CN113260862A

CN113260862A - Homogeneous assay using particle aggregation or disaggregation

Info

Publication number: CN113260862A
Application number: CN201980066465.8A
Authority: CN
Inventors: 斯蒂芬·Y·周; 丁惟; 李骥
Original assignee: Essenlix Corp
Current assignee: Shanghai Yisheng Biotechnology Co ltd; Yewei Co ltd
Priority date: 2018-08-16
Filing date: 2019-08-16
Publication date: 2021-08-13
Also published as: WO2020037289A1; JP2021536563A; US20210255177A1; US11709162B2

Abstract

The present invention discloses devices and methods for performing biological and chemical assays, such as immunoassays and nucleic acid assays, and more particularly, homogeneous assays using aggregation and disaggregation processes of microparticles or nanoparticles, without the use of washing steps.

Description

Homogeneous assay using particle aggregation or disaggregation

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application No.62/719,062, filed on 8/16/2018, the contents of which are hereby incorporated by reference in their entirety. The entire disclosure of any publication or patent document referred to herein is incorporated by reference in its entirety.

Technical Field

The present disclosure relates to devices and methods for performing biological and chemical assays, such as immunoassays and nucleic acid assays.

Background

In biological and chemical assays (e.g., diagnostic tests), homogeneous assays that do not require washing are generally preferred. The present disclosure provides apparatus and methods for achieving these goals.

Disclosure of Invention

In one or more embodiments, the present disclosure provides devices and methods for homogeneous assays employing analyte-induced particle aggregation, devices and methods for homogeneous assays employing analyte-induced particle deagglomeration, and systems for measuring analyte-induced aggregated or deagglomerated particles.

Drawings

The drawings are for illustration purposes only. The figures may or may not be drawn to scale.

Fig. 1 shows an exemplary embodiment in which certain particles have been aggregated in a liquid sample.

Fig. 2 shows several exemplary embodiments of how the sample is imaged and analyzed for optimal results.

Fig. 3 shows an exemplary flow chart and illustrates the process of the disclosed aggregate particle assay.

Figure 4 shows open and closed plate configurations for aggregate particle assays.

Fig. 5 shows the biochemical process of aggregate particle assay.

Fig. 6 shows an image from a CRP aggregate particle assay.

Detailed Description

The following detailed description illustrates some embodiments of the invention by way of example and not by way of limitation. The section headings and any subtitles used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. The content under the section title and/or the sub-title is not limited to the section title and/or the sub-title but applies to the entire description of the present disclosure.

It is an object of the present disclosure to perform homogeneous assays in a "one-step process". By "one-step" assay is meant an assay in which a drop of sample is placed in the assay and then a signal is read, with no other steps in between (e.g., no washing between steps). Assays include, for example, protein assays and nucleic acid assays.

One aspect of the present disclosure is to allow homogeneous assays to be performed in a "one-step process" without the use of any washing. In a "one-step" assay, which uses two plates that are movable relative to each other, a sample with an analyte is dropped onto one or both of the plates, the two plates are pressed against each other to compress at least a portion of the sample into a thin layer, and then the signal is read from the plates without any washing.

Another significant feature of the method can be, for example, that in certain embodiments, the two plates measured are pressed by a human hand, and at least a portion of the sample has a uniform thickness by using a particular set of plates and spacers as specified herein.

To achieve a one-step assay for detecting an analyte in a sample, a key method of the present disclosure is to analyze the analyte in the assay by aggregating and/or disaggregating particles in solution after addition of the analyte, and then using an image or a one-time lumped optical signal.

In one embodiment, the present disclosure provides a device for homogeneous assays employing particle aggregation, comprising:

a first plate, a second plate, and a spacer, wherein:

i. the panels are movable relative to one another into different configurations, including an open configuration and a closed configuration;

each of the plates has a sample contacting area on its respective inner surface for contacting a sample containing or suspected of containing a sample;

one or both of the plates comprises one or more spacers having a predetermined substantially uniform height within the sample contact area; and is

One or both of the plates comprises a plurality of isolated particles on the respective inner surface, wherein the particles have a capture agent immobilized thereon, wherein the capture agent is capable of binding and immobilizing an analyte and causing aggregation of the isolated particles upon binding to the analyte;

wherein in the open configuration the two plates are partially or fully separated, the spacing between the plates is not adjusted by spacers, and the sample is deposited on one or both of the plates; and is

Wherein in the closed configuration, the closed configuration is configured after deposition of the sample in the open configuration: at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness, and the uniform thickness of the layer is defined by the sample contacting surfaces of the plates and is accommodated by the plates and spacers.

a first plate, a second plate, and a spacer, wherein:

One or both of the plates comprises a plurality of aggregated particles on the respective inner surface, wherein the particles have a binding agent attached to each other; and wherein the analyte is capable of disaggregating the aggregated particles when the analyte contacts the particles;

wherein in the open configuration the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacers, and the sample is deposited on one or both of the plates; and

In one embodiment, the present disclosure provides a system for measuring aggregated or disaggregated particles caused by an analyte, comprising: any existing device, a light source that emits light, and an imager, wherein the imager is configured to measure light transmitted through, scattered from, or reflected from the aggregated or deaggregated particles, or any combination thereof.

In one embodiment, the present disclosure provides a method of performing a homogeneous assay employing particle aggregation, comprising the steps of:

(a) obtaining a sample suspected of containing an analyte;

(b) obtaining a first plate and a second plate, wherein:

each of the plates has a sample contacting area on its respective inner surface for contacting a sample.

One or both of the plates comprises spacers, at least one of the spacers being within the sample contact area, and the spacers having a predetermined substantially uniform height; and

one or both of the plates comprises a plurality of isolated particles or aggregated particles on the respective inner surface;

(c) depositing a sample on one or both of the plates when the plates are in an open configuration, wherein the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacer; and

(d) after (c), bonding the two plates together and pressing the plates into a closed configuration, wherein at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness, the uniform thickness of the layer being defined by the sample surfaces of the two plates and being adjusted by the spacers and the plates; and

(e) particles in a layer of uniform thickness are detected and analyzed by an image or a disposable lumped optical signal when the plate is in the closed configuration.

The apparatus and method according to any of the above embodiments, wherein the particles differ in their optical properties selected from the group consisting of: photoluminescence, electroluminescence, and electrochemiluminescence, light absorption, reflection, transmission, diffraction, scattering, diffusion, surface raman scattering, and any combination thereof.

According to the apparatus and method of any of the above embodiments, the particles in the present disclosure may be, for example, biological/non-biological, organic/non-organic, magnetic/non-magnetic, metallic/non-metallic, or luminescent/non-luminescent.

In some embodiments, the particles comprise natural particles or artificial particles, or a combination or mixture thereof. For example, a particle includes a natural biological entity, such as, but not limited to, a cell fragment, a macromolecule (e.g., a polysaccharide, a protein, or a nucleic acid), a cell aggregate, a tissue, or a viral particle. In some embodiments, the particles comprise man-made objects such as, but not limited to, polymer particles, metal particles, magnetic particles, or semiconductor particles, or combinations or mixtures thereof.

In some embodiments, the particles (e.g., beads) of the present disclosure can include a polymer, such as, but not limited to, polystyrene, polypropylene, polycarbonate, latex, or any combination thereof.

In some embodiments, the particles (e.g., beads) of the present disclosure comprise a metal, such as, but not limited to, gold, silver, copper, and platinum.

