibition assays, or serum assays, e.g., as discussed in U.S. Pat. No. 4,703,017 to Campbell et al.; M. Sajid, A.-N. Kawde, and M. Daud, (2015) “Designs, formats and applications of lateral flow assay: A literature review,” J. Saudi Chem. Soc., Vol. 19, pp. 689-705, which is incorporated herein by reference.

The amount of sample that can be put on an LFA is often limited by the physical design of the LFA, rather than by the available sample volume. For example, the physical dimensions and absorptivity of the materials forming the LFA define the amount of fluid that can be handled by the LFA. Overall sensitivity of the LFA can be increased by concentrating samples to reduce the volume of fluid containing the analyte of interest. Another factor that limits the sensitivity of LFAs is interference from substances other than the target analyte that may be present in the sample. This interference can result in background signal from non-specific binding, reduction of detection signal to noise ratio, etc.

The capture, concentration, and washing approaches described herein make it possible to increase the sensitivity of various diagnostic assays, chiefly LFAs, by producing cleaner, more highly concentrated samples. Concentration of analytes (e.g. biomarkers) is achieved by preprocessing dilute samples to capture biomarkers from large-volume samples and performing filtration concentration to obtain a smaller volume sample suitable for application to a LFA. In an aspect, capture, concentration, and washing steps are performed separately from the assay, such that conditions for these steps can be optimized without being limited to conditions required for the assay itself. Originally collected samples (e.g. urine) containing analytes of interest may be highly variable in composition and may include additional components that produce background signal if applied directly to an assay. Washing and elution steps allow the analyte of interest to be delivered to the assay in a clean, concentrated sample from which undesired components have been removed, thus reducing background interference caused by undesired components.

In liquid phase capture methods, target antigens are usually first captured with nanoparticles conjugated (coated) with an antibody or other ligand in the liquid phase. Process parameters are optimized by adjustment of incubation time and/or agitation and mixing in order to overcome kinetic and mass transfer limitations and prevent clumping or aggregation. This is followed by suitable concentration and wash steps.

For example, “magnetic particle capture and elution” is a widely used platform. Magnetic beads functionalized with antibodies to an analyte of interest can be used to capture the analyte of interest, and magnetophoresis used to separate the magnetic beads and captured analyte from bulk liquid to concentrate the sample. However, because magnetic beads tend to clump they are not typically applied directly to a LFA. An elution step is often used to release the captured antigens from the magnetic beads, and the eluent is run on the LFA. However, using size exclusion filtration separation rather than magnetic separation results in fewer material and size requirements for capture beads. Magnetic beads are typically made of dense paramagnetic materials and need to be relatively large (˜<1 um) in order to exhibit sufficient magnetic dipole moment to be manipulated by external magnetic fields easily. In addition, the possibility to use smaller, less dense bead materials may reduce gravity settling issues during incubation and hence less need for mixing.

SUMMARY

In an aspect, a sample filtration container includes, but is not limited to, a base defining a bottom of the sample filtration container; at least one side wall contiguous with the base, the at least one side wall enclosing an interior of the sample filtration container; an opening at a top of the sample filtration container, the opening adapted to receive a sample including a fluid component and a particulate material carried in the fluid component; a divider located within the interior of the sample filtration container and dividing the interior of the sample filtration container into an upper portion and a lower portion, the divider including a size exclusion filter, wherein the size exclusion filter has a first side communicating with the upper portion of the sample filtration container and a second side communicating with the lower portion of the sample filtration container, wherein the size exclusion filter has a pore size adapted to allow passage of the fluid component of the sample while blocking passage of the particulate material; and a capillary medium within the lower portion of the sample filtration container, the capillary medium adapted to draw the fluid component of the sample through the size exclusion filter from the upper portion to the lower portion of the sample filtration container. In addition to the foregoing, other aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

In an aspect, a lateral flow assay device includes, but is not limited to, a loading region adapted to receive a fluid containing a functionalized nanoparticle-captured analyte complex including one or more functionalized nanoparticle and an analyte of interest in a carrier fluid, the loading region including a sample pad; and a filter element overlying the sample pad, wherein the filter element includes pores small enough to block passage of the functionalized nanoparticle through the filter element but large enough to permit passage of the carrier fluid and unbound analyte of interest through the filter element to the sample pad; and a lateral flow membrane downstream of the sample pad and including one or more capture components adapted to capture the analyte of interest. In addition to the foregoing, other aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

In an aspect, a lateral flow assay device includes, but is not limited to, a support layer; an absorbent pad disposed on the support layer; a movable framework configured to fit closely and removably over the absorbent pad; a first filter element supported by the movable framework, the first filter element configured for fluid communication with the absorbent pad through one or more apertures in the movable framework, wherein the first filter element includes pores small enough to block passage of a functionalized nanoparticle-captured analyte complex through the first filter element but large enough to permit passage of a carrier fluid through the first filter element to the absorbent pad; a sample pad supported by the support layer, wherein the sample pad is configured so that the movable framework can be fit closely over the sample pad; and a lateral flow membrane downstream of the sample pad and including one or more capture components specific to the analyte of interest. In addition to the foregoing, other aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

In an aspect, a filtration-concentration device includes, but is not limited to, a filter membrane having a first side and a second side, the filter membrane having pores small enough to block passage of a functionalized nanoparticle-captured analyte complex from the first side to the second side but large enough to permit passage of fluid or unbound analyte from the first side to the second side; a housing configured to contain the filter membrane, the housing having an upstream chamber in fluid communication with the first side of the filter membrane and downstream chamber in fluid communication with the second side of the filter membrane; an inlet port in fluid communication with the upstream chamber, the inlet port adapted to receive a fluid sample containing a functionalized nanoparticle-captured analyte complex in a first volume of the fluid; a fluid outlet port in fluid communication with the downstream chamber, the fluid outlet port configured to permit fluid including a portion of the first volume of fluid to exit the filtration concentration device; and a retentate removal port in communication with the upstream chamber, the retentate removal port configured to allow removal of a retentate from the upstream chamber; wherein the filter membrane is chemically inert with respect to the functionalized nanoparticle-captured analyte complex and the fluid and exhibits little or no non-specific binding to materials in the fluid; wherein the upstream chamber has a volume sufficient to contain a second volume of fluid, wherein the second volume is less than the first volume. In addition to the foregoing, other aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

In an aspect, a capture concentration device includes, but is not limited to, a straight-walled container having an interior surface, a first end, a second end, and an opening at the first end, the straight-walled container adapted to receive a fluid sample including an analyte of interest and a fluid component; and a plunger including a sieve element configured to slidably engage with the interior surface of the straight-walled container and to support a stationary phase medium functionalized with at least one capture ligand adapted to bind an analyte of interest in the fluid sample, the sieve element having openings small enough to block passage of the stationary phase medium but large enough to permit passage of unbound analyte of interest and the fluid component; and a shaft attached to the sieve element and configured to transmit force to the sieve element to drive sliding movement of the sieve element within the straight-walled container. In addition to the foregoing, other aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

In an aspect, a TB LAM filtration device includes, but is not limited to, a stationary phase medium functionalized with at least one lectin adapted to bind a glycan of TB LAM to capture TB LAM from a fluid sample, the fluid sample including the TB LAM and a fluid component; and a sieve element having openings small enough to block passage of the stationary phase medium but large enough to permit passage of unbound TB LAM and the fluid component, wherein the sieve element is formed of a mildly hydrophilic, chemically inert material having minimal nonspecific binding to components of the fluid sample. In addition to the foregoing, other aspects are described in the claims, drawings, and text forming a part of the disclosure set forth herein.

DETAILED DESCRIPTION

Abbreviations and Definitions

As used herein, the abbreviations and terms listed below have the following meanings:

Half strip LFA—an LFA strip that includes assay flow path (lateral flow membrane and capture chemistry) but does not include sample pad, conjugate pad, or functionalized particles.

Functionalized particles—nanoparticles or beads functionalized with capture antibodies, or some other ligand with affinity to target biomarkers or other analytes.

Methods and systems are described which relate to processing of a liquid sample containing a biomarker or other analytes to concentrate the sample prior to further processing, e.g. for detection of the biomarker on a lateral flow assay. For example, sample concentration can be used to improve the sensitivity and effectiveness of a diagnostic assay. In in aspect, methods and systems described herein are used to detect biomarkers (e.g. TB-LAM) that can be bound by lectins. In an aspect, lectins are used as capture ligands. In other aspects, any ligand having selective affinity to a target analyte can be used, including but not limited to antibodies.

In an aspect, concentration is achieved by capturing an analyte from a sample that includes a large volume of a fluid component, separating the captured analyte from the fluid component, and releasing the captured analyte by elution into a smaller volume of a secondary fluid component, which may be applied to a downstream assay. The captured analyte can be washed prior to elution to remove components of the sample that could interfere with detection or contribute background noise.

In various aspects, the analyte is an immunogenic antigen, and the downstream diagnostic assay is a lateral flow assay (LFA). However, methods and devices described herein can be used for pre-processing samples for non-LFA assays and with non-immunogenic biomarkers, provided they can be captured on some sort of suspended solid phase such as nanoparticles, and in some instances be released by use of suitable elution buffers. The foregoing invention addresses these problems by carrying out antigen conjugation to create functionalized particle-captured analyte complexes off-LFA in the bulk liquid phase, followed by filter concentration and wash steps. The output of this process is clean, highly concentrated functionalized particle-captured analyte complexes that can be put directly on a modified version of a half strip LFA. In cases where free biomarkers are needed, they can be eluted from the capture beads with a suitable release buffer, yielding purified and concentrated biomarkers which can then be put on a suitable assay such as an LFA.

Two broad categories of capture-release approaches can be used. In a first approach, a capture ligand is immobilized onto a freely dispersed carrier phase having a large total surface area, e.g. the carrier phase can include nano/micro beads or other particles. This approach enables easy dispersion of the capture phase into the entire volume of the sample, thus reducing mass transfer limitations. However, it requires additional steps to manipulate the freely dispersed phase, e.g. to separate and/or concentrate it. In a second approach, a capture ligand is immobilized in or on a stationary phase which resides in a fixed part of a device and the sample containing analyte is flowed through the stationary phase one or more times. Alternatively, in some aspects the sample containing analyte is incubated with the stationary phase in the absence of flow. In an aspect, the stationary phase includes an open pore capture bed, such as a sponge, and the capture ligand is immobilized in the 3-D bulk of the open pore capture bed. In another aspect, the stationary phase includes a thin membrane (e.g., a coarse filter) that permits flow through of the fluid component but not the carrier phase, and the capture ligand is immobilized on the 2-D surface of the thin membrane.

Analyte Capture Using Particulate Capture Medium:

As noted above, in one approach, analyte of interest is captured using particles functionalized with capture ligands.

FIGS. 1A-1Cillustrates in schematic form components used in capture of analytes.FIG. 1Adepicts analyte100, capture ligand102, and particle104. Analyte100is an analyte of interest that is to be isolated, detected and/or quantified from a fluid sample. In an aspect, analyte100is a biomarker. Capture ligand102is a ligand having specific binding affinity for analyte100. Capture ligand102may be, for example, a lectin or an antibody. Particle104is a particle (e.g., a microbead, a microparticle, a nanoparticle, or a nanobead) that functions as a carrier phase. Particle104may be, for example, a latex, polystyrene, cellulose, e.g., NanoAct™ cellulose nanobeads (Asahi Kasei Corporation), glass, silica, gold, or gold-coated particle. In general, nanoparticles include particles having size typically specified in nanometers (indicating particle diameter) rather than molecular weight. For example, in various aspects, nanoparticles have diameters of between about 10 nm and 800 nm, between about 20 nm and 400 nm, between about 20 nm and about 100 nm, between about 100 nm and about 400 nm, between about 100 nm and about 200 nm, or between about 200 nm and about 400 nm. In various aspects, particles are suitable for use in lateral flow assay or other bead-based assays. As shown inFIG. 1B, in an aspect, particle104is functionalized with capture ligand102to form functionalized particle106. As shown inFIG. 1C, analyte100can bind to functionalized particle106to form functionalized particle-captured analyte complex108.

General Method for Analyte Capture Using Particulate Capture Medium

FIGS. 2A-2Hillustrate a generalized method for capture and concentration of an analyte. InFIG. 2A, a volume of a sample200containing an analyte100is collected. In an aspect, the sample is large (e.g. 20 ml) relative to the volume of fluid that can be readily analyzed on a lateral flow assay (e.g. 100 μl). Sample200includes analyte100in fluid component204. Sample200is collected in sample container206(e.g., a sample collection cup) and mixed with functionalized particles106, as shown inFIG. 2B. Functionalized particles106are as described in connection withFIGS. 1A-1C. In an aspect, functionalized particles106are present in sample container206prior to collection of sample200. In other aspects, functionalized particles106are added to sample container206at the same time as sample200, or subsequent to addition of sample200. Sample200is incubated with functionalized particles106for an incubation period and under an incubation condition sufficient to produce binding of analyte100to the functionalized particles to produce functionalized particle-captured analyte complex108, as depicted inFIG. 2C. By performing the capture steps inFIGS. 2B-2Cprior to applying the sample to an LFA, the volume of sample that can be exposed to the functionalized particles is much greater than the volume that could be put on an ordinary LFA. Accordingly, as much of the dilute analyte as possible is captured on the functionalized particles to form functionalized particle-captured analyte complexes, and the volume of sample that can be processed is not constrained by the specific design of the LFA. Various incubation parameters can be selected to optimize complex formation (e.g., incubation time, presence of agitation/mixing, buffer chemistry, etc.). It will be appreciated that by performing the incubation prior to applying the sample to the LFA, there are fewer constraints on how the incubation is performed.

