CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/105,128, filed on October 23, 2020, the contents of which are relied upon and incorporated herein by reference in their entirety. The entire disclosure of any publication or patent document mentioned herein is entirely incorporated by reference.

FIELD

The present disclosure relates to devices, and methods for achieving high sensitivity assay simply, rapidly (as short as 60 seconds) with low cost and by a non-professional.

BACKGROUND

In achieving a high sensitive assay, the beads are often used to capture an analyte in a liquid sample. Then the beads with captured analytes will be further treated in other liquid (such as being mixed with a liquid of labeled detection agent) and will be analyzed. For a high sensitivity, prior to the bead analysis, often the beads with analytes are separated from the original liquid sample and/or the liquid with labeled detection agent. The separation is often accomplished by washing, a magnetic separation, a centrifuge separation, or a combination. Such separation step(s) needs multiple operation steps and time consuming.

One of the aspects of the present invention is the devices and methods that simplify the steps (hence the time and cost) in an assay that uses beads to capture an analyte, and in achieving a high sensitivity.

SUMMARY

The present invention is related to devices and methods for high sensitivity assay simply, rapidly (as short as 60 seconds) at extremely low cost and used by a non-professional.

One aspect of the present invention is to achieve a high sensitivity assay in a simple low-cost device with minimum steps.

Another aspect of the present invention is that the barrier selector assay self-separates the beads that capture an analyte in a liquid sample from the liquid sample. Another aspect of the present invention is to wash away the unbounded labels (e.g. detection labels) in a sample using a simple method. The washing away allows an increase the analyte detection sensitivity significantly.

Another aspect of the present invention is to use a selective barrier and a lateral flow of washing solution to separate the captured analyte and the unbonded labels.

Another aspect of the present invention is to use beads to capture the analytes.

Another aspect of the present invention is to make the gap between the two plates with multiple size (i.e. multiple gaps), which is achieved by having a multi height on the plate surface.

In some embodiments, the method of separate the beads from the unbounded labels using washing with a lateral flow and a selective-barrier (WLS) can be used for analyte a cell. Under proper conditions, the device will control the cells similar to the beads.

In some embodiments, the prevent invention provides a device for assaying an analyte in a liquid sample, comprising, a first plate, a second plate, beads, and a barrier selector for selecting beads; wherein: the first plate and the second plate form a microfluidic channel, having an inlet for the liquid sample; the microfluidic channel comprising two sections, wherein the first section of the microfluidic channel has one end as the liquid sample inlet and the other end connected to the second section of the microfluidic channel; the beads capture the analyte in the liquid sample; the first section has a channel height selected to allow the beads flow in the first section microfluidic channel; and the second section has a channel height selected to not allow the beads to flow; thereby the interface between the first section and the second section forms a barrier selector for selecting beads, that, as the liquid sample flow from the first section to the second section, allows the liquid sample flowing through but stops the beads at the barrier selector.

In some embodiments, the device further comprises more than one channel, each has a first section and a second and the bead barrier at the interface between the first section and the second section; and (b) more than one types of beads, each type has a diameter and capture a type of analyte; wherein each the first sections has a channel height selected to allow the beads flow in the first section microfluidic channel; and each of the second sections has a channel height selected to not allow the beads to flow.

In some embodiments, the device further comprises an imager that images the beads by taking one or more images.

In some embodiments, the images are bright field image, dark field image, fluorescence image, or any combination thereof.

In some embodiments, the beads capture the analyte using a capture agent that is selected to specifically capture the analyte. In some embodiments, the device further comprises a detection agent in the liquid sample.

In some embodiments, the device further comprises a detection agent that coated on the inner surface of the first channel section.

In some embodiments, the device further comprises a hydrophilic surface that generate a capillary force that suck the liquid sample from the inlet of the microfluidic channel into the first channel section first and then second channel section.

In some embodiments, the device further comprises a hydrophilic surface that generate a capillary force that suck the liquid sample from the inlet of the microfluidic channel into the first channel section first and then second channel section, wherein the area of the second channel section is selected to be large enough, so that all liquid sample eventually will be the second channel section.