In some embodiments, the particles (e.g., beads) of the present disclosure include gold nanoparticles, gold nanoshells, gold nanotubes, and the like.

In some embodiments, the particles (e.g., beads) of the present disclosure include a semiconductor, such as, but not limited to, CdSe, CdS and CdS or CdSe coated with ZnS. In some embodiments, the particles (e.g., beads) of the present disclosure may include magnetic materials such As, but not limited to, magnetite, and magnetized ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, and GaAs.

In some embodiments, the particles may have any shape, for example, spherical (commonly referred to as beads) or rod-like, or irregular shapes, and the population of particles may have particles of the same shape or size or particles of different shapes or sizes. The particles may be nanoparticles having a size (average diameter of rods or spheres) at the nanometer level and the particles may be microparticles having a size of the micrometer scale. In some embodiments, the beads in the population of beads may have a uniform diameter or different diameters.

In some embodiments, the particles may be about 5nm to about 10 μm in size. In some embodiments, the particle size is less than 2nm, 5nm, 10nm, 20nm, 50nm, 100nm, 200nm, 500nm, 1um, 2um, 5um, 10um, 20um, 50um, 100um, 200um, 500um, 1mm, 2mm, 5mm, 10mm, 20mm, 50mm, or 100mm, or within a range between any two values. In some embodiments, the beads can have a size of 100nm, 500nm, 1 μm, 5 μm, 50 μm, 500 μm, 1mm, or a range between any two of the values, and a preferred range of 1 μm to 10 μm.

In some embodiments, the particle size is preferably from about 5nm to about 10 nm.

In some embodiments, the particle size is preferably from about 10nm to about 50 nm.

In some embodiments, the particle size is preferably from about 50nm to about 100 nm.

In some embodiments, the particle size is preferably from about 100nm to about 500 nm.

In some embodiments, the particle size is preferably from about 500nm to about 1000 nm.

In some embodiments, the particle size is preferably from about 1 μm to about 5 μm.

In some embodiments, the particle size is preferably from about 5 μm to about 10 μm.

In some embodiments, the particle size is less than 2nm, 5nm, 10nm, 20nm, 50nm, 100nm, 200nm, 500nm, 1um, 2um, 5um, 10um, 20um, 50um, 100um, 200um, 500um, 1mm, 2mm, 5mm, 10mm, 20mm, 50mm, or 100mm, or within a range between any two values. In some embodiments, the beads can have a size of 100nm, 500nm, 1 μm, 5 μm, 50 μm, 500 μm, 1mm, or a range between any two of the values, and a preferred range of 1 μm to 10 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 1.5 μm and 2.5 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 2.5 μm and 4 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 4 μm and 6 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 6 μm and 10 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 10 μm and 15 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 15 μm and 25 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 25 μm and 35 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 35 μm and 50 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 50 μm and 100 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 100 μm and 150 μm.

In a preferred embodiment, the spacer height, the spacing between the plates, and/or the sample thickness is between 150 μm and 200 μm.

In some embodiments, the particles are coated or derivatized with a reagent in addition to the binding agent to enhance binding of the selected analyte. For example, the particles may include a silica coating or be derivatized with streptavidin.

In some embodiments, aggregation of the particles of the present disclosure may be induced by binding between particles or between agents located on the surface of the particles.

In some embodiments, the binding of the agent is the result of a biological, biochemical, chemical, or physical (e.g., magnetic) effect. For example, the binding between agents may be antibody-antigen binding, complementary binding of nucleic acids (e.g., complementary strands of DNA, RNA, or other nucleic acids), binding between a catalyst and its substrate, binding or aptamer to its target, binding of an RNA interference sequence to its target, ligand-receptor binding, or binding between an agent and its agonist or antagonist.

In some embodiments, aggregation occurs naturally or by induction, for example by binding a binding agent to the particle.

In some embodiments, approximately 100% of the particles in the sample aggregate prior to contacting the analyte. In some embodiments, the percentage of aggregated particles prior to contacting the analyte is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or in a range between any two of the values. In certain embodiments, the percentage of aggregated particles prior to contacting with the analyte is greater than 60%, 70%, 80%, 90%, 95%, or 99%, or in a range between any two of the values.

In some embodiments, after contacting the analyte, approximately 100% of the particles in the sample aggregate. In some embodiments, the percentage of aggregated particles after contacting the analyte is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or in a range between any two of the values. In certain embodiments, the percentage of aggregated particles after contacting the analyte is greater than 60%, 70%, 80%, 90%, 95%, or 99%, or in a range between any two of the values.

Referring to the drawings, FIG. 1 shows an exemplary embodiment of the invention in which certain particles have been aggregated in a liquid sample. Fig. 1 (a) shows a perspective view of the liquid sample, and fig. (B) shows a top view of the liquid sample. As shown in fig. 1, the particles may aggregate to different levels, resulting in different aggregate sizes and different signal intensities than the aggregates.

In some embodiments, the sample is imaged and the image is analyzed to measure the concentration of the target analyte.

In some embodiments, the sample is imaged, and particle aggregation in the image is counted, graded, and analyzed to measure the concentration of the target analyte.

In some embodiments, the sample is imaged, and individual particles and particle aggregates in the image are counted, classified, and analyzed to measure the concentration of the target analyte.

In some embodiments, instead of imaging the sample, the disposable lumped optical signal through the device, e.g., transmittance, absorbance wavelength shift, color, is analyzed to measure the concentration of the target analyte.

In certain embodiments, aggregation increases with increasing analyte concentration.

In certain embodiments, aggregation decreases with increasing analyte concentration.

In some embodiments, higher concentrations of analyte may result in larger aggregate sizes. For example, when the analyte is at a low concentration, while more particles do not aggregate (resulting in aggregates comprising only one particle) or only moderately aggregate (resulting in aggregates comprising a small number of particles, e.g., 2 or 3), a higher concentration of analyte induces more aggregation, e.g., resulting in larger aggregates comprising a higher average number of particles per aggregate.

Fig. 2 shows several exemplary embodiments of how the sample is imaged and analyzed for optimal results. In panel (a) of fig. 2, the sample is in a thin layer, but not attached to any plate; in panel (B) of fig. 2, the sample is a thin layer on the panel; in panel (C) of fig. 2, the sample is in a thin layer compressed between two panels (e.g., the first panel and the second panel of a QMAX device).

Fig. 2 illustrates a sample preparation and imaging method: (A) the sample is in a thin layer, but not attached to any plate; (B) the sample is in a thin layer on the plate; (C) the sample is in a thin layer compressed between two plates; (D) the imaging device may capture one or more images of the layer of the sample (arrows indicate imager positioning, not shown); and (E) the sample can be imaged by a three-dimensional (3-D) scanning technique that captures all aggregates throughout the thickness of the sample (arrows indicate imager positioning, not shown).

Figure 4 shows an open and closed plate configuration for aggregate particle assay: A) is an open configuration, wherein the first plate is coated with antibody-conjugated beads; and B) in a closed configuration. When the sample is added, both plates are closed. The beads form aggregates in the presence of analyte from the sample.

Fig. 5 shows the biochemical process of aggregate particle assay. Beads were coated with polyclonal antibodies. In the presence of an analyte, multiple epitopes on the analyte can bind to the polyclonal antibody to form simple single or complex aggregates.