Once the functionalized particle-captured analyte complexes are formed within the fluid volume including the original sample combined with the functionalized particles, they are concentrated down to a much smaller volume that is compatible with the LFA. This is accomplished by size exclusion filtration that retains the functionalized particle-captured analyte complexes but allows passage of the remaining fluid component. A concentrated dispersion of functionalized particle-captured analyte complexes suspended in a small amount of residual fluid component is obtained. As shown inFIG. 2D, fluid component204, containing functionalized particle-captured analyte complex108, as well as any unbound analyte100and/or unbound functionalized particles106is transferred to separation structure208. Separation structure208includes filter210which has a pore size large enough to permit passage of fluid component204and unbound analyte100but not functionalized particle-captured analyte complex108. Accordingly, separation structure208is used to separate functionalized particle-captured analyte complex108, as well as any unbound functionalized particles106(not shown inFIG. 2D) from fluid component204, and unbound analyte100carried in fluid component204. In an aspect separation structure208also includes walls212which help to contain fluid component204. As shown inFIG. 2D, a small amount of fluid component204may remain with functionalized particle-captured analyte complex108in separation structure208after separation, but the amount of analyte (bound in functionalized particle-captured analyte complex108) within separation structure208is significantly concentrated relative to in the original sample200. The fluid component in which the functionalized particle-captured analyte complexes are suspended may contain background producing substances that cause interference and negatively affect the assay sensitivity. Accordingly, in some aspects it is desirable to perform a wash step. This may be done by adding a volume of a compatible buffer to the functionalized particle-captured analyte complexes and again perform size exclusion filtration to retain the functionalized particle-captured analyte complexes on the filter while wash buffer along with any background producing substances passes through the filter and can be discarded.FIG. 2Edepicts a wash step in which wash buffer214is added to separation structure208, to wash away remaining fluid component204from functionalized particle-captured analyte complex108. One or more wash steps can be carried out, with excess wash buffer214passing through filter210, leaving functionalized particle-captured analyte complex108surround by a small amount of residual wash buffer214, as depicted inFIG. 2F. InFIG. 2G, a small amount of elution buffer216is added to separation structure208, to replace wash buffer214. Elution buffer216is incubated with functionalized particle-captured analyte complex108for an elution period and under an elution condition sufficient to elute analyte100from functionalized particle106, such that, as shown inFIG. 2H, elution buffer216containing analyte100can pass through filter210and be separated from functionalized particle106, which remains in separation structure208.

Concentrated sample218, made up of analyte100in elution buffer216, is significantly concentrated relative to the originally collected sample200. Concentrated sample218is clean and in a small volume suitable for application to a lateral flow assay or other analysis device.

It will be appreciated the relative sizes of the first volume of sample200and the second volume of concentrated sample218determine how much the sample is concentrated. For example, if the first volume of sample fluid is twice the second volume of elution buffer, the concentration of TB LAM in the elution buffer will be twice the concentration of TB LAM in the sample fluid.

The total number of molecules=V1C1=V2C2

Where V1=volume of the body fluid sample containing TB LAM, C1is the concentration of TB LAM in the body fluid sample, V2is the volume of elution buffer containing eluted TB LAM, and C2is the concentration of TB LAM in elution buffer.

The following equation can be used to describe the concentration obtained:

As an example, if the body fluid sample is urine, a typical sample size is roughly 10 ml to roughly 50 ml. It may be possible to load up to 5 ml of fluid onto a lateral flow assay, but it is more typical that lateral flow assays can accommodate smaller samples, e.g. between about 50 μl and about 400 μl, or between about 100 μl and about 200 μl. Thus, it is desirable to concentrate an analyte from a urine sample anywhere from about 50-fold to about 500-fold.

In some aspects, analytes are eluted into an elution buffer, as shown inFIGS. 2A-2H, to produce a concentrated sample218that can be applied to an assay as described in connection withFIGS. 3A-3C. Alternatively, as will be described in greater detail in connections with e.g.FIGS. 5A-5C, functionalized particle-captured analyte complex108can be removed from separation structure208and applied directly to a downstream assay. Such an approach is dependent on the use of particles suitable for application to a LFA or other bead-based assay.

FIGS. 3A, 3B, and 3Cillustrate an example of processing of concentrated sample218(obtained through the approach illustrated inFIGS. 2A-2H) in a sandwich assay performed in a lateral flow format. InFIG. 3A, a concentrated sample218containing analyte of interest100in elution buffer216is applied at first end304of lateral flow assay device306, and moves via capillary forces to second end308, in the direction indicated by the heavy black arrow. Lateral flow assay device306includes a conjugate region310containing conjugate antibodies312specific to analyte100. Conjugate antibodies312are conjugated to one or more detectable component314, which may be, for example, latex beads, colloidal gold particles, other colloidal metals, colloidal carbon, fluorescent or luminescent labels, quantum dots, upconverting phosphores, bioluminescent markers, enzymes, magnetic or paramagnetic particles, dyes, electroactive compounds, or other suitable labels or markers. (Peter Chun, “Chapter 5. Colloidal Gold and Other Labels for Lateral Flow Immunoassays”, in Lateral Flow Immunoassay, Raphael C. Wong and Harley Y. Tse, Editors, © 2009 ISBN: 978-1-58829-908-6 e-ISBN: 978-1-59745-240-3 DOI 10.1007/978-1-59745-240-3, and “Rapid Lateral Flow Test Strips: Considerations for Product Development,” Lit. No. TB500ENOOEM Rev. C 12/13, © 2013, EMD Millipore Corporation, Billerica, Mass., both of which are incorporated herein by reference). In an aspect, detectable components are contained within and subsequently released from liposomes. Lateral flow assay device306also includes a test line316, containing antibodies318immobilized on the material forming the assay flow path, and a control line320containing antibodies322, which are specific to conjugate antibodies312.

FIG. 3Bdepicts lateral flow assay device306after sufficient time has passed for elution buffer216to spread to fill first end304of lateral flow assay device and travel downstream through lateral flow assay device306, to the location of flow front326. As elution buffer216passes conjugate region310, it solubilizes conjugate antibodies312and interacts with them to form bound conjugate antibodies328, which are bound to analyte100. Some of conjugate antibodies312remain unbound.

FIG. 3Cdepicts lateral flow assay device306after flow front326of elution buffer216has travelled to second end308of lateral flow assay device306. As elution buffer216travels past test line316, analyte100with bound conjugate antibodies328is bound by antibodies318. Unbound conjugate antibodies312bind to antibodies322at control line320. Detectable component314conjugated to bound conjugate antibodies328at test line316, and unbound conjugate antibodies312at control line320, produces a detectable signal that indicates presence of analyte100(at test line316) and presence of properly functioning assay components (at control line320), respectively.

It will be appreciated that for operation of the assay as depicted inFIGS. 3A-3C, elution buffer216is preferably suitable for eluting analyte100from functionalized particle106(inFIG. 2H) and also compatible with the detection chemistry on the lateral flow assay device306. As an alternative, if the elution buffer is not optimized for the assay chemistry, an additional running buffer having properties better suited to the assay chemistry may be applied to the lateral flow assay device for moving analyte100through the lateral flow assay device to test line316.

All diagnostic assays have constraints relating to the nature and the volume of the sample that is introduced to them, as well as the manner in which they are introduced. For example, in a typical lateral flow assay, a small amount of liquid sample (concentrated sample218inFIGS. 3A-3C), having a volume of between about 50 μl and about 100 μl, is applied to a sample pad at first end304, sometimes followed by a running buffer. Sample then wicks along the device, encountering different regions along the way. A typical LFA contains nanoparticles (detectable component314) functionalized with conjugate antibodies312specific to the target analyte being tested. If the target analyte is present in the sample, it is captured onto the functionalized nanoparticles to form functionalized particle-captured analyte complexes. Nanoparticles carrying the target antigens then flow downstream and are captured and concentrated at test line316. A suitable signal (visual contrast, fluorescence, thermal response etc.) that is generated in the presence of functionalized particle-captured analyte complexes at the test line is measured/observed to establish the presence of the analyte in the original sample.

Syringe Filter Concentration Device for Use with Particulate Capture Medium

As noted herein above, one approach to obtaining concentrated, purified analyte, as depicted generally inFIGS. 2A-2H, is to capture it with functionalized beads, wash the functionalized particle-captured analyte complexes to remove extraneous materials while bound to the beads, and elute the analyte from the beads so that washed, concentrated, unbound analyte can be applied to an LFA.

An example system and its use in concentration, washing, and elution steps is depicted inFIGS. 4A-4M. This system is carried out with a luer lock syringe in combination with a small disk filter. InFIG. 4A, sample solution400containing analyte402in fluid component404, is placed in container406, as are functionalized particles408made up of particles410functionalized with capture ligand412, and incubated atFIG. 4B, which is generally as described in connection withFIGS. 2A-2C. Fluid component404, containing functionalized particle-captured analyte complex414(as well as any unbound analyte402and/or unbound functionalized particles408) is drawn into syringe420from container406, as shown inFIG. 4C. Syringe420includes removable plunger422which provides easy access to barrel424.

A depicted inFIG. 4D, syringe420mates with inlet port426on filter housing428of filtration concentration device430. Filtration-concentration device430includes a filter membrane432having a first side434and second side436. Filter membrane432has pores small enough to block passage of a functionalized nanoparticle-captured analyte complex414from first side434to second side436but large enough to permit passage of fluid or unbound analyte from first side434to second side436. Filtration-concentration device430also includes housing440, which is configured to contain filter membrane432. Housing440has an upstream chamber442in fluid communication with the first side434of filter membrane432and downstream chamber444in fluid communication with second side436of filter membrane432. Filter membrane432is designed with an optimum holdup (dead) volume to yield the right sample volume for the downstream assay (e.g. 100 μl). Filter membrane432can be formed from various materials, including, for example, at least one of organic membranes made up of one or more polymers, as cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber, having pore size selected to block passage of functionalized particles408.

Inlet port426is in fluid communication with upstream chamber442and is adapted to receive fluid sample containing a functionalized nanoparticle-captured analyte complex414in a first volume of fluid component404. Filtration concentration device430includes fluid outlet port450in fluid communication with downstream chamber444. Fluid outlet port450is configured to permit fluid including a portion of the first volume of fluid to exit filtration concentration device430. In addition, filtration concentration device430includes retentate removal port448in communication with upstream chamber442. Retentate removal port448is configured to allow removal of a retentate from the upstream chamber. For example, this can be used to recover functionalized particles408for reuse. In an aspect, retentate removal port448includes valve452which remains closed except when it is desired to remove materials via retentate removal port448. Fluid outlet port450and retentate removal port448may include luer lock connectors or other connectors that allow for convenient connection of filtration-concentration device430with syringe420, or with other components. Filter membrane432is chemically inert with respect to the functionalized nanoparticle-captured analyte complex414and fluid component404and exhibits little or no non-specific binding to materials in fluid component404. Upstream chamber442has a volume sufficient to contain a second volume of fluid, wherein the second volume is less than the first volume. The fluidic design of the filtration-concentration device430, especially the upstream side compartment, may be such that it maximizes the recovery of the concentrated (and optionally also washed) functionalized particle-captured analyte complexes.

InFIG. 4F, syringe420(or alternatively, another syringe device) containing wash buffer460is connected to filtration concentration device430. Plunger422is depressed to push wash buffer460through functionalized nanoparticle-captured analyte complex414on filter membrane432, driving residual fluid component404through fluid outlet port450, until as shown inFIG. 4G, functionalized nanoparticle-captured analyte complex414on filter membrane432is cleaned and surrounded by wash buffer460. Wash buffer460can be, for example, phosphate buffered saline (PBS). As depicted inFIGS. 4H and 4I, a syringe420can be used to push air464through filtration concentration device430to drive remaining wash buffer460out of upstream chamber442and out of fluid outlet port450, leaving substantially dry functionalized nanoparticle-captured analyte complex414on filter membrane432in upstream chamber442.

In cases where the target analyte needs to be freed from the functionalized particle-captured analyte complexes414following concentration and washing as described above, an elution or release buffer can next be pushed through the bed of functionalized particle-captured analyte complexes releasing the analyte. InFIGS. 4J and 4K, syringe420is used to push a small volume of elution buffer466through filtration concentration device430. Analyte402is eluted from functionalized particles408, and carried through filter membrane432and exits filtration concentration device430via fluid outlet port450, carried in elution buffer466. Elution buffer466with analyte402can be applied to a lateral flow assay or other assay for detection, as described elsewhere herein.

FIGS. 4L and 4Millustrate retrieval of functionalized particles408for reuse. A first syringe420is connected to inlet port426, and a second syringe468is connected to retentate removal port448. Fluid outlet port450is closed, e.g. by closing a valve470. Suitable carrier fluid (e.g., wash buffer460) is injected into upstream chamber442using first syringe420, plunger472of second syringe468is simultaneously withdrawn to draw functionalized particles408in wash buffer460into second syringe468, and captured for reuse.

In some aspects, functionalized particles408are suitable to be used in a lateral flow assay or other bead-based assay, in that they include a detectable component (e.g., latex beads, colloidal gold particles, other colloidal metals, colloidal carbon, fluorescent or luminescent labels, quantum dots, upconverting phosphores, bioluminescent markers, enzymes, magnetic or paramagnetic particles, dyes, electroactive compounds, or other suitable labels or markers, as described above in connection withFIGS. 3A-3C). Thus it is not necessary to elute analyte402off of the functionalized particles. In such applications, functionalized particle-captured analyte complexes414can be removed from filtration concentration device430following washing, e.g. atFIG. 4G or 4I, and a dual syringe setup like that used inFIGS. 4L and 4Mcan be used to retrieve functionalized particle-captured analyte complexes414in a buffer suitable for application to a lateral flow assay.

Application of functionalized particle-captured analyte complexes414directly to a half-strip lateral flow assay device500is illustrated inFIGS. 5A-5C. Half-strip lateral flow assay device500is a lateral flow assay without the sample pad or conjugate pad or functionalized particles present. For example, functionalized particle-captured analyte complexes414(made up of functionalized particle408and captured analyte402) can be removed from separation structure208, following removal of wash buffer214, e.g., as depicted inFIG. 2F, orFIG. 4G or 4I. InFIG. 5A, sample502containing functionalized particle-captured analyte complexes414in carrier fluid504(which may be wash buffer or running buffer) is applied at first end506of half strip lateral flow assay device500, and moves via capillary forces to second end508, in the direction indicated by the heavy black arrow. In this case, half strip lateral flow assay device500does not include a conjugate pad containing antibodies specific to analyte402; instead, functionalized particles408in functionalized particle-captured analyte complexes414include a detectable component, as noted above. Half strip lateral flow assay device500does, however, contain antibodies318that are specific to analyte402, immobilized on the material forming the assay flow path, which may be localized at a test line316as depicted inFIG. 5A. In an aspect, half strip lateral flow assay device500also includes a control line320containing antibodies322, which are specific to functionalized particles408.