In some embodiments, the channel width is 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, 200 urn, 300 urn, 500 urn, 800 urn, 1 mm, 2 mm, 3 mm, 5 mm, 10 mm, 15 mm, 20 mm, 20 mm, 50 mm, and any values between the two.

In some embodiments, the first section and the second section comprise respectively spacers that regulate the channel height in each of the section.

In some embodiments, the channel height of first section is 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, 150 urn or in a range between any two of the values, while the channel of second section is 1 urn, 2 urn, 3 urn, 5 urn, 10 urn or in a range between any two of the values.

In some embodiments, the channel height of first section is 10 urn, 20 urn, 30 urn, 50 urn, or in a range between any two of the values, while the channel height of second section is 2 urn, 3 urn, 5 urn, 8 urn, 9 urn or in a range between any two of the values.

In some embodiments, the comprises the channel height of each section is selected from 0.5 urn, 1 urn, 2 urn, 3 urn, 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, 150 urn or in a range between any two of the values.

In some embodiments, the preferred spacing of one section is 2 urn, 3 urn, 5 urn, 10 urn, or in a range between any two of the values.

In some embodiments, the preferred spacing of one section is 10 urn, 30 urn, 50 urn, 100 urn, or in a range between any two of the values.

In some embodiments, the difference between two spacing of each section is 0.5 urn, 1 urn, 2 urn, 3 urn, 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, or in a range between any two of the values.

In some embodiments, the difference between two spacing of each section is 3 urn, 5 urn, 10 urn, 20 urn, or in a range between any two of the values. In some embodiments, the beads has a size of 0.5 urn, 1 urn, 2 urn, 3 urn, 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, or in a range between any two of the values.

In some embodiments, the device further comprises the beads haves a preferred size of 5 urn, 6 urn, 8 urn, 10 urn, 20 urn, or in a range between any two of the values.

In some embodiments, the channel height the capture agent includes proteins (e.g., antibodies, antigens), and nucleic acids (e.g., oligonucleotides, DNA, RNA including aptamers).

In some embodiments, the channel height the capture agent is anti-CRP antibody for CRP detection, anti-COVID virus antibody for COVID virus detection, COVID antigen for COVID antibody detection.

In some embodiments, the channel height the spacing between the spacer is 5 urn, 10 urn, 30 urn, 50 urn, 80 urn, 100 urn, 200 urn, 300 urn, 500 urn, 1 mm or in a range between any two of the values.

In some embodiments, the channel height the spacing between the spacer is 50 urn, 80 urn, 100 urn, 200 urn, or in a range between any two of the values

In some embodiments, the present invention provides a method of assaying an analyte in a liquid sample, comprising: obtaining any device of prior claim; flowing the liquid sample from the inlet into the first channel section and then second channel section; making the liquid sample only in the second channel section; and observing the beads trapped at the barrier selector for the bead selection.

A method of claim 25, further comprising, after flowing the liquid sample, a step of flowing a washing solution into the channel from the inlet.

A method of claim 25, further comprising, after flowing the liquid sample, a step of flowing a washing solution into the channel from the inlet.

A method of claim 25, wherein the device detect the analyte captured by the beads without a washing step.

A method of claim 25, wherein the beads are imaged by an imager by taking one or more images.

A method of claim 25, wherein the image are bright field image, dark field image, fluorescence image, or any combination thereof.

A method of claim 25 further comprising one more steps of flowing a second liquid after flowing the liquid sample, wherein the second liquid contains a reagent.

Brief Description of the Drawings

The drawings, described below, are for illustration purposes only. In some Figures the drawings are in scale and not to scale in other Figures. For clarity purposes, some elements are enlarged when illustrated in the Figures. The drawings are in scale in some figures, but not to scale in other Figures. The drawings are not intended to limit the scope of the disclosure. The drawings assist in understanding embodiments or aspects of embodiments of the present invention.