In some embodiments, the antibodies coated on the particles can be, for example, one type of polyclonal antibody, a mixture of polyclonal antibodies, or a mixture of monoclonal and polyclonal antibodies.

In some embodiments, the binding between the reagents in the assay may be, for example, antibody-antigen binding, complementary binding of nucleic acids (e.g., complementary strands of DNA, RNA, or other nucleic acids), binding between a catalyst and its substrate, binding or aptamer to its target, binding of an RNA interference sequence to its target, ligand-receptor binding, or binding between an agent and its agonist or antagonist.

In some embodiments, aggregation of the particles may be induced, for example, by addition of an agonist.

In some embodiments, aggregation of the particles may be reversed, for example, by the addition of an antagonist.

In some embodiments, the imaged region can be, for example, a single layer, multiple layers, or the entire 3D space of the liquid sample.

In some embodiments, imaging may be accomplished, for example, by bright field focusing or plasmon resonance shifting.

In some embodiments, the sample may be directly imaged if the thickness of the sample is less than a predetermined value (e.g., about, 0.5 μm, 1 μm, 2 μm, 5 μm, or 10 μm), or if the ratio between the thickness of the sample and the size of the particles is less than a predetermined value (e.g., about, 50, 20, 10, 5, or 2 microns), because any significant overlap of aggregates in the sample is unlikely. This overlap depends on the thickness of the sample and the ratio between the thickness of the sample and the particle size. The probability of overlap is also lower when the thickness is lower and/or when the ratio is lower. In some embodiments, the ratio may be, for example, less than 1000, 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5, or 1.2, or within a range between any two values recited.

In some embodiments, to improve the accuracy of the results, more than a certain percentage of the lateral area of the sample needs to be imaged.

For example, in some embodiments, the ratio of imaged area to total sample area can be, e.g., greater than 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or within a range between any two of the values. In certain embodiments, the ratio of the imaged area to the total sample area may be, for example, in the range of about 1% to about 10%.

In graphs (D) and (E) of FIG. 2, the thickness of the sample is greater than a predetermined value (e.g., 10 μm, 20 μm, 50 μm, or 100 μm). In some embodiments, alternative methods of imaging of the sample and measurement of the analyte may be employed when the sample thickness is greater than a predetermined value, or when the ratio between the sample thickness and the particle size is above a predetermined value (e.g., 10, 20, 50, or 100). For example, as shown in diagram (D) of fig. 2, the imaging device may capture one or more images of a layer of the sample, where the images may be used to assess the total concentration of the analyte in the sample. In certain embodiments, as shown in diagram (E) of fig. 2, the sample may be imaged by a three-dimensional (3D) scanning technique that captures all aggregates throughout the thickness of the sample without reducing any overlap. The three-dimensional scanning technique used in the present invention may be any contact or non-contact three-dimensional technique that produces a result of an overall scan of the sample. For example, the 3D scanning technique may be a time-of-flight scan or a triangulation scan, or any variations or modifications thereof.

Other principles and methods:

in some embodiments, aggregation assays may use, for example, gold nanoparticles or microparticles to form aggregates by binding to the analyte.

In some embodiments, the gold particles are coated with a reagent having a binding affinity for the target analyte. The size of the particles may be, for example, from about 5nm to about 10 μm. The shape may be, for example, spherical (commonly referred to as beads) or rod-like, or irregular, and the population of particles may have particles of the same shape and size or particles of different shapes or sizes.

In some embodiments, coating may be achieved, for example, by covalent bonds or passive absorption. The gold particles may be, for example, pure gold or have a gold shell surrounding a core particle of other material, such as latex or silica. The thickness of the gold shell may be from about 1nm to about 10 μm.

In some embodiments, the coated gold particles may be printed or sprayed and dried on QMAX cards.

In some embodiments, the aggregation may be formed as follows: add liquid sample to QMAX card and close the card. The deposited gold particles are released from the QMAX cards in a liquid. If the sample contains a target analyte, the coated gold particles can bind to the target and form aggregates at multiple locations on the target surface. Aggregation may be simple aggregation in which multiple gold particles bind to a single analyte, or complex aggregation in which a "network" is formed between multiple gold particles and multiple analytes.

In some embodiments, the formed particle aggregates may be imaged, for example, at visible wavelengths or a specified range of wavelengths.

In some embodiments, analyte concentration and particle aggregation may be analyzed, for example, from wavelength shifts of the particles.

In some embodiments, the particles may be analyzed for analyte concentration and aggregation, e.g., based on the size of the aggregated particles.

In some embodiments, the particles may be analyzed for analyte concentration and aggregation, e.g., based on the size of the disaggregated particles.

In some embodiments, the color (absorption wavelength range, fluorescence wavelength range), size, and number of aggregates can be determined by image analysis.

Alternatively, in some embodiments, the size or aggregation may be determined, for example, by plasmon resonance displacement of gold particle aggregation.

In some embodiments, in a disaggregation assay, an aggregation of particles present dissociates in the presence of an analyte.

In some embodiments, the particles are coated with an agent having a binding affinity for a particular agent. The coated gold particles are pre-mixed with a specific reagent so that aggregates have formed prior to the assay.

In some embodiments, the nature of dissociation of the aggregate by the target analyte can be cleavage of the pre-binding agent (e.g., enzymatic cleavage of nucleic acids), removal or destruction of the pre-binding agent (e.g., denaturation of the pre-binding protein), or competition with the pre-binding agent. When such an analyte is present in the sample, the preformed aggregates will dissociate.

In some embodiments, dissociation of the aggregates can be accomplished, for example, by removing or disrupting the pre-binding agent (e.g., denaturing the pre-bound protein).

The device, kit, system, smartphone system, and method of any preceding embodiment, wherein the particles are made of a material selected from the group consisting of: polystyrene, polypropylene, polycarbonate, PMMG, PC, COC, COP, glass, resin, aluminum, gold, or other metal or any other material whose surface may be modified to associate with a capture agent.

The device, kit, system, smartphone system, and method of any preceding embodiment, wherein the beads are treated with a protein stabilizing agent.

The device, kit, system, smartphone system, and method of any preceding embodiment, wherein the capture agent is conjugated to a bead.

The device, kit, system, smartphone system, and method of any preceding embodiment, wherein the beads are prepared by: activation with N-hydroxysuccinimide (NHS); blocking by using BSA solution; incubation with a capture agent solution.

The device, kit, system, smartphone system, and method of any preceding embodiment, wherein the liquid sample is a biological sample selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, endolymph, perilymph, stool, breath, gastric acid, gastric juice, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and any combination thereof.

The device, kit, system, smartphone system, and method of any preceding embodiment wherein the sample is an environmental liquid sample from a source selected from the group consisting of: rivers, lakes, ponds, oceans, glaciers, icebergs, rainwater, snow, sewage, reservoirs, tap water or drinking water; solid samples from soil, compost, sand, rock, concrete, wood, brick, dirt, and the like, and any combination thereof.

Examples of the invention

Homogeneous particle aggregation for human CRP (C-reactive protein)

Here, we describe a homogeneous QMAX immunoassay experiment of human CRP according to one embodiment of the present disclosure.

In this experiment, the device for immunoassay comprises a first plate and a second plate. A conventional glass slide was used as the first plate and an X plate with 10 μm spacers was used as the second plate, as shown in fig. 4. An iPhone with a bright field adapter was used as the detector.