FIG. 5Bdepicts half strip lateral flow assay device500after sufficient time has passed for carrier fluid504to spread to fill first end506of lateral flow assay device and travel downstream through lateral flow assay device500, to the location of flow front526.

FIG. 5Cdepicts half strip lateral flow assay device500after flow front526of carrier fluid504has travelled to second end508of half strip lateral flow assay device500. As carrier fluid504travels past test line316, functionalized particle-captured analyte complexes414are bound by antibodies318. Unbound functionalized particles408bind to antibodies322at control line320. Functionalized particle-captured analyte complexes414captured at test line316, and functionalized particles408at control line320both include detectable components that produce a detectable signal that indicates presence of analyte402(at test line316) and presence of properly functioning assay components (at control line320), respectively.

Sample Filtration Container for Use with Particulate Capture Medium

FIGS. 6A-6Bdepicts a sample filtration container that can be used to concentrate nanoparticles with captured antigen, which can then be transferred to a half strip LFA as illustrated inFIGS. 5A-5C.FIG. 6Adepicts a sample filtration container600that includes a base602defining a bottom of sample filtration container600, at least one side wall604. Side wall604is contiguous with base602, and encloses interior606of sample filtration container600. Sample filtration container600includes opening608at a top610of sample filtration container600. Opening608is adapted to receive a sample614including a fluid component616and a particulate material618carried in the fluid component, as depicted inFIG. 6A. Particulate material618may include, for example, functionalized particles that can be incubated with an analyte-containing sample fluid to form functionalized particle-captured analyte complexes, previously-formed functionalized particle-captured analyte complexes, or other types of particulate materials, without limitation. Sample filtration container600includes divider620located within the interior of the sample filtration container, which divides the interior of sample filtration container600into an upper portion622and lower portion624. Divider620includes a size exclusion filter626, wherein the size exclusion filter626has a first side628communicating with upper portion622of sample container600and a second side630communicating with lower portion624of sample container600. Size exclusion filter626has a pore size adapted to allow passage of the fluid component616of sample614while blocking passage of the particulate material618. In an aspect, size exclusion filter626is formed from various materials, including, for example, at least one of organic membranes made up of one or more polymers, as cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber, having pore size selected to block passage of functionalized particles408. Sample filtration container600also includes a capillary medium632within lower portion624of sample container600. Capillary medium632is adapted to draw fluid component616of sample614through size exclusion filter626from the upper portion622to the lower portion624of the sample container. Capillary medium632may retain the fluid component within the lower portion of the sample container. Capillary medium632includes, for example, a cellulosic material, a fiber-based material, a glass fiber, a sponge, a resin, a superabsorbent polymer (e.g., sodium polyacrylate or potassium polyacrylate), or a hydrogel.

In an aspect, a flow control feature634is located between upper portion622and lower portion624, either above or below size exclusion filter626. InFIG. 6A, flow control feature634is shown below size exclusion filter626. In the example ofFIG. 6A, flow control feature634is a dissolvable sacrificial layer (formed from, e.g., a sugar or pullulan). Alternative, the flow control feature may be a mechanically actuated valve or a mechanism that keeps upper portion622and lower portion624physically separate and clicks them together on demand. Flow control feature634is used when it is desirable to keep upper portion622and lower portion624physically separate so that fluid component616is retained in upper portion622long enough to allow incubation for analyte capture to occur. In the event that capture incubation occurs rapidly, or if it the incubation step is performed in another container and already-formed capture bead-analyte complex is poured into upper portion622, then flow control feature634may be omitted.

As shown inFIG. 6B, after flow control feature634has dissolved (or been opened in some other manner), fluid component616flows through size exclusion filter626into lower portion624, drawn by capillary forces exerted by capillary medium632, while particulate material618remains in upper portion622, concentrated into a small residual volume of fluid component616. If desired, a wash buffer can be added to sample filtration container600. The wash buffer will similarly be drawn into the capillary medium632leaving concentrated, cleaned particles in upper portion622. Particles can be manually removed for further processing, e.g. for elution of analyte.

As depicted inFIGS. 7A and 7B, in an aspect a sample filtration container700, includes sample collection region702located at a bottom of the upper portion704of the sample filtration container700, where sample collection region702has a reduced cross-sectional area relative to the upper portion704as a whole. Upper portion704is separated from lower portion706by divider710, which in this case is substantially planar and oriented at an oblique angle relative to side wall712. Sample collection region702is bounded by the at least one side wall and the divider at a location714(circled inFIG. 7A) at which the divider710forms an acute angle with side wall712. It should be noted that as shown inFIGS. 7A and 7B, divider710is substantially but not absolutely planar; it is formed by the combination of size exclusion filter716and wall extension718, which in this example are not co-planar. As shown inFIG. 7B, fluid component720is drawn across size exclusion filter716into capillary medium722, while particulate material724collects in sample collection region702. Particulate material724can be removed, for example, by pipetting.FIG. 7Cdepicts an alternative sample filtration container750in which size exclusion filter752functions as a divider. Capillary medium is indicated at754. Sample filtration containers600,700and750depicted inFIGS. 6A-6B and 7A-7Care depicted only in cross section. Containers600and750may take the form of a cylinder or rectangular prism. Container700may have a rectangular, circular, or ovoid cross-section at section lines A-A or B-B. The particular dimensions of the containers can be selected for ease of manufacture, storage, or handling, and/or to provide desired absolute and relative volumes of the upper and lower portions of the containers. For example, container700provides for a relatively larger volume of capillary medium722in lower portion706compared to the volume of fluid that can be contained in upper portion704. This may be useful, for example, if capillary medium722is intended to have the capacity to absorb fluid components of both sample and wash buffer.

In other aspects, not shown, a divider may include a shaped depression, with the sample collection region located at the bottom of the shaped depression. For example, the divider may have an inverted conical shape, in which vertex of the cone forms the depression in which particulate material collects. Various other configurations of divider710can be utilized to form a reduced cross-section sample collection region. In some aspects, the divider can be configured to be removed from the sample filtration container, and can serve as a transfer device for the concentrated beads.

Method of Using Sample Filtration Container

FIG. 8depicts a method of using a sample filtration container of the type depicted inFIGS. 6A-6B and 7A-7C. In an aspect, a method800of filtering a sample includes adding a sample including a fluid component and a particulate material into a sample filtration container, the sample filtration container including a base defining a bottom of the sample filtration container; at least one side wall contiguous with the base, the at least one side wall enclosing an interior of the sample container; a divider located within the interior of the sample container and dividing the interior of the sample container into an upper portion and a lower portion, the divider including a size exclusion filter, wherein the size exclusion filter has a first side communicating with the upper portion of the sample container and a second side communicating with the lower portion of the sample container, wherein the size exclusion filter has a pore size adapted to allow passage of the fluid component of the sample while blocking passage of the particulate material; a sample collection region located at a bottom of the upper portion of the sample container; and a capillary medium within the lower portion of the sample container, the capillary medium adapted to draw the fluid component of the sample through the size exclusion filter from the upper portion to the lower portion of the sample container, as indicated at802; allowing the fluid component of the sample to be drawn through the size exclusion filter and into the capillary medium, as indicated at804; and removing filtrate including the particulate material from the sample collection region located at a bottom of the upper portion of the sample container, as indicated at806.

Methods of Filtering Particulates Applied to LFA

In an aspect, after analyte of interest is capture on a functionalized nanoparticle, functionalized nanoparticle-captured analyte complexes are applied directly to an LFA, rather than first eluting the analyte from the functionalized nanoparticle and applying a solution containing the eluted analyte to the LFA.FIG. 9is a flow diagram of such a method. As shown inFIG. 9, a method900of detecting a biomarker of interest from a fluid sample includes collecting a fluid sample including a fluid component containing a biomarker, as indicated at902; incubating the fluid sample with a plurality of functionalized nanoparticles for an incubation period sufficient to produce binding of at least a portion of the biomarker with the functionalized nanoparticles to form functionalized nanoparticle-captured biomarker complexes, the functionalized nanoparticles including nanoparticles functionalized with one or more ligands having an affinity to the biomarker, as indicated at904; filtering the fluid sample and functionalized nanoparticle-captured biomarker complexes with a size-exclusion filter to separate the functionalized nanoparticle-captured biomarker complexes from the fluid component, as indicated at906; transferring the functionalized nanoparticle-captured biomarker complexes to a lateral flow assay device, as indicated at908; and detecting the biomarker at a test line of the lateral flow assay device, the test line including antibodies specific to the biomarker and adapted to bind the biomarker at the test line, as indicated at910. In an aspect, method900includes washing the functionalized nanoparticle-captured biomarker complexes on the size-exclusion filter with a wash buffer prior to transferring the functionalized nanoparticle-captured biomarker complexes to the lateral flow assay device, for example as described generally in connection withFIGS. 2E and 2F.

In aspect, the lateral flow assay device is a half-strip lateral flow assay device, and wherein detecting the biomarker at the test line includes detecting the functionalized nanoparticle-biomarker complex at the test line, wherein the functionalized nanoparticle includes a detectable component, as indicated at912. This approach is illustrated inFIGS. 5A-5C). In this approach, the same nanoparticles used for capture-concentration of the biomarker are also used for detection on the lateral flow assay.

In another aspect, functionalized nanoparticle-biomarker complex is applied to an LFA and elution of analyte is performed on the LFA.FIG. 10depicts such an approach, which is a variant of the general method shown inFIG. 9. Method steps902-908are as discussed in connection withFIG. 9. Method1000includes eluting the biomarker from the functionalized nanoparticle on the lateral flow assay device, as indicated at1002. In general, this approach involves filtration of functionalized nanoparticle-biomarker complex using a filter attached to or built into the LFA cassette, and elution of analyte from nanoparticles performed on the LFA. For example, following elution of analyte on the LFA, the method includes exposing the eluted biomarker to an antibody specific to the biomarker on the lateral flow assay device, wherein the antibody specific to the biomarker is conjugated to a detectable component, and wherein detecting the biomarker at a test line includes detecting the detectable component.

In an aspect, the lateral flow assay device includes a size exclusion filter overlying a sample pad, the exclusion filter having pore size sufficient to permit passage of the biomarker but not the functionalized nanoparticle, and wherein transferring the functionalized nanoparticle-captured biomarker complexes to the lateral flow assay device includes applying the functionalized nanoparticle-captured biomarker complexes to the size exclusion filter, as indicated at1004.

In another aspect of method1000, the lateral flow assay device includes a filter element overlying an absorbent pad, the filter element supported by a movable framework and having pore size sufficient to permit passage of fluid and unbound analyte but not the functionalized nanoparticle, and transferring the functionalized nanoparticle-captured biomarker complexes to the lateral flow assay device includes applying the functionalized nanoparticle-captured biomarker complexes to the filter element; and wherein the method further includes allowing excess fluid associated with the functionalized nanoparticle-captured biomarker complexes to pass through first filter element and be absorbed by the absorbent pad, and transferring the filter element on the movable framework to a sample pad of the lateral flow assay device; wherein eluting the biomarker from the functionalized nanoparticle includes eluting the biomarker into the sample pad through the filter element, as indicated at1006.

FIG. 11depicts additional aspects of a related method1100(in which steps902-908and1002are as discussed herein above). In an aspect of method1100, the lateral flow assay device includes a first filter element overlying an absorbent pad, the first filter element supported by a movable framework attached to a hinge element, the first filter element and having pore size sufficient to permit passage of fluid but not the functionalized nanoparticle, and wherein transferring the functionalized nanoparticle-captured biomarker complexes to the lateral flow assay device includes applying the functionalized nanoparticle-captured biomarker complexes to the first filter element; and wherein the method further includes allowing excess fluid associated with the functionalized nanoparticle-captured biomarker complexes to pass through the first filter element and be absorbed by the absorbent pad, and moving the first filter element from a position over the absorbent pad to a position over a second filter element overlying a sample pad of the lateral flow assay device by rotating the movable framework around an axis of the hinge element to flip the first filter element over, wherein eluting the biomarker from the functionalized nanoparticle includes applying an elution buffer to the first filter element and allowing the elution buffer to pass through the first filter element, eluting the biomarker into the sample pad through the second filter element, as indicated at1102.

In various aspects, the eluent flows from the filter to the LFA via a flow path that is connected manually (e.g., by sliding, hinging, clicking, depressing, pull tab) or automatically, e.g. through swelling of a wet absorbing material, removal of a dissolvable flow barrier (complex sugar or such), or opening of a surface tension valve.

LFA with Particle Filter

As discussed herein above, in some cases the concentrated functionalized particle-captured analyte complex are placed directly onto a specially designed LFA having a coarse membrane on the sample pad to retain the beads but permit passage of the liberated analyte.

FIGS. 12A-12Cdepict an LFA which includes a concentration/separation portion adapted for receiving functionalized particle-captured analyte complex, e.g. from a sample filtration container as depicted inFIGS. 6A and 6BandFIGS. 7A-7C. Lateral flow assay device1200includes a loading region1202including sample pad1204. Loading region1202is adapted to receive a fluid1206containing a functionalized nanoparticle-captured analyte complex1208including one or more functionalized nanoparticle1210and an analyte of interest1212in a carrier fluid1214.

Lateral flow assay device1200includes filter element1216overlying sample pad1204, wherein the filter element1216includes pores small enough to block passage of the functionalized nanoparticle1210through the filter element1216but large enough to permit passage of the carrier fluid1214and unbound analyte of interest1212through filter element1216to sample pad1204. In an aspect, filter element1216is formed from various materials, including, for example, at least one of organic membranes made up of one or more polymers, as cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber. In addition, lateral flow assay device1200includes lateral flow membrane1220downstream of sample pad1204and including one or more capture components1222adapted to capture analyte of interest1212. In an aspect, lateral flow assay device1200also includes conjugate pad1224, wick1226, and backing1228. In an aspect, lateral flow membrane1220includes test line1230including the one or more capture components1222. In some aspects, lateral flow membrane1220also includes control line1232.