Fig. 1 shows one embodiment of the devices and methods from (a) top-view and (b) crosssection view. The device comprises a first plate and a second plate. The sample is between the first plate and second plate. The device has at least two areas (area 1 and area 2) with two spacing heights (e.g. sub-diameter spacing 1 and over-diameter spacing 2). Liquid sample with beads is flowed into the device from the sample entrance at spacing 2 area to spacing 1 area. In one embodiment, the spacing-1 has a size smaller than bead size, and bead size is smaller than spacing-2. Once beads arriving interface or boundary line of the two areas, the beads are trapped and enriched at the line, while liquid are continuously sucked into spacing- 1 area.

Fig. 2 shows one method of trapping and enriching beads at the interface boundary line between the two areas with sub-diameter spacing-1 and over-diameter spacing-2, (a) Add sample with beads into spacing-2 area from sample entrance; (b) Liquid in sample are sucked into spacing-1 area, while leaving beads at the interface of the two areas; (c) In some embodiment, all the liquid is sucked into spacing-1 area, leaving all the beads at the boundary of the two areas. In some other embodiment, by controlling the total area of spacing-1 , not all the liquid is sucked into spacing-1 area.

Fig. 3 shows one embodiment of the devices for multiplexing assay with 3 samples (Sample- 1 , Sample-2 and Sample-3) containing 3 beads (Bead-1 , Bead-2 and Bead-3) flowing into 3 channels (Channel-1 , Channel-2 and Channel-3), (a) Three samples are added into three spacing-2 areas separately. The spacing-2 area has 3 separated branches in this device, (b) All liquid is sucking into spacing-1 , while leaving beads in 3 separated boundary lines for analyzing.

Fig. 4 shows an example of device using multi spacing device controlling beads and performing assay, (a) shows the top view and (b) shows the cross-section schematic of 2 area arrangement with spacing 1 and spacing 2 in one device, (c) shows one example microscopy photo of such device testing samples containing 10 urn size beads. Spacing-1 is around 5 urn while spacing-2 is around 30 urn. The samples with beads are added from spacing-2 area and flowing into spacing-1 area. After flowing, the beads are trapped and enriched at the interface or boundary line of two areas.

Fig. 5 shows an example of device using multi spacing device controlling beads and performing assay, (a) shows the bright field image of device at interface line with trapped and enriched beads, (b) shows the fluorescence image of device at interface line with trapped and enriched beads. The beads have sandwich immunoassay (capture antibody, antigen, and labeled detection antibody) on it with green fluorescence label.

Fig. 6 shows an example of device added at least two kinds of liquid into it. Liquid-1 has beads, (a-c) Same as Fig.2, after flowing liquid-1 into the device from the sample entrance at spacing- 2 area, the beads are trapped and enriched at the boundary of two area, (d) Add liquid-2 as washing buffer solution into the device, (e) After liquid-2 reaching the boundary, the liquid is further sucked into spacing-1 passing through the beads, (f) Finally, all liquid is sucked into spacing-1 area. In some other embodiment, by controlling the total area of spacing-1 , not all the liquid is sucked into spacing-1 area. With above process, the beads are washed by the liquid-2.

Fig. 7 shows another example of device added at least two kinds of liquids into it. Liquid-1 has beads, (a) After flowing liquid-1 into the device from the sample entrance at spacing-2 area, by controlling the liquid volume, the liquid does not touch the interface line of two areas, thus the beads are not yet trapped and enriched at the boundary of two area, (b) Add liquid-2 as washing buffer solution into the device, (c) After liquid 1 reaching the boundary, the liquid is further sucked into spacing-1 passing through the beads, until all liquid is sucked into spacing- 1 area, leaving beads on the boundary of two areas. With above process, the beads are washed by the liquid-2.

Detailed Description

The following detailed description illustrates certain embodiments of the invention by way of example and not by way of limitation. If any, 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 contents under a section heading and/or subtitle are not limited to the section heading and/or subtitle, but apply to the entire description of the present invention.

Definition

The terms “liquid”, “solution” and “fluidic” are interchangeable.

The terms “liquid”, “sample” and “liquid sample” are interchangeable.