The experiment was performed according to the following procedure:

1. and (4) antibody conjugation. Rabbit polyclonal anti-crp (abcam) was conjugated to 2 μm protein a polystyrene beads (Invitrogen) according to the manufacturer's manual. The conjugated beads were then blocked with 4% BSA in PBS overnight at 4 ℃.

2. The panels were coated. 1 μ L of conjugate beads were dropped onto the substrate card of Q card and air dried.

3. The sample is added. 3 μ L of CRP solution (in PBS) with different concentrations were added to the substrate card and then covered with an X plate (2 μm column height).

4. After imaging for 1 minute, the bright field of the Q card was imaged by iPhone 6s and bright field adapter.

Fig. 6 shows an image from a CRP aggregate particle assay. CRP with different concentrations is detected, and bright field images are taken after 1 min. Note that the aggregate size is clearly correlated with analyte concentration.

Compression Regulated Open Flow (CROF)

Manipulation of the sample or reagent in the assay may result in improved assays. Such manipulations include, but are not limited to, manipulating the geometry and location of the sample and/or reagent, mixing or binding of the sample and reagent, and the contact area of the reagent sample with the plate.

Many embodiments of the present invention manipulate the geometry, location, contact area, and mixing of samples and/or reagents using a method known as "Compression Regulated Open Flow (CROF)" and an apparatus that performs CROF.

The term "Compressive Open Flow (COF)" refers to a method of changing the shape of a flowable sample deposited on a plate by: (i) placing another plate on top of at least a portion of the sample, and (ii) then compressing the sample between the two plates by pushing the two plates towards each other; wherein the compression reduces the thickness of at least a portion of the sample and causes the sample to flow into the open spaces between the plates.

The term "compression-regulated open flow" or "CROF" (or "self-calibrating compression open flow" or "SCOF" or "SCCOF") refers to a specific type of COF in which the final thickness of part or the entire sample after compression is "regulated" by a spacer placed between two plates.

The term "final thickness of part or the whole sample is adjusted by the spacer" in the CROF process means that once a certain sample thickness is reached, the relative movement of the two plates and thus the change in the sample thickness stops in the CROF process, wherein the certain thickness is determined by the spacer.

One embodiment of a CROF method comprises:

(a) obtaining a flowable sample;

(b) obtaining a first plate and a second plate that are movable relative to each other into different configurations, wherein each plate has a substantially flat sample contacting surface, wherein one or both of the plates contains a spacer and the spacer has a predetermined height, and the spacer is located on the respective sample contacting surface;

(c) depositing a sample on one or both of the plates when the plates are configured in an open configuration; wherein the open configuration is one in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacers; and

(d) after (c), spreading the sample by bringing the plates into a closed configuration, wherein in the closed configuration the plates face each other, the spacer and the associated volume of the sample are between the plates, the thickness of the associated volume of the sample being adjusted by the plates and the spacer, wherein the associated volume is at least a part of the total volume of the sample, and wherein during spreading of the sample flows laterally between the two plates.

Unless otherwise indicated, the term "plate" refers to a plate used in a CROF process, which is a solid having a surface that can be used with another plate to compress a sample placed between the two plates to reduce the thickness of the sample.

The term "plate" or "plate pair" refers to two plates in a CROF process.

The term "first plate" or "second plate" refers to a plate used in a CROF process.

The term "plates facing each other" refers to the situation in which pairs of plates at least partially face each other.

Unless otherwise specified, the term "spacer" or "stop" refers to a mechanical object that, when placed between two plates, sets a limit on the minimum spacing between the two plates, which limit can be reached when the two plates are compressed together. That is, during compression, the spacer will stop the relative movement of the two plates to prevent the plate separation from becoming less than a preset (i.e., predetermined) value. There are two types of spacers: an "open spacer" and a "closed spacer".

The term "open spacer" means that the spacer has a shape that allows liquid to flow around the entire perimeter of the spacer and through the spacer. For example, the posts are open spacers.

The term "occlusive spacer" refers to a spacer that has a shape such that liquid cannot flow through the entire perimeter of the spacer and cannot flow through the spacer. For example, the annulus spacer is a closed spacer for liquid within the annulus, wherein the liquid within the annulus spacer remains within the annulus and cannot flow to the outside (outer periphery).

The terms "spacer has a predetermined height" and "spacer has a predetermined spacer pitch" mean that the values of the spacer height and spacer pitch, respectively, are known prior to the CROF process. If the values of the spacer height and spacer spacing are not known prior to the CROF process, then they are not predetermined. For example, in the case of beads sprayed as spacers on a plate, the spacer pitch is not predetermined in the case where the beads fall on random positions of the plate. Another example of an unpredictable spacer pitch is a spacer that moves during the CROF process.

In the CROF process, the term "spacers are fixed on their respective plates" means that the spacers are attached to the plates at a position, and the attachment to that position is maintained in the CROF process (i.e., the position of the spacers on the respective plates does not change). An example of "the spacer is fixed with its respective plate" is that the spacer is made integrally from one piece of material of the plate, and the position of the spacer relative to the plate surface does not change during the CROF. An example of "the spacer is not fixed with its corresponding plate" is that the spacer is bonded to the plate by an adhesive, but during use of the plate, during CROF, the adhesive cannot hold the spacer in its original position on the plate surface, and the spacer moves away from its original position on the plate surface.

The term "the spacer is integrally fixed to the plate" means that the spacer and the plate behave like a single piece of object, wherein during use the spacer does not move or separate from its original position on the plate.

The term "open configuration" of two plates in a CROF process refers to a configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers.

The term "closed configuration" of two plates in a CROF process means a configuration in which the plates face each other, the spacer and the associated volume of sample are between the plates, the thickness of the associated volume of sample being adjusted by the plates and the spacer, wherein the associated volume is at least a portion of the entire volume of the sample.

In the CROF process, the term "sample thickness is adjusted by the plate and the spacer" means that, for a given condition of the plate, the sample, the spacer and the plate compression method, the thickness of at least one port of the sample in the closed configuration of the plate can be predetermined according to the properties of the spacer and the plate.

In a CROF apparatus, the term "inner surface" or "sample surface" of a plate refers to the surface of the plate that contacts the sample, while the other surface of the plate (not contacting the sample) is referred to as the "outer surface".

The term "X-plate" of a CROF apparatus refers to a plate comprising spacers located on a sample surface of the plate, wherein the spacers have a predetermined spacer pitch and spacer height, and wherein at least one of the spacers is within a sample contact area.

The term "CROF device" refers to a device that performs a CROF process. The term "croned" refers to the use of CROF processing. For example, the term "the sample is croned" means that the sample is placed within a CROF apparatus, subjected to a CROF process, and, unless otherwise specified, maintained in the final configuration of the CROF.

The term "CROF board" refers to two boards used to perform a CROF process.

The term "surface smoothness" or "surface smoothness variation" of a flat surface refers to the average deviation of a flat surface from a perfectly flat plane over a short distance of about or less than a few microns. Surface smoothness is different from surface flatness variations. A flat surface may have good surface flatness, but poor surface smoothness.

The term "surface flatness" or "surface flatness variation" of a flat surface refers to the average deviation of a flat surface from a perfectly flat plane over a long distance of about or greater than 10 μm. Surface flatness variation is different from surface smoothness. A flat surface may have good surface smoothness but poor surface flatness (i.e., greater surface flatness variation).

The term "relative surface flatness" of a plate or sample is the ratio of the change in plate surface flatness to the final sample thickness.