As shown inFIG. 12A, functionalized particle-captured analyte complex1208in carrier fluid1214is applied to filter element1216. Carrier fluid1214may be a wash buffer from a previous processing step, for example. Carrier fluid1214passes through filter element1216and is absorbed into sample pad1204, while functionalized particle-captured analyte complex1208remains on top of filter1216. InFIG. 12B, elution buffer1224is applied to functionalized particle-captured analyte complex1208on filter element1216. As shown inFIG. 12C, analyte1212is eluted from functionalized particle1236and carried into sample pad1204by elution buffer1234. Analyte1212in elution buffer1224travels through conjugate pad1224and lateral flow membrane1220where it is captured at test line1230. Elution buffer1234(or other running buffer) and any uncaptured analyte1212eventually travel toward and into wick1226. Sample pad1204functions to control the rate at which sample fluid enters conjugate pad1224. Sample pad1204may contain proteins, detergents, viscosity enhancers, buffers, salts, or other materials that improve the properties of the sample fluid. In an aspect, the one or more capture components1222include one or more capture component adapted to capture TB LAM. In an aspect, the one or more capture components1222include one or more antibody adapted to capture TB LAM. In an aspect, the sample pad1204is capable of absorbing about 5 ml. In various aspects, the sample pad1204includes at least one of cellulose, glass fiber, cotton, rayon, a woven mesh, and a synthetic non-woven material (see, e.g., Brendan O'Farrell, “Chapter 1. Evolution in Lateral Flow-Based Immunoassay Systems”, in Lateral Flow Immunoassay, Raphael C. Wong and Harley Y. Tse, Editors, © 2009 ISBN: 978-1-58829-908-6 e-ISBN: 978-1-59745-240-3 DOI 10.1007/978-1-59745-240-3, and “Rapid Lateral Flow Test Strips: Considerations for Product Development,” Lit. No. TB500ENOOEM Rev. C 12/13, © 2013, EMD Millipore Corporation, Billerica, Mass., both of which are incorporated herein by reference). For example, suitable materials include, but are not limited to, SureWick® glass fiber pads and cellulose pads from EMD Millipore, Billerica, Mass. and CF1 to CF7 100% cotton linter pads from GE Healthcare Biosciences, Pittsburgh, Pa.). In an aspect, the filter element1216has a pore size of between about 0.1 μm and about 0.4 μm. Choice of a suitable pore size is dependent on the size of functionalized nanoparticle1210. In an aspect, the filter element1216is formed of a chemically inert material having minimal nonspecific binding to components of the fluid. In an aspect, the filter element1216is formed of a mildly hydrophilic material. In an aspect, conjugate pad1224contains dried conjugate (e.g., conjugate antibodies and detectable component as discussed in connection withFIGS. 3A-3C). In various aspects, conjugate pad1224is formed from glass fiber, polyesters, cotton, or rayon, e.g. as discussed in “Rapid Lateral Flow Test Strips: Considerations for Product Development,” Lit. No. TB500ENOOEM Rev. C 12/13, © 2013, EMD Millipore Corporation, Billerica, Mass., which is incorporated herein by reference. In an aspect, lateral flow membrane1220is a porous membrane with well-defined capillary flow properties that provides a uniform and controlled flow of fluid to test line1230and control line1232. In various aspects, lateral flow membrane materials include nitrocellulose, polyvinylidene fluoride, charge-modified nylon, polyether sulfone, nitrocellulose acetate, glass fiber, cellulose, paper, silica, a porous synthetic polymer, polyester, nylon, cotton, a sintered material, a woven material, or a non-woven material. Lateral flow membrane materials may be treated with surfactant to improve the wettability of the membrane. (See, e.g. Michael A. Mansfield, “Chapter 6. Nitrocellulose Membranes for Lateral Flow Immunoassays: A Technical Treatise”, in Lateral Flow Immunoassay, Raphael C. Wong and Harley Y. Tse, Editors, © 2009 ISBN: 978-1-58829-908-6 e-ISBN: 978-1-59745-240-3 DOI 10.1007/978-1-59745-240-3; E. J. Flynn, J. Arndt, L. Brothier, and M. A. Morris (2013), “Control of pore structure formation in cellulose nitrate polymer membranes,” Advances in Chem. Science., Vol. 2, Issue 2, June 203, pp. 9-18; and “Rapid Lateral Flow Test Strips: Considerations for Product Development,” Lit. No. TB500EN00EM Rev. C 12/13, © 2013, EMD Millipore Corporation, Billerica, Mass., each of which is incorporated herein by reference, for discussion of lateral flow membrane and conjugate pad materials).

In an aspect, test line1230contains immobilized antibodies specific to the analyte of interest, bound irreversibly to lateral flow membrane1220, and a control line1232contains immobilized antibodies specific to conjugate antibodies. Wick1226(an absorbent pad) is located at the downstream end of lateral flow membrane1220. In various aspects, wick1226includes at least one of cellulose, high-density cellulose, glass, polyester, nylon, cotton, mono-component fiber, or bi-component fiber (see, e.g., Brendan O'Farrell, “Chapter 1. Evolution in Lateral Flow-Based Immunoassay Systems”, in Lateral Flow Immunoassay, Raphael C. Wong and Harley Y. Tse, Editors, © 2009 ISBN: 978-1-58829-908-6 e-ISBN: 978-1-59745-240-3 DOI 10.1007/978-1-59745-240-3, and “Rapid Lateral Flow Test Strips: Considerations for Product Development,” Lit. No. TB500ENOOEM Rev. C 12/13, © 2013, EMD Millipore Corporation, Billerica, Mass., both of which are incorporated herein by reference).

In an aspect, sample pad1204, conjugate pad1224, lateral flow membrane1220, and wick1226are formed on backing1228. In various aspects, backing1228includes a non-porous plastic film or card, including one or more of polystyrene, vinyl (poly vinyl chloride or PVC), or polyester. In various aspects, backing1228includes an adhesive, which may be covered by a release liner. Thickness of the backing may be for example 0.0005 to 0.015 inches, with thicker materials typically used for stand-alone test strips, while thinner materials may be used in a holder or housing (see, e.g. Jennifer S. Ponti, “Chapter 3. Material Platform for the Assembly of Lateral Flow Immunoassay Test Strips”, in Lateral Flow Immunoassay, Raphael C. Wong and Harley Y. Tse, Editors, © 2009 ISBN: 978-1-58829-908-6 e-ISBN: 978-1-59745-240-3 DOI 10.1007/978-1-59745-240-3, and “Rapid Lateral Flow Test Strips: Considerations for Product Development,” Lit. No. TB500ENOOEM Rev. C 12/13, © 2013, EMD Millipore Corporation, Billerica, Mass., both of which are incorporated herein by reference). Although it is typical that lateral flow assay devices are formed on a backing, in some cases the materials forming the lateral flow assay device are sufficiently self-supporting that the backing can be omitted. Unless otherwise noted, materials and construction of other lateral flow assay devices described herein are similar to the materials and construction discussed of the device described and depicted in connection withFIGS. 12A-12C.

LFA with Translating Particle Transfer Mechanism

FIGS. 13A-13Ddepict a lateral flow assay device1300that includes a concentration/separation device and a particle transfer mechanism. Lateral flow assay device1300includes a support layer1302(e.g., similar to backing1228inFIG. 12A-12C), an absorbent pad1304disposed on support layer1302, a movable framework1306configured to fit closely and removably over the absorbent pad1304, a first filter element1308supported by movable framework1306, sample pad1310, and lateral flow membrane1312. First filter element1308is configured for fluid communication with the absorbent pad1304through one or more apertures in movable framework1306, wherein the first filter element1308includes pores small enough to block passage of a functionalized nanoparticle-captured analyte complex1316through the first filter element1308but large enough to permit passage of a carrier fluid1320through first filter element1308to absorbent pad1304. Sample pad1310is supported by support layer1302and configured so that movable framework1306can be fit closely over sample pad1310. Lateral flow membrane1312is located downstream of sample pad1310and includes one or more capture components1320specific to analyte of interest1322. In an aspect, the first filter element1308includes pores large enough to permit passage of unbound analyte1322through first filter element1308to absorbent pad1304. Pore size thus depends on the size of the functionalized nanoparticles to be captured. In an aspect, the first filter element1308has a pore size between about of about 0.1 μm and about 0.4 μm.

In an aspect, first filter element1308is formed of a chemically inert material having minimal nonspecific binding to components of the fluid sample. In an aspect, the first filter element1308is formed of a mildly hydrophilic material. Possible materials for first filter element1308includes, for example, at least one of organic membranes made up of one or more polymers, as cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber. In an aspect, one or more capture components1320include one or more capture component adapted to capture TB LAM, e.g. one or more antibody adapted to capture TB LAM. In an aspect, lateral flow membrane1312includes a test line1324including the one or more capture components1320. Lateral flow membrane1312may also include control line1326. In an aspect, movable framework1306is configured to fit over sample pad1310with the first filter element1308in fluid communication with sample pad1310through one or more apertures in the movable framework. In an aspect, LFA device1300also includes conjugate pad1332between sample pad1310and lateral flow membrane1312, and wick1334downstream of lateral flow membrane1312.

InFIG. 13A, movable framework1306carrying first filter element1308is positioned over absorbent pad1304. Functionalized nanoparticle-captured analyte complex1316(made up of analyte of interest1322and functionalized nanoparticles1338) in carrier fluid1320is applied to first filter element1308. As depicted inFIG. 13B, carrier fluid1320travels through first filter element1308and into absorbent pad1304, while functionalized nanoparticle-captured analyte complex1316remains on top of first filter element1308. Divider1330is a region of non-fluid-conductive material between absorbent pad1304and sample pad1310, which prevents fluid from traveling from absorbent pad1304to sample pad1310, and in an aspect also supports movable framework1306as it is moved from absorbent pad1304to sample pad1310. As indicated by the black arrow inFIG. 13B, movable framework1306can be moved by translation until it rests over sample pad1310, as depicted inFIG. 13C. This can be accomplished by sliding movable framework1306, although alternatively, movable framework1306could be picked up and then placed on sample pad1310. Elution buffer1336is applied to functionalized nanoparticle-captured analyte complex1316on first filter element1308. As shown inFIG. 13D, analyte of interest1322is eluted and travels through first filter element1308, through openings in movable framework1306, and into sample pad1310in elution buffer1336, while functionalized nanoparticles1338remain on top of first filter element1308. Analyte of interest1322travels through conjugate pad1332and lateral flow membrane1312to test line1324, where it is detected by capture components1320, e.g. as described elsewhere herein. In an aspect, elution buffer continues through lateral flow membrane1312to wick1334, where it is absorbed.

FIGS. 14A and 14Billustrates components of the device ofFIGS. 13A-13Din greater detail.FIG. 14Adepicts movable framework1306carrying first filter element1308positioned over absorbent pad1304, also showing divider1330and sample pad1310. It can be seen that movable framework1306is configured to fit closely over absorbent pad1304, in that top1500of movable framework1306is slightly wider than absorbent pad1304, and sides1502of movable framework1306extend downward from top1500adjacent the sides of absorbent pad1304to hold movable framework1306in position over absorbent pad1304. Sample pad1310has a width substantially the same as that of absorbent pad1304, such that top1500of movable framework1306is slightly wider than sample pad1310, and sides1502of movable framework1306extend downward from top1500adjacent the sides of sample pad1310to hold movable framework1306in position over sample pad1310, as depicted inFIG. 14B.

FIG. 15Ais a cross-sectional view of first filter element1308, movable framework1306, and absorbent pad1304taken at section line A-A inFIG. 14A, illustrating the fit of top1500and sides1502with regard to absorbent pad1304.FIG. 15Bis a cross-sectional view of a related embodiment, in which sides1502of movable framework1306include elongated projections1504which extend into grooves106in absorbent pad1304(or alternatively, in a rigid housing containing absorbent pad1304, not shown), allowing movable framework to be slid with respect to absorbent pad1304without being lifted.FIG. 15Cis a cross-sectional view, taken at section line C-C inFIG. 15B(at a location like that of section line B-B inFIG. 14B) of features that allow movable framework1306to be secured in place once it has been slid into the proper position over sample pad1310. InFIG. 15C, sides1502of movable framework1306include elongated projections1504, as depicted inFIG. 15B. In addition, each elongated projection1504also includes an angled tooth1508, which fits into detent1510to lock movable framework in place with respect to sample pad1310(or a rigid housing containing sample pad1310). In an aspect, side1502flexes outward slightly to allow angled tooth1508to slide along side1512of sample pad1310, and snap inward into place when detent1510is reached.

LFA with Hinged Particle Transfer Mechanism

FIGS. 16A-16Ddepict a lateral flow assay device1600similar to that depicted inFIGS. 13A-13D, but having an alternative mechanism for transferring functionalized nanoparticle-captured analyte complex1316from absorbent pad1304to sample pad1310. Lateral flow assay device1600includes support layer1302, absorbent pad1304, first filter element1308, sample pad1310, lateral flow membrane1312, capture components1320, test line1324, control line1326, conjugate pad1332, and wick1334which are as described above in connection withFIGS. 13A-13D. Housing1620around absorbent pad1304is also depicted. First filter element1308is supported by a movable framework1602, which in this case is hinged and flipped over rather than translated in order to move first filter element1308to sample pad1310. As depicted inFIG. 16A, lateral flow assay device1600includes a second filter element1604located over the sample pad1310. Second filter element1604is similar to first filter element1308, in that it includes pores small enough to block passage of the functionalized nanoparticle-captured analyte complex1316, as well as functionalized nanoparticle without captured analyte, through second filter element1604but large enough to permit passage of a second carrier fluid and unbound analyte through the second filter element1604to sample pad1310. First filter element1308and second filter element1604can be formed from various materials, including, for example, at least one of organic membranes made up of one or more polymers, as cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber. Lateral flow assay device1600includes hinge element1606disposed on support layer1302between absorbent pad1304and sample pad1310and attached to moveable framework1602such that moveable framework1602can be rotated around an axis of the hinge element1606to flip first filter element1308over to move first filter element1308from a position over the absorbent pad1304to a position over second filter element1604, so that first filter element1308is in fluid communication with sample pad1310through the second filter element1604.