The terms “inlet” and “entrance” of a microfluidic channel are interchangeable.

The terms “a section of the microfluidic channel” and “a channel section” of a microfluidic channel are interchangeable. The terms “the gap” between the two plates” and “the channel height” of a microfluidic channel formed by two plate are interchangeable.

The term “inner surface” of a plate refers to the surface of the plate that is facing another plate, wherein the two plates form a microfluidic channel.

The term “specific binding” refers to an agent (e.g. capture agent or detection agent) binds to a target analyte through an interaction that is sufficient to permit the agent to bind and concentrate the target molecule from a heterogeneous mixture of different molecules. The binding interaction is typically mediated by an affinity region of the capture agent. Typical capture agents include any moiety that can specifically bind to a target analyte. Certain capture agents specifically bind a target molecule with a dissociation constant (Kn) of less than about 10' ⁶ M ( e.g., less than about 10’ ⁷ M, less than about 10’ ⁸ M, less than about 10’ ⁹ M, less than about 10' ¹⁹ M, less than about 10' ¹¹ M, less than about 10’ ¹² M, to as low as 10’ ¹⁶ M, or a range between any two of the values) without significantly binding to other molecules.

Devices

According to the present invention, as illustrated in Fig 1 , one embodiment of the devices for assaying an analyte in a liquid sample using beads, microfluidic channel, and a barrier based bead selector, comprising a first plate and a second plate, forming a lateral flow microfluidic channel. The microfluidic channel has an inlet (or entrance). A channel height is defined by the separation between the inner surfaces of the plates. The device also comprises the beads that capture the analyte in the liquid sample. The microfluidic channel comprising two sections. The first section is the section of the microfluidic channel next to the inlet. The first section has a channel height selected to allow the beads flow in the first section microfluidic channel. The second section of the microfluidic channel is connected after the first section. The second section has a channel height selected to not allow the beads to flow. Therefore, when a bead in a liquid sample flows in the first section of the microfluidic channel towards to the second section of the microfluidic channel, the bead will stop at the interface between the first section and the second section of the microfluidic channel. If the lateral flow in the microfluidic channel is caused by capillary force of the microfluidic channel, then the second section of the microfluidic channel can, due to smaller channel height and a stronger capillary force, suck the liquid sample out of the first section of the microfluidic channel, leaving the bead stopped at the interface between the first section and the second channel and leaving the less or no liquid sample in the first channel section. With less liquid or liquid in the first section of the microfluidic channel can enhance the signal of the bead, because less background signals from the liquid. According to the present invention, a device for assaying an analyte in a liquid sample, comprising, a first plate, a second plate, beads, and a barrier selector for selecting beads; wherein:

(a) the first plate and the second plate form a microfluidic channel, having an inlet for the liquid sample;

(b) the microfluidic channel comprising two sections, wherein the first section of the microfluidic channel has one end as the liquid sample inlet and the other end connected to the second section of the microfluidic channel;

(c) the beads capture the analyte in the liquid sample;

(d) the first section has a channel height selected to allow the beads flow in the first section microfluidic channel; and

(e) the second section has a channel height selected to not allow the beads to flow; thereby the interface between the first section and the second section forms a barrier selector for selecting beads, that, as the liquid sample flow from the first section to the second section, allows the liquid sample flowing through but stops the beads at the barrier selector.

The microfluidic channel has a width in plane of the plates. The channel width can be defined by confining a liquid flow using solid wall, or a trench on the plate surface (using capillary force) or a combination. The channel width is at least three times of the bead diameter.

The channel width is 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, 200 urn, 300 urn, 500 urn, 800 urn, 1 mm, 2 mm, 3 mm, 5 mm, 10 mm, 15 mm, 20 mm, 20 mm, 50 mm, and any values between the two.

In some embodiments, the device can detect the analyte captured by the beads without a washing step.

In some embodiments, the beads are imaged by an imager by taking one or more images.

In some embodiments, the image are bright field image, dark field image, fluorescence image, or any combination thereof.