The term "final sample thickness" in the CROF process refers to the sample thickness at the closed configuration of the plate in the CORF process, unless otherwise indicated.

The term "compression method" in CROF refers to a method of bringing two plates from an open configuration to a closed configuration.

The term "target area" or "area of interest" of a plate refers to an area of the plate that is relevant to the function performed by the plate.

The term "at most" means "equal to or less than". For example, the spacer height is at most 1 μm, which means that the spacer height is equal to or less than 1 μm.

The term "sample area" refers to the area of the sample in a direction substantially parallel to the space between the plates and perpendicular to the thickness of the sample.

The term "sample thickness" refers to the sample dimension in a direction perpendicular to the surfaces of the plates facing each other (e.g., the direction of the spacing between the plates).

The term "plate spacing" refers to the distance between the inner surfaces of two plates.

The term "deviation in final sample thickness" in CROF means the difference between a predetermined spacer height (determined by the manufacture of the spacer) and the average of the final sample thickness, where the final sample thickness is averaged over a given area (e.g., the average of 25 different points (4 mm apart) over a 1.6cm x 1.6cm area).

The term "uniformity of the measured final sample thickness" in the CROF process means the standard deviation (e.g., standard deviation from the mean) of the final sample thickness measured over a given sample area.

The terms "relevant volume of sample" and "relevant area of sample" in the CROF process refer to the volume and area, respectively, of a partial or total volume of sample deposited on a plate in the CROF process, which is related to the function performed by the corresponding method or device, wherein the function includes, but is not limited to, reducing the binding time of an analyte or entity, detecting an analyte, quantifying the volume, quantifying the concentration, mixing a reagent or controlling the concentration (analyte, entity or reagent).

Unless specifically stated otherwise, the terms "some embodiments," "in some embodiments," "one embodiment," "another embodiment," "certain embodiments," "many embodiments," etc., in the present disclosure refer to embodiments that apply to the entire disclosure (of the invention).

Unless otherwise specified, the term "height" or "thickness" of an object in a CROF process refers to the dimension of the object in a direction perpendicular to the surface of the plate. For example, the spacer height is a dimension of the spacer in a direction perpendicular to the plate surface, and the spacer height and the spacer thickness mean the same thing.

Unless otherwise specified, the term "region" of an object in a CROF process refers to a region of the object that is parallel to the surface of the plate. For example, the spacer region is a spacer region parallel to the plate surface.

The term "lateral" in a CROF process refers to a direction parallel to the plate surface, unless otherwise specified.

Unless otherwise specified, the term "width" of a spacer in a CROF process refers to the lateral dimension of the spacer.

The term "spacer inside the sample" means that the spacer is surrounded by the sample (e.g., a column spacer inside the sample).

The term "critical bending span" of a plate in a CROF process refers to the span (i.e., distance) of the plate between two supports at which the bending of the plate is equal to the allowed bending for a given compliant plate, sample, and compressive force. For example, for a given flexible plate, sample and compressive force, if the allowed bending is 50nm and the critical bending span is 40 μm, the bending of the plate between two adjacent spacers spaced apart by 40 μm will be 50nm, and if the two adjacent spacers are less than 40 μm, the bending will be less than 50 nm.

The term "flowable" as used with respect to a sample means that the lateral dimension increases as the thickness of the sample decreases. For example, a fecal sample is considered flowable.

In some embodiments of the invention, the sample of the CROF process cannot flow to benefit from the process, as long as the sample thickness can be reduced by the CROF process. For example, to stain tissue by placing a dye on the surface of a CROF plate, the CROF process can reduce tissue thickness and thus accelerate the saturation incubation time for dye staining.

"CROF Card (or Card)", "COF Card", "QMAX-Card", "Q Card", "CROF device", "COF device", "QMAX device", "CROF board", "COF board", and "QMAX board" are interchangeable, but in some embodiments the COF Card does not contain a spacer; and the term refers to a device comprising a first plate and a second plate that are movable relative to each other into different configurations (including open and closed configurations) and comprising spacers (except for some embodiments of COFs) that adjust the spacing between the plates. The term "X-board" refers to one of the two boards in a CROF card, with spacers fixed to the board. Further description of COF cards, CROF cards and X boards is described in provisional application serial No. 62/456065 filed on 7.2.2017, which is incorporated herein in its entirety for all purposes.

A further aspect of the present disclosure includes a CROF apparatus that includes a plurality of capture agents that each bind a plurality of analytes in a sample, i.e., a multiplexed CROF apparatus. In this case, the CROF device containing multiple capture agents may be configured to detect different types of analytes (proteins, nucleic acids, antibodies, pathogens, etc.). Different analytes can be distinguished from each other on the array based on location within the array, emission wavelength of the detectable label bound to the different analytes, or a combination of the above.

Health conditions that may be diagnosed or measured by the methods, devices and systems of the present invention include, for example: chemical equilibrium; nourishing and health care; exercising; fatigue; sleeping; stress; pre-diabetes; allergies; aging; exposure to environmental toxins, pesticides, herbicides, synthetic hormone analogs; pregnancy; female climacteric; the male is in climacteric.

In certain embodiments, the relative levels of nucleic acid in two or more different nucleic acid samples can be obtained and compared using the methods described above. In these examples, the results obtained from the above methods are typically normalized to the total amount of nucleic acid in the sample (e.g., constitutive RNA) and compared. This may be done by comparing ratios or by any other means. In particular embodiments, the nucleic acid profiles of two or more different samples can be compared to identify nucleic acids associated with a particular disease or disorder.

In some examples, the different samples may consist of an "experimental" sample (i.e., the target sample) and a "control" sample, to which the experimental sample may be compared. In many embodiments, the different samples are paired cell types, or portions thereof, one cell type being a target cell type, e.g., abnormal cells, and the other being control cells, e.g., normal cells. If two parts of a cell are compared, the parts are typically the same part from each of the two cells. However, in certain embodiments, two portions of the same cell may be compared. Exemplary pairs of cell types include, for example, cells isolated from tissue biopsies (e.g., from tissue with a disease, such as colon cancer, breast cancer, prostate cancer, lung cancer, skin cancer, or tissue infected with a pathogen, etc.), as well as normal cells from the same tissue, typically from the same patient; cells grown in tissue culture are immortalized (e.g., cells with a proliferative mutation or an immortalizing transgene), infected with a pathogen, or treated (with environmental or chemical agents, e.g., peptides, hormones, altered temperature, growth conditions, physical stress, cellular transformation); and normal cells (e.g., the same cells as the experimental cells except that it is not immortal, infected, or treated, etc.); cells isolated from a mammal having cancer, a disease, an elderly mammal, or a mammal exposed to a condition, and cells from the same species, preferably from a healthy or young mammal of the same family; and differentiated and undifferentiated cells from the same mammal (e.g., one cell is a progenitor cell of the other in a mammal). In one embodiment, different types of cells, neuronal and non-neuronal cells, or cells of different states (e.g., before and after stimulation of the cells) may be used. In another embodiment of the invention, the test material is a cell susceptible to infection by a pathogen, such as a virus, e.g., human immunodeficiency virus (HIV infection), etc., and the control material is a cell resistant to infection by the pathogen. In another embodiment of the invention, the sample pair is represented by undifferentiated cells, such as stem cells and differentiated cells.