FIG. 16Adepicts functionalized nanoparticle-captured analyte complex1316in carrier fluid1320being applied to first filter element1308.FIG. 16Bdepicts functionalized nanoparticle-captured analyte complex1316on top of first filter element1308, while carrier fluid1320has traveled through first filter element1308and into absorbent pad1304. Movable framework1602is rotated around the axis of hinge element1606to flip first filter element1308over to move first filter element1308from a position over the absorbent pad1304to a position over second filter element1604, in the direction indicated by the curving arrow.FIG. 16Cshows movable framework1602and first filter element1308positioned over second filter element1604, with functionalized nanoparticle-captured analyte complex1316located between first filter element1308and second filter element1604. As depicted inFIG. 16C, elution buffer1336is applied, and passes through movable framework1602, first filter element1308, second filter element1604, and into sample pad1310. As depicted inFIG. 16D, elution buffer1336carries analyte1322with it into sample pad1310but leaving functionalized nanoparticles1338on top of second filter element1604. Analyte1322moves through sample pad1310, conjugate pad1332, and lateral flow membrane1312for detection at test line1324, as described above. Second filter element1604may be similar to or the same as first filter element1308, e.g. formed of the same types of materials and having similar pore size. In an aspect, second filter element1604is formed of a chemically inert material having minimal nonspecific binding to components of the fluid sample. In an aspect, second filter element1604is formed of a mildly hydrophilic material.

FIGS. 17A-17Billustrate in greater detail the hinging transfer mechanism described in connection withFIGS. 16A-16D. Absorbent pad1304, and sample pad1310are carried on support layer1302. Movable framework1602is connected to hinge element1606by arms1702.FIG. 17Ashows first filter element1308supported on movable framework1602. InFIG. 17B, movable framework1602has been rotated around hinge element1606to flip first filter element1308onto second filter element1604, which is positioned over sample pad1310. As can be seen movable framework1602includes frame1704and grid1706which supports first filter element1308and includes apertures1708that allow for passage of fluid.

Analyte Capture Using Stationary Phase Medium

As discussed above, one approach for capturing and concentrating analytes is to us a functionalized stationary substrate rather than functionalized particles. In this approach, a capture ligand is immobilized in or on a stationary phase that resides in a fixed part of a device and the sample containing analyte is flowed through the stationary phase one or more times. Depending on interplay of kinetics and device design, passing sample through the stationary phase once may be sufficient to capture the analyte; alternatively, it may be preferable to pass sample containing analyte through the stationary phase multiple times, in some cases with repeated back and forth flow, to provide sufficient opportunity for the analyte to be captured. Similarly, wash buffer can be flowed through one or multiple times as needed to reduce background. Eventually, a small volume of release buffer can be added to release analyte in concentrated form. Several examples using a stationary phase medium are provided below.

Capture Concentration with Functionalized Membrane

FIGS. 18A-18Gdepict use of a filtration device1800in which a capture ligand is immobilized on the 2-D surface of a stationary phase that includes a thin, substantially incompressible membrane. In the example ofFIGS. 18A-18G, filtration device1800is a TB LAM filtration device in which a lectin is used as a capture ligand. It will be appreciated that such a device could be implemented with different capture ligands in order to capture other analytes. In an aspect, filtration device1800includes a stationary phase medium1802functionalized with at least one lectin1804adapted to bind a glycan of TB LAM to capture TB LAM1806from fluid sample1808. Fluid sample1808includes TB LAM1806and a fluid component1810. Filtration device1800includes a sieve element1816having openings small enough to block passage of the stationary phase medium1802but large enough to permit passage of unbound TB LAM1806and the fluid component1810. Sieve element1816is formed of a mildly hydrophilic, chemically inert material having minimal nonspecific binding to components of the fluid sample.

In an aspect, TB LAM filtration device1800includes a housing1820configured to receive sieve element1816and stationary phase medium1802. For example, housing1820may be a simple cup-like structure, as depicted in cross-section inFIGS. 18A-18G. Alternatively, housing1820may be similar in form to a syringe filter, e.g., similar to filtration-concentration device430depicted inFIG. 4D. In an aspect, filtration device1800includes a connector adapted to connect to the housing to a downstream vacuum source (not shown, but similar to fluid outlet port450inFIG. 4D, for example). In an aspect, TB LAM filtration device1800includes a connector adapted to connect to the housing to a upstream positive pressure source (not shown, but similar to inlet port426inFIG. 4D, for example). In an aspect, housing1820is configured for orienting sieve element1816and stationary phase medium1802such that gravity draws fluid through sieve element1816and away from stationary phase medium1802. Housing1820may be so configured by including features that allow it to be placed on a support surface or attached to a supporting structure such as a receptacle for connecting waste fluid. Such features could include, for example, a level base region or several legs or tabs that support it with respect to the support surface, or a threaded region that permits it to be screwed onto corresponding threads on a jar or similar fluid receptacle.

In an aspect, stationary phase medium1802has a bed volume of between about 50 μl and about 400 μl. In an aspect, stationary phase medium1802has a bed volume less than about 200 μl. In an aspect, stationary phase medium1802has a bed volume less than about 300 μl.

In an aspect, stationary phase medium1802includes at least one of a resin, a gel, a hydrogel, a sponge, a fibrous material, a fiber mat, a cellulosic material, a cellulose pad, a polymer, a nanofiber, an electrospun polylactic acid, agarose, POROS® bioprocessing resin, Sepharose® gel filtration media, or Sephadex® gel filtration media. In an aspect, stationary phase medium1802includes a membrane, e.g. at least one of nitrocellulose, nylon, glass fiber, polytetrafluoroethylene, polyvinylidene difluoride, or Immunodyne® ABC membrane (available from PALL Corporation).

Sieve element1816can be constructed from a metal, a polymer, glass, fabric, a ceramic, a sintered material, or a felted material, for example. Sieve element1816should be chemically inert. Sieve element1816can take the form of a mesh, a plate having perforations therein, or a porous material.

In an aspect, lectin1804includes at least one lectin configured to bind TB LAM in a dose-dependent manner. In various aspects, lectin1804includes at least one lectin capable of binding specifically to mannose or at least one lectin capable of binding specifically to arabinose. For example, lectin1804may includeGalanthus nivalislectin,Hippeastrumhybrid lectin, orLens culinarisagglutinin, or a combination thereof, e.g., a combination ofGalanthus nivalislectin andHippeastrumhybrid lectin. In an aspect, lectin1804is configured (e.g., by appropriate choice of a lectin, such as the examples provided above) to release the analyte of interest under a mildly acidic condition (e.g., about pH 4). In an aspect, lectin1804is configured (e.g., by appropriate choice of a lectin, such as the examples provided above) to release the analyte of interest under a chaotropic condition. In an aspect, lectin1804is configured (e.g., by appropriate choice of a lectin, such as the examples provided above) to release the analyte of interest when exposed to an elution buffer compatible with downstream detection and quantification of the TB LAM with an α LAM-antibody.

Filtration device1800is used in a manner similar to that described and depicted inFIGS. 2A-2H. InFIG. 18A, fluid sample1808is added to filtration device1800. InFIG. 18B, fluid sample1808is exposed to lectin1804in filtration device1800to allow TB LAM1806to bind to lectin1804. Passing fluid sample1808through the stationary phase medium1802once may be sufficient to capture the TB LAM1806. Alternatively, it may be preferable to pass fluid sample1808through stationary phase medium1802multiple times to provide sufficient opportunity for TB LAM1806to be captured.

InFIG. 18C, fluid component1810and unbound TB LAM1806pass through stationary phase medium1802and sieve element1816, while TB LAM1806bound to lectin1804is retained on stationary phase medium1802. InFIG. 18D, wash buffer1824is added to filtration device1800. As shown inFIG. 18E, wash buffer1824passes through stationary phase medium1802and sieve element1816, removing materials that contribute to background, while TB LAM1806bound to lectin1804remains on stationary phase medium1802. As noted previously, wash buffer1824can be passed through stationary phase medium1802in multiple batches, allowing for the use of a large amount of wash buffer1824to accomplish thorough washing of TB LAM1806. InFIGS. 18F and 18G, a small volume of elution buffer1826is added to filtration device1800. TB LAM1806is eluted from lectin1804and passes through stationary phase medium1802and sieve element1816, exiting filtration device1800as concentrated sample1830, made up of TB LAM1806in elution buffer1826. The final volume of concentrated sample1830is dependent upon the dead volume of the system, i.e., the amount of fluid that can be retained in stationary phase medium1802.

Capture Concentration with Stationary Phase Functionalized Throughout its Bulk

FIGS. 19A-19G, depict a filtration device1900, similar to that depicted inFIGS. 18A-18G, however, in this case, the stationary phase medium1902includes an open pore capture bed, such as a sponge, with capture ligand immobilized in the 3-D bulk of the open pore capture bed. Again, the filtration device1900is illustrated as a TB LAM filtration device that uses a lectin1804(as described herein above) as a capture ligand, but the device could be implemented with different capture ligands in order to capture other analytes. In an aspect, filtration device1900includes a stationary phase medium1902functionalized with at least one lectin1804adapted to bind a glycan of TB LAM to capture TB LAM1806from fluid sample1808(which, as discussed above, includes TB LAM1806and a fluid component1810). Filtration device1900includes a sieve element1816having openings small enough to block passage of the stationary phase medium1902but large enough to permit passage of unbound TB LAM1806and the fluid component1810. Sieve element1816is formed of a mildly hydrophilic, chemically inert material having minimal nonspecific binding to components of the fluid sample. In an aspect, filtration device1900includes housing1920, which may be similar to housing1820inFIG. 18A-18G, for example.

In an aspect, the stationary phase medium1902includes a resin such as agarose, POROS® bioprocessing resin (ThermoFisher Scientific), Sepharose® gel filtration Media (Millipor-Sigma), or Sephadex® gel filtration media (Millipor-Sigma), a gel, a hydrogel, a sponge, a fibrous material (e.g., a fiber mat), a cellulosic material (e.g., cellulose pad), a polymer, a nanofiber, electrospun polylactic acid, for example.

In an aspect, stationary phase medium1902includes a compressible structure, e.g. formed from, e.g., a porous material and/or a compressible material. For example, in an aspect a compressible structure can be formed from a porous material, a fibrous material, or an open cell foam, for example.

InFIG. 19A, fluid sample1808is added to filtration device1900. InFIG. 19B, fluid sample1808is exposed to lectin1804in stationary phase medium1902to allow TB LAM1806to bind to lectin1804. Passing fluid sample1808through the stationary phase medium1902once may be sufficient to capture the TB LAM1806. Alternatively, it may be preferable to pass fluid sample1808through stationary phase medium1902multiple times to provide sufficient opportunity for TB LAM1806to be captured.

InFIG. 19C, fluid component1810and unbound TB LAM1806pass through stationary phase medium1902and sieve element1816, while TB LAM1806bound to lectin1804is retained in stationary phase medium1902. InFIG. 19D, wash buffer1824is added to filtration device1900. InFIG. 19E, wash buffer1824passes through stationary phase medium1902and sieve element1816, removing materials that contribute to background, while TB LAM1806bound to lectin1804remains in stationary phase medium1902. As noted previously, wash buffer1824can be passed through stationary phase medium1902in multiple batches, allowing for the use of a large amount of wash buffer1824to accomplish thorough washing of TB LAM1806. InFIGS. 19F and 19G, a small volume of elution buffer1826is added to filtration device1900. TB LAM1806is eluted from lectin1804and passes through stationary phase medium1902and sieve element1816, exiting filtration device1900as concentrated sample1830, made up of TB LAM1806in elution buffer1826. The final volume of concentrated sample1830is dependent upon the dead volume of the system, i.e., the amount of fluid that can be retained in stationary phase medium1902.

The dead volume of the system influences the final volume of the concentrated sample1830. This approach is useful when the initial sample volume to be processed is much larger than that dead volume. If stationary phase medium1902is formed from a compressible material/structure, the dead volume can be reduced by compressing it, e.g. like squeezing out a sponge. Stationary phase medium1902can be kept in its uncompressed state while being contacted with fluid sample1808, wash buffer1824, or elution buffer1826(e.g. during steps depicted inFIGS. 19B, 19D, and 19F), but compressed during fluid removal steps (e.g., inFIGS. 19C, 19E, and 19G).FIGS. 20A-20Gdepict an example of a device that can be used in such an approach.

Capture Concentration Device

FIGS. 20A-20Gillustrates a capture concentration device2000which can be used in connection with stationary phase medium2002containing capture ligand2004immobilized within its 3-D bulk.

As shown inFIG. 20A, a capture concentration device2000includes a straight-walled container2006, which has an interior surface2008, a first end2010, a second end2012, and an opening2014at first end2010. The straight-walled container2006is adapted to receive a fluid sample2020including an analyte of interest2022and a fluid component2024. Capture concentration device2000also includes plunger2026, which includes sieve element2028and shaft2030. Sieve element2028is configured to support a stationary phase medium2002, which is functionalized with at least one capture ligand2004adapted to bind an analyte of interest2022in the fluid sample2020. Sieve element2028has openings small enough to block passage of stationary phase medium2002but large enough to permit passage of unbound analyte of interest2022and the fluid component2024. As shown inFIG. 20B, sieve element2028is configured to slidably engage2032with the interior surface2008of straight-walled container2006. Shaft2030is attached to sieve element2028and configured to transmit force to sieve element2028to drive sliding movement of sieve element2028within straight-walled container2006. In an aspect, sieve element2028may be constructed from a mesh or screen-like material, a woven or felted material, or a rigid or semi-rigid plate or frame having apertures or openings formed therein. In an aspect, sieve element2028is configured to form a slidable seal with the interior surface2008of straight-walled container2006, for example sieve element2008may include a gasket around its periphery, or its periphery may be sufficiently smooth to substantially seal with interior surface2008. In an aspect, the slidable seal is a fluid-tight seal. In another aspect, the slidable seal blocks passage of stationary phase medium2002but may permit passage of fluid.

In an aspect, straight-walled container2006is substantially cylindrical. As depicted inFIGS. 20A-20G, straight-walled container2006is closed at second end2012. In an alternative embodiment, opening2014at first end2010is a first opening, and the straight-walled container includes a second opening at the second end2012(not depicted inFIGS. 20A-20G), such that the straight walled container2006is similar in form to a syringe.