Control and measurement of sample thickness without the use of spacers

In some embodiments of the invention, the spacer for adjusting the sample or the relevant volume of the sample is replaced by (a) a positioning sensor capable of measuring the inter-plate spacing and (b) a device capable of controlling the position of the plate and moving the plate to the desired inter-plate spacing based on the information provided by the sensor. In some embodiments, all spacers are replaced by translation stages, monitoring sensors, and feedback systems.

The pitch and/or sample thickness is measured using optical methods. In some embodiments, the measuring (f) of the spacing between the inner surfaces comprises using optical interference. Multiple wavelengths may be used for optical interference. For example, an optical signal generated due to interference of light reflected at the inner surfaces of the first and second plates oscillates with the wavelength of the light. From the oscillations, the spacing between the inner surfaces can be determined. To enhance the interference signal, one or both of the interior surfaces may be coated with a light reflective material.

In some embodiments, the measuring (f) of the spacing between the inner surfaces includes acquiring optical imaging (e.g., acquiring 2D (two-dimensional) or 3D (three-dimensional) images of the sample, and the acquired images may be multiple times with different viewing angles, different wavelengths, different phases, and/or different polarizations, and image processing.

The entire sample area or volume is measured using optical methods. In some embodiments, the measurement (f) of the entire sample area or volume includes acquiring optical imaging (e.g., acquiring 2D (two-dimensional) or 3D (three-dimensional) images of the sample, and the acquired images may be multiple times with different viewing angles, different wavelengths, different phases, and/or different polarizations and image processing.

The speed was measured. In some embodiments, the release time controlling material is coated with or mixed with the reagent on the plate, wherein the release time controlling material delays the time for the reagent to be released to the sample. In some embodiments, the release time control material delays the release of the dry reagent into the blood sample by at least 3 seconds, such as at least 5 seconds or at least 10 seconds, or at least 20 seconds, or at least 60 seconds, or at least 90 seconds, or a value within a range of both.

In some embodiments, the device is configured to analyze the sample after a value in a range of 60 seconds or less, 90 seconds or less, 120 seconds or less, 240 seconds or less, 300 seconds or less, or both.

In some embodiments, in the closed configuration, the final sample thickness device is configured to analyze the sample in 60 seconds or less.

In some embodiments, in the closed configuration, the final sample thickness device is configured to have a reagent to sample saturation time of a value in a range of 10 seconds or less, 30 seconds or less, 60 seconds or less, 90 seconds or less, 120 seconds or less, 240 seconds or less, 300 seconds or less, or both.

Further examples of QMAX cards. The method and apparatus of any of the above embodiments, wherein the spacer has a cylindrical shape and a nearly uniform cross-section.

The method or device of any of the above embodiments, wherein a spacer pitch (SD) is equal to or less than about 120 μ ι η (microns).

The method or device of any of the above embodiments, wherein a spacer pitch (SD) is equal to or less than about 100 μ ι η (microns).

The method and apparatus of any of the above embodiments, wherein the spacer spacing (ISD) is divided by the thickness (h) of the flexible sheet and the young's modulus (E) (ISD)⁴/(hE)) 5X10⁶um³a/GPa or less.

The method and apparatus of any of the above embodiments, wherein the spacer spacing (ISD) is divided by the thickness (h) of the flexible sheet and the young's modulus (E) (ISD)⁴/(hE)) 5X10⁵um³a/GPa or less.

The method and apparatus of any of the above embodiments, wherein the spacers have a columnar shape, a substantially flat top surface, a predetermined substantially uniform height, and a predetermined constant spacer pitch that is at least about 2 times greater than the size of the analyte, wherein the young's modulus of the spacers multiplied by the fill factor of the spacers is equal to or greater than 2MPa, wherein the fill factor is the ratio of the spacer contact area to the total plate area, and wherein for each spacer the ratio of the lateral dimension of the spacer to its height is at least 1 (one).

The method and apparatus of any of the above embodiments, wherein the spacers (e.g., posts) are either fixedly spaced or non-fixedly spaced.

The method and apparatus of any of the above embodiments, wherein the spacer has a columnar shape, a substantially flat top surface, a predetermined substantially uniform height, and a predetermined constant heightDetermining a spacer spacing, the spacer spacing being at least about 2 times greater than the size of the analyte, wherein the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 2MPa, wherein the fill factor is the ratio of the spacer contact region to the total plate region, and wherein for each spacer the ratio of the lateral dimension of the spacer to its height is at least 1 (one), wherein the spacer spacing (ISD) is the fourth power divided by the thickness (h) of the flexible plate and the Young's modulus (E) (ISD)⁴/(hE)) 5X10⁶μm³a/GPa or less.

The method and apparatus of any of the above embodiments, wherein a ratio of a spacer pitch to an average width of the spacers is 2 or more, and a filling factor of the spacers multiplied by a young's modulus of the spacers is 2MPa or more.

The method and apparatus of any of the above embodiments, wherein the analyte is a protein, a peptide, a nucleic acid, a synthetic compound, or an inorganic compound.

The method and device according to any of the preceding embodiments, wherein the sample is selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, and urine.

The method and apparatus of any of the above embodiments, wherein the spacer has a shape of a pillar, and a ratio of a width to a height of the pillar is equal to or greater than 1.

The method and apparatus of any of the above embodiments, wherein the sample deposited on one or both plates has an unknown volume.

The method and apparatus of any of the above embodiments, wherein the spacer has a shape of a post, and the post has a substantially uniform cross-section.

The method and apparatus of any of the above embodiments, wherein the sample is used to detect, purify, and quantify chemical compounds or biomolecules associated with the stage of certain diseases.

The method and device according to any of the above embodiments, wherein the sample is related to infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, psychiatric disorders, neuropsychiatric disorders, pulmonary diseases, renal diseases, and other and organic diseases.

The method and apparatus of any of the above embodiments, wherein the sample is involved in the detection, purification, and quantification of the microorganism.

The method and apparatus of any of the above embodiments, wherein the sample relates to viruses, fungi and bacteria from the environment (e.g., food, water, soil).

The method and apparatus of any of the above embodiments, wherein the sample relates to the detection, quantification of a chemical compound or biological sample (e.g., toxic waste, anthrax) that poses a hazard to food safety or national safety.

The method and apparatus of any of the above embodiments, wherein the sample is associated with quantification of a vital parameter in a medical or physiological monitor.

The method and apparatus of any of the above embodiments, wherein the sample is associated with glucose, blood, oxygen levels, complete blood cell count.

The method and apparatus of any of the above embodiments, wherein the sample involves the detection and quantification of specific DNA or RNA from a biological sample.

The method and apparatus of any of the above embodiments, wherein the sample involves sequencing and comparison of genetic sequences in DNA in chromosomes and mitochondria for genomic analysis.

The method or device of any of the above embodiments, wherein the sample involves detection of a reaction product, e.g., during drug synthesis or purification.

The method and apparatus of any of the above embodiments, wherein the sample is a cell, a tissue, a body fluid, and a stool.

The method and apparatus of any of the above embodiments, wherein the sample is a sample in the human, veterinary, agricultural, food, environmental, and pharmaceutical testing fields.

The method and device of any of the above embodiments, wherein the sample is a biological sample selected from hair, nails, ear wax, breath, connective tissue, muscle tissue, nerve tissue, epithelial tissue, cartilage, cancerous sample, or bone.

The method and apparatus of any of the above embodiments, wherein the spacer pitch is in a range of 5 μ ι η to 120 μ ι η.

The method and apparatus of any of the above embodiments, wherein the inter-spacer distance is in a range of 120 μm to 200 μm.

The method and apparatus of any of the above embodiments, wherein the flexible sheet has a thickness in a range of 20 μm to 250 μm and a young's modulus in a range of 0.1GPa to 5 GPa.

The method and apparatus of any of the above embodiments, wherein for the flexible sheet, the thickness of the flexible sheet times the Young's modulus of the flexible sheet is in the range of 60 to 750GPa- μm.

The method and apparatus of any of the above embodiments, wherein the uniform thickness sample layer is at least 1mm²Is uniform in lateral area.

The method and apparatus of any of the above embodiments, wherein the uniform thickness sample layer is at least 3mm²Is uniform in lateral area.

In some embodiments, measuring the sample area or volume by imaging includes (a) calibrating the image scale by using a sample of known area or volume (e.g., the imager is a smartphone and the size of the image taken by the phone can be calibrated by comparing images of samples of known size taken on the same phone); (b) comparing the image to graduated markings (rulers) disposed on or near the first and second plates (discussed further herein), and (c) combinations thereof.

As used herein, light may include visible light, ultraviolet light, infrared light, and/or near-infrared light. The light may include wavelengths in the range of 20nm to 20,000 nm.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise, e.g., when the word "single" is used. For example, reference to "an analyte" includes a single analyte and a plurality of analytes, reference to "a capture agent" includes a single capture agent and a plurality of capture agents, reference to "a detection agent" includes a single detection agent and a plurality of detection agents, reference to "a reagent" includes a single reagent and a plurality of reagents, and reference to "a camera" includes a single camera and a plurality of cameras.

As used herein, the terms "adapted" and "configured" mean that an element, component, or other subject matter is designed and/or intended to perform a given function. Thus, use of the terms "adapted" and "configured" should not be read to mean that a given element, component, or other subject matter is simply "capable" of performing a given function. Similarly, subject matter recited as being configured to perform a particular function may additionally or alternatively be described as being operable to perform that function.

As used herein, the phrase "for example," when used in reference to one or more components, features, details, structures, embodiments, and/or methods in accordance with the present disclosure, is intended to convey that the described components, features, details, structures, embodiments, and/or methods are illustrative, non-exclusive examples of components, features, details, structures, embodiments, and/or methods in accordance with the present disclosure. Accordingly, the described components, features, details, structures, embodiments, and/or methods are not intended to be limiting, required, or exclusive/exhaustive; as well as other components, features, details, structures, embodiments, and/or methods, including structurally and/or functionally similar and/or equivalent components, features, details, structures, embodiments, and/or methods, are also within the scope of the present disclosure.

As used herein, the phrases "at least one" and "one or more" in reference to a list of more than one entity refer to any one or more of the entities in the list of entities and are not limited to each and at least one of each entity specifically listed in the list of entities. For example, "at least one of a and B" (or, equivalently, "at least one of a or B," or, equivalently, "at least one of a and/or B") can refer to a alone, B alone, or a combination of a and B.

As used herein, the term "and/or" disposed between a first entity and a second entity refers to one of (1) the first entity, (2) the second entity, and (3) the first entity and the second entity. The use of "and/or" listed plural entities should be read in the same way, i.e., "one or more" of the entities so combined. In addition to the entities specifically identified by the "and/or" clause, other entities, whether related or unrelated to those specifically identified, may optionally be present. Thus, as a non-limiting example, in some embodiments, when used in conjunction with an open language such as "including," references to "a and/or B" may refer to a only (optionally including entities other than B); in some embodiments, only B (optionally including entities other than a); in still other embodiments, reference is made to both a and B (optionally including additional entities). These entities may refer to elements, acts, structures, steps, operations, values, etc.

If any patent, patent application, or other reference is incorporated by reference herein and (1) the manner in which a term is defined is inconsistent with an unincorporated portion of this disclosure or other incorporated reference and/or (2) is otherwise inconsistent with an unincorporated portion of this disclosure or other incorporated reference, the unincorporated portion of this disclosure shall control and the term or disclosure incorporated therein shall only control the reference in which the first definition of the term and/or the incorporated disclosure first appears.

It is believed that the following claims particularly point out certain combinations and subcombinations that are directed to one of the disclosed inventions and are novel and non-obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed through amendment of the present claims or presentation of new claims in this or a related application. Such amended or new claims, whether they are directed to a different invention or directed to the same invention, whether different, broader, narrower or equal in scope to the original claims, are also regarded as included within the subject matter of the inventions of the present disclosure.

Claims

1. An apparatus for homogeneous assays employing particle aggregation, comprising:

a first plate, a second plate, and a spacer, wherein:

the panels are movable relative to one another into different configurations, including an open configuration and a closed configuration;

each of said plates having on its respective inner surface a sample contacting region for contacting a sample containing or suspected of containing a sample,

one or both of the plates contain one or more spacers having a predetermined substantially uniform height within the sample contact area; and is

One or both of the plates comprises a plurality of isolated particles on respective inner surfaces, wherein the particles have a capture agent immobilized on the surface of the particles, wherein the capture agent is capable of binding and immobilizing the analyte and causing aggregation of the isolated particles upon binding to the analyte;

wherein,

in the open configuration, the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacers, and the sample is deposited on one or both of the plates; and is

In the closed configuration, the closed configuration is configured after deposition of a sample in the open configuration: at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness, and the uniform thickness of the layer is defined by the inner surfaces of the plates and is accommodated by the plates and the spacers.

2. An apparatus for homogeneous assays employing particle aggregation, comprising:

a first plate, a second plate, and a spacer, wherein:

one or both of the plates contain one or more spacers having a predetermined substantially uniform height within the sample contact area;

one or both of the plates comprises a plurality of aggregated particles on the respective inner surface, wherein the particles have a binder attached to the surface of the particles; and wherein when the analyte contacts the particles, the analyte disaggregates the aggregated particles;

wherein,

in the open configuration, the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacers, and the sample is deposited on one or both of the plates; and

3. A system for measuring aggregated or disaggregated particles caused by an analyte, comprising: any prior device, a light source that emits light, and an imager, wherein the imager is configured to measure light transmitted through, scattered from, or reflected from the aggregated or deaggregated particles, or any combination thereof.

4. A method of performing a homogeneous assay employing particle aggregation, comprising the steps of:

(a) obtaining a sample containing or suspected of containing an analyte;

(b) obtaining a first plate and a second plate, wherein:

each of the plates has a sample contacting area on its respective inner surface for contacting the sample.

One or both of the plates comprises spacers, at least one of the spacers being within the sample contact area and the spacers having a predetermined substantially uniform height;

one or both of the plates comprises a plurality of isolated particles on respective inner surfaces;

(c) depositing the sample on one or both of the plates when the plates are in the open configuration, wherein in the open configuration the two plate portions are either completely separated and the spacing between the plates is not adjusted by the spacer;

(d) after (c), bonding the two panels together and pressing the panels into the closed configuration, wherein in the closed configuration: at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness defined by the sample surfaces of the two plates and adjusted by the spacer and the plates; and is

(e) The aggregated particles in the uniform thickness layer are detected and analyzed by an image or a disposable lumped optical signal when the plate is in the closed configuration.