In an aspect, plunger2026is configured to support stationary phase medium2002on a side of sieve element2028facing toward second end2012of straight-walled container2006when plunger2026is positioned within straight-walled container2006, as depicted inFIGS. 20A-20G. Thus, stationary phase medium2002is located between sieve element2028and second end2012, such that, if stationary phase medium2002is compressible, it can be compressed between sieve element2028and second end2012

Alternatively, in some embodiments, plunger2026is configured to support the stationary phase medium2002on a side of the sieve element2028facing toward the first end2010of the straight-walled container2006when plunger2026is positioned within straight-walled container2006(not illustrated).

In an aspect, sieve element2028includes a metal, polymer, ceramic, glass, or other material. In an aspect, one or both of sieve element2028and stationary phase medium2002are formed of a chemically inert material having minimal nonspecific binding to components of the fluid sample. In an aspect, the sieve element and the stationary phase medium are formed of mildly hydrophilic material. The stationary phase medium may take the form of, e.g., a disc of filter or sponge material that can be attached to the sieve element.

In an aspect, the stationary phase medium2002includes a compressible structure having the capture ligand2004immobilized within its bulk. In various aspects, the stationary phase medium includes a porous material and/or a compressible material. In an aspect, the stationary phase medium includes at least one of a resin, a gel, a hydrogel, a sponge, a fibrous material, a fiber mat, a cellulosic material, a cellulose pad), a polymer, a nanofiber, an electrospun polylactic acid, agarose, POROS® bioprocessing resin, Sepharose® gel filtration media, or Sephadex® gel filtration media.

In an aspect, the capture ligand2004includes at least one lectin, for example a lectin adapted to bind a glycan of TB LAM. For example, the lectin may be specific to mannos or arabinose. For example, the lectin may beGalanthus nivalislectin,Hippeastrumhybrid lectin, orLens culinarisagglutinin, for example as discussed herein above. In some aspects, the at least one lectin includes a combination of lectins, for example, a combination ofGalanthus nivalislectin andHippeastrumhybrid lectin. The lectin may be configured to bind TB LAM in a dose-dependent manner.

As depicted inFIG. 20B, in use, plunger2026is inserted into container2006containing analyte2022in fluid component2024. Exposure of capture ligand2004to analyte2022in fluid component2024is maximized by pushing the stationary phase medium2002back and forth through the sample, as indicated by the black arrow. After incubation with agitation, shown inFIG. 20B, plunger2026is depressed as shown inFIG. 20C, to compress stationary phase medium2002. Fluid component2024and any unbound analyte2022is pressed out of stationary phase medium2002, and passes through sieve element2028to the upper region of container2006, where it can be poured off or pipetted out of container2006. Analyte bound to capture ligand2004, to form capture ligand-analyte complexes2034, remains in stationary phase medium2002. As shown inFIG. 20C, the volume of fluid component2024remaining in stationary phase medium2002is reduced due the compression of stationary phase medium2002.

InFIG. 20D, wash buffer2038is added to container2006and plunger2026is moved up and down to move wash buffer2038through stationary phase medium2002. InFIG. 20E, plunger2026is depressed to compress stationary phase medium2002. Wash buffer2038and any interference-producing substances are pressed out of stationary phase medium2002, and pass through sieve element2028to the upper region of container2006, for removal. Capture ligand-analyte complexes2034remain in stationary phase medium2002.

InFIG. 20F, elution buffer2040is added to container2006and plunger2026is moved up and down to move elution buffer2040through stationary phase medium2002to elute analyte2022from capture ligand2004. InFIG. 20G, plunger2026is depressed to compress stationary phase medium2002. Elution buffer2040and analyte2022are pressed out of stationary phase medium2002, and pass through sieve element2028to the upper region of container2006for removal. In an aspect, the volume of elution buffer2040is smaller than the volume of fluid component2024in the original sample, such that cleaned analyte sample2042(analyte2022in elution buffer2040) is concentrated relative to the original sample.

FIGS. 20A-20Gdepict a capture concentration device2000compresses stationary phase medium2002by direct manual application of pressure via plunger2026. It will be appreciated that higher compression forces could be applied by modifying the device to include a simple mechanical advantage device if greater forces was needed.

FIGS. 20A-20Gdepicted a capture concentration device2000in which stationary phase medium2002is compressible. In related embodiments, a similar device can be constructed with a stationary phase medium that is a substantially incompressible membrane having the capture ligand immobilized on its surface. For example, incompressible membrane may be a nitrocellulose or polyvinylidene difluoride (PVDF) membrane.

FIG. 21depicts a generalized method of concentrating TB LAM. Method2100includes collecting a first volume of sample of a body fluid containing TB LAM in a sample container, the sample container containing a lectin-conjugated medium, the lectin-conjugated medium including at least one lectin adapted to bind a glycan of the TB LAM, at2102; incubating the sample with the lectin-conjugated medium for an incubation period under an incubation condition sufficient to produce binding of the TB LAM to the lectin-conjugated medium, at2104; separating a fluid component of the sample from the lectin-conjugated medium with a first filter, the lectin-conjugated medium having the TB LAM bound thereto, at2106; adding a second volume of an elution buffer to the lectin-conjugated medium, wherein the first volume is greater than the second volume, at2108; exposing the elution buffer to the lectin-conjugated medium for an elution period and under an elution condition sufficient to elute the TB LAM from the lectin-conjugated medium, at2110; and separating the elution buffer from the lectin-conjugated medium using a second filter, the elution buffer containing the TB LAM eluted from the lectin-conjugated medium, at2112. In an aspect, the incubation period is about 30 minutes, for example. It will be appreciated that in various aspects, lectin-containing medium can be added to the container before, after, or at the same time as the sample. The sample container may include a sample cup or a transfer container such as a jar, tube or vial, for example.

In some aspects, the lectin-conjugated medium includes at least one lectin configured to bind LAM in a dose-dependent manner. In various aspects, the lectin-conjugated medium includes, for example, at least one lectin capable of binding specifically to mannose (e.g.,Galanthus nivalislectin,Hippeastrumhybrid lectin,Lens culinarisagglutinin) or at least one lectin capable of binding specifically to arabinose. In an aspect, the lectin-conjugated medium includes a combination ofGalanthus nivalislectin andHippeastrumhybrid lectin.

In an aspect, the method2100further includes applying the elution buffer containing the TB LAM to a lateral flow assay configured for detection of TB LAM, as indicated at2114. For example, the lateral flow assay may be configured for antibody-based detection of TB LAM. In an aspect, the second volume (i.e., the volume of elution buffer) is between about 50 μl and about 400 μl. In some aspect, the second volume is less than about 200 μl, or less than about 300 μl.

In an aspect, method2100also includes a step of adding a wash buffer to the lectin-conjugated medium and separating the wash buffer from the lectin-conjugated medium prior to adding the second volume of the elution buffer to the lectin-conjugated medium, as indicated at2116. This could be done between steps2106and2108inFIG. 21, for example.

In an aspect, the at least one lectin is configured to release the analyte of interest under a mildly acidic (e.g., about pH 4) condition. In an aspect, the at least one lectin is configured to release the analyte of interest under a chaotropic condition. In an aspect, the at least one lectin is configured to release the analyte of interest when exposed to an elution buffer compatible with downstream detection and quantification of the TB LAM with an α LAM-antibody. In an aspect, the elution condition includes a mildly acidic condition. In another aspect, the elution condition includes a chaotropic condition, which can be obtained by including at least one of urea, acetate, or MgCl2in the elution buffer. In various aspects, if elution buffer containing analyte is to be applied directly to a lateral flow assay for detection of TB LAM, the elution buffer is compatible with downstream detection and quantification of the TB LAM with an α LAM-antibody.

In various aspects, the lectin-conjugated medium includes a resin, a membrane, particles or beads. In some aspects, the lectin-conjugated medium includes a stationary phase medium functionalized with lectins.

In an aspect, separating the fluid component of the sample from the lectin-conjugated medium with the first filter, at2106, includes applying the sample fluid with the lectin-conjugated medium to the first filter and causing the sample fluid to pass through the filter while the lectin-conjugated medium is retained on the first filter. Fluid can be caused to pass through the filter by applying vacuum to a downstream side of filter, applying pressure to an upstream side of filter, orienting the filter such that gravity draws fluid through filter, or drawing fluid through filter with capillary pressure, for example.

Similarly, in an aspect, separating the elution buffer from the lectin-conjugated medium with the second filter, at2112, includes applying the elution buffer with the lectin-conjugated medium to the second filter and causing the elution buffer to pass through the second filter while the lectin-conjugated medium is retained on the second filter. Again, fluid can be caused to pass through the filter by applying vacuum to a downstream side of filter, applying pressure to an upstream side of filter, orienting the filter such that gravity draws fluid through filter, or drawing fluid through filter with capillary pressure, for example.

In various aspects, at least one of the first filter and the second filter includes an inline filter, a gravity filter, or a filter built into the sample container. In some aspects, the first filter is the same as the second filter. In other aspects, the first filter is distinct from the second filter.

Prophetic Example Application 1: Lectin Beads in Urine Cup for Capture of TB-LAM

Devices and methods described herein can be used, for example, in the concentration and detection of TB-LAM.Mycoplasma-specific membrane glycolipid LAM is released into bodily fluids in small amounts in infected individuals with active TB infection. Isolation, detection and quantification ofMycoplasmaLAM is important in diagnosing TB, for example using an immunoassay.

Concentration of LAM is necessary for its detection and quantification in high sensitivity assays. Mechanical concentration can be used but is labor- and equipment-intensive process that is not easy to integrate with lateral flow immunoassays. Therefore, a method can be formed that includes the following steps:1. Collect urine sample having a volume of e.g. 10-50 ml in urine collection cup (see, e.g.FIG. 2A)2. Allow the sample to incubate with GNL-conjugated beads for 30 minutes (see, e.g.FIGS. 2B-2C)3. Remove the supernatant by running the sample through an inline filter to capture the beads (see, e.g.FIG. 2D)4. Add small amount (e.g. 200 μL-300 μL) of elution and running buffer (see, e.g.FIG. 2G)5. Transfer buffer containing eluted LAM (see, e.g.FIG. 2H) to lateral flow immunoassay for antibody-based detection (seeFIG. 3A-3C)

Lectin from the snowdrop flowerGalanthus nivalisLectin (GNL) binds LAM in a dose-dependent manner. The kinetics of GNL-LAM interaction are comparable to activity of many antibodies, making the system feasible for use in assays. LAM can be removed (eluted) from GNL under mildly acidic (about pH 4) and chaotropic conditions (e.g. conditions that break hydrogen bonds, for example obtainable by using urea, acetate buffer, or MgCl2in the buffer). GNL binds mannose, and as an alternative, other mannose-specific lectins (such asHippeastrumhybrid lectin (HHL)) can be used. For example, S. Mitra and H. R. Das described the use of a mannose specific lectin isolated from the fish Labeo rohita for detecting LAM in an ELISA format (Indian Journal of Clinical Biochemistry, 2001, 16(2), 181-184). Chinese Patent CN101974099A describes the use of flat lentil lectin to purify LAM to create a reagent suitable for detecting anti-LAM antibodies. Both references are incorporated herein by reference. In other aspects, arabinose-specific Lectins can be used to bind LAM. In some aspects, combinations of lectins can be used.

Isolation and purification of LAM using lectins according to methods described herein is cheap and compatible with the downstream immunodetection. Buffers used for both binding and elution of LAM are compatible with downstream detection and quantification of LAM using an α-LAM antibody, for example in a lateral flow assay.

Prophetic Example Application 2

Devices and methods described herein can be used in various other applications involving washing and concentration of particular materials, as well. For example, the filtration-concentration device430depicted inFIGS. 4D-4Mcan be used to concentrate and wash parasite eggs in preparation for detection. For example,Schistosoma(blood flukes) are parasitic flatworms. Their eggs can be found in human feces. A stool sample could be diluted in a sample cup similar to container406inFIG. 4C, the diluted sample drawn up into e.g. syringe420, and injected into filtration concentration device430. Multiple syringe volumes can be injected into filtration concentration device for filtration, as needed. After initial filtration, wash solution can be injected into filtration concentration device430, to wash the concentrated eggs. Following washing steps, concentrated eggs can be removed from upstream chamber442via retentate removal port448, using a method similar to that depicted inFIGS. 4L and 4M.

In a general sense, the various embodiments described herein can be implemented, individually and/or collectively, by various types of mechanical and/or electro-mechanical systems having a wide range of electrical components such as hardware, software, firmware, or virtually any combination thereof, and a wide range of components that can impart mechanical force or motion such as rigid bodies, spring or torsional bodies, hydraulics, and electro-magnetically actuated devices, or virtually any combination thereof. Consequently, as used herein “electro-mechanical system” includes, but is not limited to, electrical circuitry operably coupled with a transducer (e.g., an actuator, a motor, a piezoelectric crystal, etc.), electrical circuitry having at least one discrete electrical circuit, electrical circuitry having at least one integrated circuit, electrical circuitry having at least one application specific integrated circuit, electrical circuitry forming a general purpose computing device configured by a computer program (e.g., a general purpose computer configured by a computer program which at least partially carries out processes and/or devices described herein, or a microprocessor configured by a computer program which at least partially carries out processes and/or devices described herein), electrical circuitry forming a memory device (e.g., forms of random access memory), electrical circuitry forming a communications device (e.g., a modem, communications switch, or optical-electrical equipment), and any non-electrical analog thereto, such as optical or other analogs. Those skilled in the art will also appreciate that examples of electro-mechanical systems include but are not limited to a variety of consumer electrical systems, as well as other systems such as motorized transport systems, factory automation systems, security systems, and communication/computing systems. Those skilled in the art will recognize that electro-mechanical as used herein is not necessarily limited to a system that has both electrical and mechanical actuation except as context can dictate otherwise.

This disclosure has been made with reference to various example embodiments. However, those skilled in the art will recognize that changes and modifications can be made to the embodiments without departing from the scope of the present disclosure. For example, various operational steps, as well as components for carrying out operational steps, can be implemented in alternate ways depending upon the particular application or in consideration of any number of cost functions associated with the operation of the system; e.g., one or more of the steps can be deleted, modified, or combined with other steps.