5. A method of performing a homogeneous assay employing particle deagglomeration, comprising the steps of:

(a) obtaining a sample containing or suspected of containing an analyte;

(b) obtaining a first plate and a second plate, wherein:

each of the plates has a sample contacting area on its respective inner surface for contacting the sample;

one or both of the plates comprises a plurality of aggregated particles on the respective inner surface;

(e) Disaggregated particles in the uniform thickness layer are detected and analyzed by an image or a disposable lumped optical signal when the plate is in the closed configuration.

6. The device, system and method according to any preceding claims, wherein the particles are different in their optical properties selected from the group consisting of: photoluminescence, electroluminescence, and electrochemiluminescence, light absorption, reflection, transmission, diffraction, scattering, diffusion, surface raman scattering, and any combination thereof.

7. A device, system and method according to any preceding claim wherein the particles are biological/non-biological, organic/non-organic, magnetic/non-magnetic, metallic/non-metallic, luminescent/non-luminescent, or combinations thereof.

8. The device, system and method according to any of the preceding claims, wherein said particles are natural, man-made, or mixtures thereof.

9. The device, system and method of any of the above claims, wherein the particles are natural biological entities selected from cells, cell fragments, macromolecules, polysaccharides, proteins, nucleic acids, cell aggregates, tissues, viral particles or mixtures thereof.

10. The device, system and method according to any of the preceding claims, wherein said particles are selected from polymer particles, metal particles, magnetic particles, semiconductor particles, or combinations or mixtures thereof.

11. The device, system and method according to any of the preceding claims, wherein the particles are a polymer selected from polystyrene, polypropylene, polycarbonate, latex or any combination thereof.

12. A device, system and method according to any preceding claim wherein the particles are metal particles selected from silver, copper, platinum or mixtures thereof.

13. The device, system and method according to any of the preceding claims, wherein said particles are gold nanoparticles, gold nanoshell particles, gold nanotube particles or mixtures thereof.

14. The device, system and method according to any of the preceding claims, wherein the particles are a semiconductor selected from CdSe, CdS coated with ZnS, CdSe coated with ZnS or mixtures thereof.

15. The device, system and method according to any of the preceding claims, wherein the particles are magnetic particles selected from magnetite, magnetized ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, GaAs or mixtures thereof.

16. The device, system and method according to any of the preceding claims, wherein the particles have a shape selected from spheres, beads, rods, irregular shapes or combinations thereof.

17. An apparatus, system and method according to any preceding claim wherein the population of particles has particles of the same size or shape, particles of different shapes or sizes, or mixtures thereof.

18. The device, system and method according to any of the preceding claims, wherein said particles are nanoparticles having a size in the nanometer range; the particles are microparticles having micron-sized dimensions or mixtures thereof.

19. The device, system and method according to any of the preceding claims, wherein the particles are beads in a population of beads and have a uniform diameter, different diameters, or mixtures thereof.

20. The device, system and method of any of the above claims, wherein the particle size is from about 5nm to about 10 um.

21. The device, system, and method of any of the above claims, wherein the particle size is less than 2nm, 5nm, 10nm, 20nm, 50nm, 100nm, 200nm, 500nm, 1um, 2um, 5um, 10um, 20um, 50um, 100um, 200um, 500um, 1mm, 2mm, 5mm, 10mm, 20mm, 50mm, or 100mm, or within a range between any two values.

22. A device, system and method according to any preceding claim wherein the particles are beads and have a size of 100nm, 500nm, 1 μm, 5 μm, 50 μm, 500 μm, 1mm, or a range between any two of the values.

23. A device, system and method according to any preceding claim wherein the particles are beads and have a size of from 1 to 10 μm.

24. The device, system and method according to any of the preceding claims, wherein the particle size is from about 5nm to about 10 nm.

25. The device, system and method according to any of the preceding claims, wherein the particle size is from about 10nm to about 50 nm.

26. The device, system and method according to any of the preceding claims, wherein the particle size is from about 50nm to about 100 nm.

27. The device, system and method according to any of the preceding claims, wherein the particle size is from about 100nm to about 500 nm.

28. The device, system and method according to any of the preceding claims, wherein the particle size is from about 500nm to about 1000 nm.

29. The device, system and method according to any of the preceding claims, wherein the particle size is from about 1nm to about 5 nm.

30. The device, system and method according to any of the preceding claims, wherein the particle size is from about 5nm to about 10 nm.

31. The device, system, and method of any of the above claims, wherein the particle size is less than 2nm, 5nm, 10nm, 20nm, 50nm, 100nm, 200nm, 500nm, 1um, 2um, 5um, 10um, 20um, 50um, 100um, 200um, 500um, 1mm, 2mm, 5mm, 10mm, 20mm, 50mm, or 100mm, or within a range between any two values; in some embodiments, the beads can have a size of 100nm, 500nm, 1 μm, 5 μm, 50 μm, 500 μm, 1mm, or a range between any two of the values, and a preferred range of 1 μm to 10 μm.

32. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 1.5 μ ι η to 2.5 μ ι η.

33. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 2.5 μ ι η to 4 μ ι η.

34. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 4 μ ι η to 6 μ ι η.

35. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 6 μ ι η to 10 μ ι η.

36. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 10 μ ι η to 15 μ ι η.

37. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 15 μ ι η to 25 μ ι η.

38. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 25 μ ι η to 35 μ ι η.

39. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 35 μ ι η to 50 μ ι η.

40. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 50 μ ι η to 100 μ ι η.

41. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 100 μ ι η to 150 μ ι η.

42. The device, system and method of any of the above claims, wherein the spacer height, spacing between the plates, the sample thickness, or any combination thereof is from 150 μ ι η to 200 μ ι η.

43. The device, system and method of any preceding claim, wherein the binding agent and the particles are coated or surface modified with a reagent to enhance binding of a selected analyte.

44. The device, system and method of any preceding claim, further comprising a silica coating, streptavidin, or a combination thereof on the surface of the particles.

45. A device, system and method according to any preceding claim wherein aggregation of the particles is induced by binding between the particles or between agents located on the surface of the particles.

46. The device, system and method according to any of the preceding claims, wherein the binding of the agent is the result of a biological, biochemical, chemical, physical or magnetic effect.

47. The device, system and method according to any of the preceding claims, wherein the binding between the agents is selected from antibody-antigen binding, complementary binding of nucleic acids, binding between a catalyst and its substrate, binding or aptamer to its target, binding of RNA interference sequences to its target, ligand-receptor binding, or binding between an agent and its agonist or antagonist.

48. The device, system and method according to any of the preceding claims, wherein said particle aggregation occurs with binding of a binding agent to said particles and is native or induced.

49. A device, system and method according to any preceding claim wherein approximately 100% of the particles in the sample aggregate prior to contacting the analyte.

50. A device, system and method according to any preceding claim wherein the percentage of aggregated particles prior to contact with the analyte is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or in a range between any two of said values.

51. A device, system and method according to any preceding claim wherein the percentage of aggregated particles prior to contacting the analyte is greater than 60%, 70%, 80%, 90%, 95% or 99%, or in a range between any two of said values.

52. A device, system and method according to any preceding claim wherein approximately 100% of the particles in the sample aggregate after contacting the analyte.

53. A device, system and method according to any preceding claim wherein the percentage of aggregated particles after contacting the analyte is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, or in a range between any two of the values.

54. A device, system and method according to any preceding claim wherein the percentage of aggregated particles after contacting the analyte is greater than 60%, 70%, 80%, 90%, 95% or 99%, or in a range between any two of the values.