Additionally, as will be appreciated by one of ordinary skill in the art, principles of the present disclosure, including components, can be reflected in a computer program product on a computer-readable storage medium having computer-readable program code means embodied in the storage medium. Any tangible, non-transitory computer-readable storage medium can be utilized, including magnetic storage devices (hard disks, floppy disks, and the like), optical storage devices (CD-ROMs, DVDs, Blu-ray discs, and the like), flash memory, and/or the like. These computer program instructions can be loaded onto a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions that execute on the computer or other programmable data processing apparatus create a means for implementing the functions specified. These computer program instructions can also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture, including implementing means that implement the function specified. The computer program instructions can also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer-implemented process, such that the instructions that execute on the computer or other programmable apparatus provide steps for implementing the functions specified.

The herein described components (e.g., steps), devices, and objects and the discussion accompanying them are used as examples for the sake of conceptual clarity. Consequently, as used herein, the specific exemplars set forth and the accompanying discussion are intended to be representative of their more general classes. In general, use of any specific exemplar herein is also intended to be representative of its class, and the non-inclusion of such specific components (e.g., steps), devices, and objects herein should not be taken as indicating that limitation is desired.

In some instances, one or more components can be referred to herein as “configured to.” The reader will recognize that “configured to” can generally encompass active-state components and/or inactive-state components and/or standby-state components, unless context requires otherwise.

Clause 1. A method of concentrating TB LAM, comprising:

collecting a first volume of sample of a body fluid containing TB LAM in a sample container, the sample container containing a lectin-conjugated medium, the lectin-conjugated medium including at least one lectin adapted to bind a glycan of the TB LAM;

incubating the sample with the lectin-conjugated medium for an incubation period under an incubation condition sufficient to produce binding of the TB LAM to the lectin-conjugated medium;

separating a fluid component of the sample from the lectin-conjugated medium with a first filter, the lectin-conjugated medium having the TB LAM bound thereto; adding a second volume of an elution buffer to the lectin-conjugated medium, wherein the first volume is greater than the second volume;

exposing the elution buffer to the lectin-conjugated medium for an elution period and under an elution condition sufficient to elute the TB LAM from the lectin-conjugated medium;

separating the elution buffer from the lectin-conjugated medium using a second filter, the elution buffer containing the TB LAM eluted from the lectin-conjugated medium.

Clause 2. The method of clause 1, further comprising applying the elution buffer containing the TB LAM to a lateral flow assay configured for detection of TB LAM.

Clause 3. The method of clause 2, wherein the lateral flow assay is configured for antibody-based detection of TB LAM.

Clause 4. The method of clause 1, further comprising adding a wash buffer to the lectin-conjugated medium and separating the wash buffer from the lectin-conjugated medium prior to adding the second volume of the elution buffer to the lectin-conjugated medium.

Clause 5. The method of clause 1, wherein the lectin-conjugated medium includes at least one lectin configured to bind LAM in a dose-dependent manner.

Clause 6. The method of clause 1, wherein the lectin-conjugated medium includes at least one lectin capable of binding specifically to mannose.

Clause 7. The method of clause 1, wherein the lectin-conjugated medium includes at least one lectin capable of binding specifically to arabinose.

Clause 8. The method of clause 1, wherein the lectin-conjugated medium includesGalanthus nivalislectin.

Clause 9. The method of clause 1, wherein the lectin-conjugated medium includesHippeastrumhybrid lectin.

Clause 10. The method of clause 1, wherein the lectin-conjugated medium includesLens culinarisagglutinin.

Clause 11. The method of clause 1, wherein the lectin-conjugated medium includes a combination ofGalanthus nivalislectin andHippeastrumhybrid lectin.

Clause 12. The method of clause 1, wherein the lectin-conjugated medium includes at least one of a resin, a gel, a hydrogel, a sponge, a fibrous material, a fiber mat, a cellulosic material, a cellulose pad, a polymer, a nanofiber, an electrospun polylactic acid, agarose, POROS® bioprocessing resin, Sepharose® gel filtration media, or Sephadex® gel filtration media.

Clause 13. The method of clause 1, wherein the lectin-conjugated medium includes a membrane.

Clause 14. The method of clause 1, wherein the lectin-conjugated medium includes particles or beads.

Clause 15. The method of clause 14, wherein separating the fluid component of the sample from the lectin-conjugated medium the first filter includes applying the sample fluid with the lectin-conjugated medium to the first filter and causing the sample fluid to pass through the filter while the lectin-conjugated medium is retained on the first filter.

Clause 16. The method of clause 14, wherein separating the elution buffer from the lectin-conjugated medium with the second filter includes applying the elution buffer with the lectin-conjugated medium to the second filter and causing the elution buffer to pass through the second filter while the lectin-conjugated medium is retained on the second filter.

Clause 17. The method of clause 1, wherein the lectin-conjugated medium includes a stationary phase medium functionalized with lectins.

Clause 18. The method of clause 1, wherein the incubation period is about 30 minutes.

Clause 19. The method of clause 1, wherein the elution condition includes a mildly acidic condition.

Clause 20. The method of clause 1, wherein the elution condition includes a chaotropic condition.

Clause 21. The method of clause 20, wherein chaotropic condition is obtained by including at least one of urea, acetate or MgCl2in the elution buffer.

Clause 22. The method of clause 1, wherein the elution buffer is compatible with downstream detection and quantification of the LAM with an α LAM-antibody.

Clause 23. The method of clause 1, wherein the sample container includes a sample cup.

Clause 24. The method of clause 1, wherein the sample container includes a transfer container.

Clause 25. The method of clause 1, wherein separating the fluid component of the sample from the lectin-conjugated medium with the first filter includes applying the sample fluid with the lectin-conjugated medium to the first filter and causing the sample fluid to pass through the first filter while the lectin-conjugated medium is retained on the first filter.

Clause 26. The method of clause 1, wherein separating the elution buffer from the lectin-conjugated medium with the second filter, includes applying the elution buffer with the lectin-conjugated medium to the second filter and causing the elution buffer to pass through the second filter while the lectin-conjugated medium is retained on the second filter.

Clause 27. The method of clause 1, wherein at least one of the first filter and the second filter includes an inline filter.

Clause 28. The method of clause 1, wherein at least one of the first filter and the second filter includes a gravity filter.

Clause 29. The method of clause 1, wherein at least one of the first filter and the second filter includes a filter built into the sample container.

Clause 30. The method of clause 1, wherein the first filter is the same as the second filter.

Clause 31. The method of clause 1, wherein the first filter is distinct from the second filter.

Clause 32. The method of clause 1, wherein the second volume is between about 50 μl and about 400 μl.

Clause 33. The method of clause 1, wherein the second volume is less than about 200 μl.

Clause 34. The method of clause 1, wherein the second volume is less than about 300 μl.

a base defining a bottom of the sample filtration container;

at least one side wall contiguous with the base, the at least one side wall enclosing an interior of the sample filtration container;

an opening at a top of the sample filtration container, the opening adapted to receive a sample including a fluid component and a particulate material carried in the fluid component;

a divider located within the interior of the sample filtration container and dividing the interior of the sample filtration container into an upper portion and a lower portion, the divider including a size exclusion filter, wherein the size exclusion filter has a first side communicating with the upper portion of the sample filtration container and a second side communicating with the lower portion of the sample filtration container, wherein the size exclusion filter has a pore size adapted to allow passage of the fluid component of the sample while blocking passage of the particulate material; and

a capillary medium within the lower portion of the sample filtration container, the capillary medium adapted to draw the fluid component of the sample through the size exclusion filter from the upper portion to the lower portion of the sample filtration container.

Clause 36. The sample filtration container of clause 35, further comprising a sample collection region located at a bottom of the upper portion of the sample container, the sample collection region having a reduced cross-sectional area relative to the upper portion as a whole.

Clause 37. The sample filtration container of clause 36, wherein the divider is substantially planar and oriented at an oblique angle relative to the at least one side wall, and wherein the sample collection region is bounded by the at least one side wall and the divider at a location at which the divider forms an acute angle with the at least one side wall.

Clause 38. The sample filtration container of clause 36, wherein the divider includes a shaped depression, and wherein the sample collection region is located at a bottom of the shaped depression in the divider.

Clause 39. The sample filtration container of clause 35, wherein the capillary medium is adapted to retain the fluid component within the lower portion of the sample container.

Clause 40. The sample filtration container of clause 35, wherein the capillary medium is adapted to retain a fluid volume of between about 10 ml and about 50 ml.

Clause 41. The sample filtration container of clause 35, wherein the capillary medium includes a cellulosic material, a fiber-based material, a glass fiber, a sponge, a resin, a superabsorbent polymer, or a hydrogel.

Clause 42. The sample filtration container of clause 35, wherein the size exclusion filter has a pore size of between about 0.1 μm and about 0.4 μm.

Clause 43. The sample filtration container of clause 35, wherein the size exclusion filter includes at least one of organic membranes made up of one or more polymers, as cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber.

Clause 44. A method of filtering a sample, comprising:

adding a sample including a fluid component and a particulate material into a sample filtration container, the sample filtration container including

a base defining a bottom of the sample filtration container;

at least one side wall contiguous with the base, the at least one side wall enclosing an interior of the sample container;

a divider located within the interior of the sample container and dividing the interior of the sample container into an upper portion and a lower portion, the divider including a size exclusion filter, wherein the size exclusion filter has a first side communicating with the upper portion of the sample container and a second side communicating with the lower portion of the sample container, wherein the size exclusion filter has a pore size adapted to allow passage of the fluid component of the sample while blocking passage of the particulate material;

a sample collection region located at a bottom of the upper portion of the sample container; and

a capillary medium within the lower portion of the sample container, the capillary medium adapted to draw the fluid component of the sample through the size exclusion filter from the upper portion to the lower portion of the sample container;

allowing the fluid component of the sample to be drawn through the size exclusion filter and into the capillary medium; and

removing filtrate including the particulate material from the sample collection region located at a bottom of the upper portion of the sample container.

Clause 45. A lateral flow assay device, comprising:

a loading region adapted to receive a fluid containing a functionalized nanoparticle-captured analyte complex including one or more functionalized nanoparticle and an analyte of interest in a carrier fluid, the loading region including

a sample pad; and

a filter element overlying the sample pad, wherein the filter element includes pores small enough to block passage of the functionalized nanoparticle through the filter element but large enough to permit passage of the carrier fluid and unbound analyte of interest through the filter element to the sample pad; and

a lateral flow membrane downstream of the sample pad and including one or more capture components adapted to capture the analyte of interest.

Clause 46. The lateral flow assay device of clause 45, wherein the one or more capture components include one or more capture component adapted to capture TB LAM.

Clause 47. The lateral flow assay device of clause 45, wherein the one or more capture components include one or more antibody adapted to capture TB LAM.

Clause 48. The lateral flow assay device of clause 45, wherein lateral flow membrane includes a test line including the one or more capture components.

Clause 49. The lateral flow assay device of clause 45, wherein the filter element has a pore size of between about 0.1 μm and about 0.4 μm.

Clause 50. The lateral flow assay device of clause 45, wherein the filter element is formed of a chemically inert material having minimal nonspecific binding to components of the fluid.

Clause 51. The lateral flow assay device of clause 45, wherein the filter element is formed of a mildly hydrophilic material.

Clause 52. A method of detecting a biomarker of interest from fluid sample, comprising:

collecting a fluid sample including a fluid component containing a biomarker;

incubating the fluid sample with a plurality of functionalized nanoparticles for an incubation period sufficient to produce binding of at least a portion of the biomarker with the functionalized nanoparticles to form functionalized nanoparticle-captured biomarker complexes, the functionalized nanoparticles including nanoparticles functionalized with one or more ligands having an affinity to the biomarker;

filtering the fluid sample and functionalized nanoparticle-captured biomarker complexes with a size-exclusion filter to separate the functionalized nanoparticle-captured biomarker complexes from the fluid component;

transferring the functionalized nanoparticle-captured biomarker complexes to a lateral flow assay device; and

detecting the biomarker at a test line of the lateral flow assay device, the test line including antibodies specific to the biomarker and adapted to bind the biomarker at the test line.

Clause 53. The method of clause 52 including washing the functionalized nanoparticle-captured biomarker complexes on the size-exclusion filter with a wash buffer prior to transferring the functionalized nanoparticle-captured biomarker complexes to the lateral flow assay device.

Clause 54. The method of clause 52, wherein the lateral flow assay device is a half-strip lateral flow assay device, and wherein detecting the biomarker at the test line includes detecting the functionalized nanoparticle-biomarker complex at the test line, wherein the functionalized nanoparticle includes a detectable component.

Clause 55. The method of clause 52, including eluting the biomarker from the functionalized nanoparticle on the lateral flow assay device.

Clause 56. The method of clause 55, wherein the lateral flow assay device includes a size exclusion filter overlying a sample pad, the exclusion filter having pore size sufficient to permit passage of the biomarker but not the functionalized nanoparticle, and wherein transferring the functionalized nanoparticle-captured biomarker complexes to the lateral flow assay device includes applying the functionalized nanoparticle-captured biomarker complexes to the size exclusion filter.

Clause 57. The method of clause 55, including exposing the eluted biomarker to an antibody specific to the biomarker on the lateral flow assay device, wherein the antibody specific to the biomarker is conjugated to a detectable component, and wherein detecting the biomarker at a test line includes detecting the detectable component.

Clause 58. The method of clause 55, wherein the lateral flow assay device includes a filter element overlying an absorbent pad, the filter element supported by a movable framework and having pore size sufficient to permit passage of fluid and unbound analyte but not the functionalized nanoparticle, and wherein transferring the functionalized nanoparticle-captured biomarker complexes to the lateral flow assay device includes applying the functionalized nanoparticle-captured biomarker complexes to the filter element; and wherein the method further includes

allowing excess fluid associated with the functionalized nanoparticle-captured biomarker complexes to pass through first filter element and be absorbed by the absorbent pad; and

transferring the filter element on the movable framework to a sample pad of the lateral flow assay device;

wherein eluting the biomarker from the functionalized nanoparticle includes eluting the biomarker into the sample pad through the filter element.

Clause 59. The method of clause 55, wherein the lateral flow assay device includes a first filter element overlying an absorbent pad, the first filter element supported by a movable framework attached to a hinge element, the first filter element and having pore size sufficient to permit passage of fluid but not the functionalized nanoparticle, and wherein transferring the functionalized nanoparticle-captured biomarker complexes to the lateral flow assay device includes applying the functionalized nanoparticle-captured biomarker complexes to the first filter element; and wherein the method further includes

allowing excess fluid associated with the functionalized nanoparticle-captured biomarker complexes to pass through the first filter element and be absorbed by the absorbent pad; and

moving the first filter element from a position over the absorbent pad to a position over a second filter element overlying a sample pad of the lateral flow assay device by rotating the movable framework around an axis of the hinge element to flip the first filter element over;

wherein eluting the biomarker from the functionalized nanoparticle includes applying an elution buffer to the first filter element and allowing the elution buffer to pass through the first filter element, eluting the biomarker into the sample pad through the second filter element.

Clause 60. A lateral flow assay device, comprising:

a support layer;

an absorbent pad disposed on the support layer;

a movable framework configured to fit closely and removably over the absorbent pad;

a first filter element supported by the movable framework, the first filter element configured for fluid communication with the absorbent pad through one or more apertures in the movable framework, wherein the first filter element includes pores small enough to block passage of a functionalized nanoparticle-captured analyte complex through the first filter element but large enough to permit passage of a carrier fluid through the first filter element to the absorbent pad;

a sample pad supported by the support layer, wherein the sample pad is configured so that the movable framework can be fit closely over the sample pad; and

a lateral flow membrane downstream of the sample pad and including one or more capture components specific to the analyte of interest.

Clause 61. The lateral flow assay device of clause 60, wherein the first filter element includes at least one of organic membranes made up of one or more polymers, as cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber.

Clause 62. The lateral flow assay device of clause 60, wherein the first filter element includes pores large enough to permit passage of unbound analyte through the first filter element to the absorbent pad.

Clause 63. The lateral flow assay device of clause 60, wherein the first filter element has a pore size of between about 0.1 μm and about 0.4 μm.

Clause 64. The lateral flow assay device of clause 60, wherein the first filter element is formed of a chemically inert material having minimal nonspecific binding to components of the fluid sample.

Clause 65. The lateral flow assay device of clause 60, wherein the first filter element is formed of a mildly hydrophilic material.

Clause 66. The lateral flow assay device of clause 60, wherein the one or more capture components include one or more capture component adapted to capture TB LAM.

Clause 67. The lateral flow assay device of clause 60, wherein the one or more capture components include one or more antibody adapted to capture TB LAM.

Clause 68. The lateral flow assay device of clause 60, wherein lateral flow membrane includes a test line including the one or more capture components.

Clause 69. The lateral flow assay device of clause 60, wherein the movable framework is configured to fit over the sample pad with the first filter element in fluid communication with the sample pad through one or more apertures in the movable framework.

Clause 70. The lateral flow assay device of clause 77, wherein the movable framework is configured to slide from the absorbent pad to the sample pad.

Clause 71. The lateral flow assay device of clause 60, further comprising

a second filter element located over the sample pad, wherein the second filter element includes pores small enough to block passage of the functionalized nanoparticle-captured analyte complex and functionalized nanoparticle through the second filter element but large enough to permit passage of a second carrier fluid and unbound analyte through the second filter element to the sample pad; and

a hinge element disposed on the support layer between the absorbent pad and the sample pad and attached to the moveable framework such that the moveable framework can be rotated around an axis of the hinge element to flip the first filter element over to move the first filter element from a position over the absorbent pad to a position over the second filter element, so that the first filter element is in fluid communication with the sample pad through the second filter element.

Clause 72. The lateral flow assay device of clause 71, wherein at least one of the first filter element and the second filter element includes at least one of organic membranes made up of one or more polymers, as cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber.

Clause 73. The lateral flow assay device of clause 71, wherein the second filter element has a pore size of between about 0.1 μm and about 0.4 μm.

Clause 74. The lateral flow assay device of clause 71, wherein the second filter element is formed of a chemically inert material having minimal nonspecific binding to components of the fluid sample.

Clause 75. The lateral flow assay device of clause 71, wherein the second filter element is formed of a mildly hydrophilic material.

Clause 76. A filtration-concentration device, comprising:a filter membrane having a first side and a second side, the filter membrane having pores small enough to block passage of a functionalized nanoparticle-captured analyte complex from the first side to the second side but large enough to permit passage of fluid or unbound analyte from the first side to the second side;a housing configured to contain the filter membrane, the housing having an upstream chamber in fluid communication with the first side of the filter membrane and downstream chamber in fluid communication with the second side of the filter membrane;an inlet port in fluid communication with the upstream chamber, the inlet port adapted to receive a fluid sample containing a functionalized nanoparticle-captured analyte complex in a first volume of the fluid;a fluid outlet port in fluid communication with the downstream chamber, the fluid outlet port configured to permit fluid including a portion of the first volume of fluid to exit the filtration concentration device; anda retentate removal port in communication with the upstream chamber, the retentate removal port configured to allow removal of a retentate from the upstream chamber;wherein the filter membrane is chemically inert with respect to the functionalized nanoparticle-captured analyte complex and the fluid and exhibits little or no non-specific binding to materials in the fluid;

wherein the upstream chamber has a volume sufficient to contain a second volume of fluid, wherein the second volume is less than the first volume.

Clause 77. The filtration-concentration device of clause 84, wherein the filter membrane includes at least one of an organic membrane, a polymer, cellulose, nitrocellulose, cellulose acetate, polysulfone, polyvinylidene fluoride, polyethersulfone, polyethylene sulfone, polyamide, polyethylene teraphthalate, or polytetrafluoroethylene, or glass fiber.

Clause 78. The filtration-concentration device of clause 84, wherein the fluid outlet port is adapted for connection to a vacuum source.

Clause 79. The filtration-concentration device of clause 84, further comprising a vacuum source connected downstream of the fluid outlet port.

Clause 80. The filtration-concentration device of clause 84, wherein the inlet port is adapted for connection to a positive pressure source.

Clause 81. The filtration-concentration device of clause 84, further comprising a positive pressure source connected upstream of the inlet port.

Clause 82. A capture concentration device, comprising:a straight-walled container having an interior surface, a first end, a second end, and an opening at the first end, the straight-walled container adapted to receive a fluid sample including an analyte of interest and a fluid component; anda plunger includinga sieve element configured to slidably engage with the interior surface of the straight-walled container and to support a stationary phase medium functionalized with at least one capture ligand adapted to bind an analyte of interest in the fluid sample, the sieve element having openings small enough to block passage of the stationary phase medium but large enough to permit passage of unbound analyte of interest and the fluid component; and

a shaft attached to the sieve element and configured to transmit force to the sieve element to drive sliding movement of the sieve element within the straight-walled container.

Clause 83. The capture concentration device of clause 82, wherein the straight-walled container is substantially cylindrical.

Clause 84. The capture concentration device of clause 82, wherein the straight-walled container is closed at the second end.

Clause 85. The capture concentration device of clause 82, wherein the opening at the first end is a first opening, and wherein the straight-walled container includes a second opening at the second end.

Clause 86. The capture concentration device of clause 82, wherein the plunger is configured to support the stationary phase medium on a side of the sieve element facing toward the first end of the straight-walled container when the plunger is positioned within the straight-walled container.

Clause 87. The capture concentration device of clause 82, wherein the plunger is configured to support the stationary phase medium on a side of the sieve element facing toward the second end of the straight-walled container when the plunger is positioned within the straight-walled container

Clause 88. The capture concentration device of clause 82, wherein the sieve element includes a metal, a polymer, glass, fabric, a ceramic, a sintered material, or a felted material.

Clause 89. The capture concentration device of clause 82, wherein the sieve element and the stationary phase medium are formed of a chemically inert material having minimal nonspecific binding to components of the fluid sample.

Clause 90. The capture concentration device of clause 82, wherein the sieve element and the stationary phase medium are formed of mildly hydrophilic material.

Clause 91. The capture concentration device of clause 82, further comprising the stationary phase medium.

Clause 92. The capture concentration device of clause 82, wherein the stationary phase medium is formed of a chemically inert material having minimal nonspecific binding to components of the fluid sample.

Clause 93. The capture concentration device of clause 82, wherein the stationary phase medium includes a substantially incompressible membrane having the capture ligand immobilized on its surface.

Clause 94. The capture concentration device of clause 93, wherein the stationary phase medium includes at least one of nitrocellulose, nylon, glass fiber, polytetrafluoroethylene, polyvinylidene difluoride, or Immunodyne® ABC membrane.

Clause 95. The capture concentration device of clause 82, wherein the stationary phase medium includes a compressible structure having the capture ligand immobilized within its bulk.

Clause 96. The capture concentration device of clause 82, wherein the second volume is between about 50 μl and about 400 μl.

Clause 97. The capture concentration device of clause 82, wherein the second volume is less than about 200 μl.

Clause 98. The capture concentration device of clause 82, wherein the second volume is less than about 300 μl.

Clause 99. The capture concentration device of clause 82, wherein the stationary phase medium includes a porous material.

Clause 100. The capture concentration device of clause 82, wherein the stationary phase medium includes a compressible material.

Clause 101. The capture concentration device of clause 82, wherein the capture ligand includes at least one lectin.

Clause 102. The capture concentration device of clause 101, wherein the at least one lectin is adapted to bind a glycan of TB LAM.

Clause 103. The capture concentration device of clause 101, wherein the at least one lectin includesGalanthus nivalislectin.

Clause 104. The capture concentration device of clause 101, wherein the at least one lectin includesHippeastrumhybrid lectin.

Clause 105. The capture concentration device of clause 101, wherein the at least one lectin includesLens culinarisagglutinin.

Clause 106. The capture concentration device of clause 101, wherein the at least one lectin includes a combination ofGalanthus nivalislectin andHippeastrumhybrid lectin.

Clause 107. The capture concentration device of c clause 101, wherein the at least one lectin includes at least one lectin configured to bind LAM in a dose-dependent manner.

Clause 108. The capture concentration device of clause 101, wherein the at least one lectin includes at least one lectin capable of binding specifically to mannose.

Clause 109. The capture concentration device of clause 101, wherein the at least one lectin includes at least one lectin capable of binding specifically to arabinose.

Clause 110. The capture concentration device of clause 101, wherein the at least one lectin is configured to release the analyte of interest under a mildly acidic condition.

Clause 111. The capture concentration device of clause 101, wherein the at least one lectin is configured to release the analyte of interest under a chaotropic condition.

Clause 112. The capture concentration device of clause 101, wherein the at least one lectin is configured to release the analyte of interest when exposed to an elution buffer compatible with downstream detection and quantification of the LAM with an α LAM-antibody.

a stationary phase medium functionalized with at least one lectin adapted to bind a glycan of TB LAM to capture TB LAM from a fluid sample, the fluid sample including the TB LAM and a fluid component; and

a sieve element having openings small enough to block passage of the stationary phase medium but large enough to permit passage of unbound TB LAM and the fluid component, wherein the sieve element is formed of a mildly hydrophilic, chemically inert material having minimal nonspecific binding to components of the fluid sample.

Clause 114. The TB LAM filtration device of clause 113, wherein the stationary phase medium includes a compressible structure having the at least one lectin immobilized within its bulk.

Clause 115. The TB LAM filtration device of clause 114, wherein the compressible structure includes a porous material.

Clause 116. The TB LAM filtration device of clause 114, wherein the compressible structure includes a compressible material.

Clause 117. The TB LAM filtration device of clause 113, wherein the stationary phase medium includes a substantially incompressible membrane having the at least one lectin immobilized on its surface.

Clause 118. The TB LAM filtration device of clause 117, wherein the substantially incompressible membrane includes at least one of nitrocellulose, nylon, glass fiber, polytetrafluoroethylene, polyvinylidene difluoride, or Immunodyne® ABC membrane.

Clause 119. The TB LAM filtration device of clause 113, wherein the stationary phase medium includes at least one of a resin, a gel, a hydrogel, a sponge, a fibrous material, a fiber mat, a cellulosic material, a cellulose pad, a polymer, a nanofiber, an electrospun polylactic acid, agarose, POROS® bioprocessing resin, Sepharose® gel filtration media, or Sephadex® gel filtration media.

Clause 120. The TB LAM filtration device of clause 113, wherein the sieve element includes a metal, a polymer, glass, fabric, a ceramic, a sintered material, or a felted material.

Clause 121. The TB LAM filtration device of clause 113, wherein the at least one lectin includes at least one lectin configured to bind LAM in a dose-dependent manner.

Clause 122. The TB LAM filtration device of clause 113, wherein the at least one lectin includes at least one lectin capable of binding specifically to mannose.

Clause 123. The TB LAM filtration device of clause 113, wherein the at least one lectin includes at least one lectin capable of binding specifically to arabinose.

Clause 124. The TB LAM filtration device of clause 113, wherein the at least one lectin includesGalanthus nivalislectin.

Clause 125. The TB LAM filtration device of clause 113, wherein the at least one lectin includesHippeastrumhybrid lectin.

Clause 126. The TB LAM filtration device of clause 113, wherein the at least one lectin includesLens culinarisagglutinin.

Clause 127. The TB LAM filtration device of clause 113, wherein the at least one lectin includes a combination ofGalanthus nivalislectin andHippeastrumhybrid lectin.

Clause 128. The TB LAM filtration device of clause 113, wherein the at least one lectin is configured to release the analyte of interest under a mildly acidic condition.

Clause 129. The TB LAM filtration device of clause 113, wherein the at least one lectin is configured to release the analyte of interest under a chaotropic condition.

Clause 130. The TB LAM filtration device of clause 113, wherein the at least one lectin is configured to release the analyte of interest when exposed to an elution buffer compatible with downstream detection and quantification of the LAM with an α LAM-antibody.

Clause 131. The TB LAM filtration device of clause 113, further comprising a housing configured to receive the sieve element and the stationary phase medium.

Clause 132. The TB LAM filtration device of clause 131, including a connector adapted to connect to the housing to a downstream vacuum source.

Clause 133. The TB LAM filtration device of clause 131, including a connector adapted to connect to the housing to a upstream positive pressure source.

Clause 134. The TB LAM filtration device of clause 131, wherein the housing is configured to for orienting the sieve element and stationary phase medium such that gravity draws fluid through the sieve element and away from the stationary phase medium.