In some embodiments, the beads capture the analyte using a capture agent that is selected to specifically capture the analyte.

In some embodiments, detection agent in the liquid sample. In some embodiments, the detection agent is coated on the inner surface of the first channel section.

In some embodiments, the first section and the second section comprising respectively spacers that regulate the channel height in each of the section.

In some embodiments, there is a detection agent in the liquid that can bind to the analyte and has a label on it. In some embodiments, the gap of first section is 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, 150 urn or in a range between any two of the values, while the gap of second section is 1 urn, 2 urn, 3 urn, 5 urn, 10 urn or in a range between any two of the values.

In some embodiments, the preferred gap of first section is 10 urn, 20 urn, 30 urn, 50 urn, or in a range between any two of the values, while the gap of second section is 2 urn, 3 urn, 5 urn, 8 urn, 9 urn or in a range between any two of the values.

In some embodiments, the gap of each section is selected from 0.5 urn, 1 urn, 2 urn, 3 urn, 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, 150 urn or in a range between any two of the values.

The device of any prior device claim, wherein the preferred spacing of one section is 2 urn, 3 urn, 5 urn, 10 urn, or in a range between any two of the values.

The device of any prior device claim, wherein the preferred spacing of one section is 10 urn, 30 urn, 50 urn, 100 urn, or in a range between any two of the values.

The device of any prior device claim, wherein the difference between two spacing of each section is 0.5 urn, 1 urn, 2 urn, 3 urn, 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, or in a range between any two of the values.

The device of any prior device claim, wherein the prefered difference between two spacing of each section is 3 urn, 5 urn, 10 urn, 20 urn, or in a range between any two of the values.

The device of any prior device claim, wherein the bead has a size of 0.5 urn, 1 urn, 2 urn, 3 urn, 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, or in a range between any two of the values.

The device of any prior device claim, wherein the beads haves a preferred size of 5 urn, 6 urn, 8 urn, 10 urn, 20 urn, or in a range between any two of the values.

In one embodiment, the capture agent includes proteins (e.g., antibodies, antigens), and nucleic acids (e.g., oligonucleotides, DNA, RNA including aptamers).

In one embodiment, the capture agent is anti-CRP antibody for CRP detection, anti- COVI D virus antibody for COVI D virus detection, COVI D antigen for COVI D antibody detection.

In one embodiment, the spacing between the spacer is 5 urn, 10 urn, 30 urn, 50 urn, 80 urn, 100 urn, 200 urn, 300 urn, 500 urn, 1 mm or in a range between any two of the values.

In one embodiment, the preferred spacing between the spacer is 50 urn, 80 urn, 100 urn, 200 urn, or in a range between any two of the values.

Microfluidic Channel Formed by Using Two Moving Plates.

According to the present invention, in some embodiments, the first plate and the second plate are movable relative to each other into different configuration, inclusing an open configuration and a closed configuration, wherein the liquid sample is dropped on the first section when the plates are an open configuration (instead on the inlet of the microfluidic channel), wherein an open configuration, in which: the two plates are partially or entirely separated apart, the spacing between the plates is not regulated by the spacers; wherein the close configuration, in which: at least part of the deposited sample is compressed by the two plates into a layer that is confined by the two plates and has a respective substantially uniform thickness.

A-1. A method of assaying an analyte in a liquid sample, comprising: a. obtaining one or more beads that comprising a capture agent immobilized on the surface of the beads, wherein the capture agent binds to the analyte in the sample that contains or is suspected of containing the analyte; b. obtaining a detection agent that binds to the analyte or the capture agent, wherein the detection agent comprises a label; c. obtaining two plates, wherein at least one of the plate surface has a different height (Fig. 1), so that the two plates can, when facing each other, form two gaps, wherein one of the gaps is a sub-diameter gap that is smaller than the diameter of the beads, and the other gap is over-diameter gap that is larger than the diameter of the beads; d. having the sample, the beads, and the detection agent between the two plates and in the area of the over-diameter gap, and having at least a part of the area of the sub-diameter gap without the sample, wherein some of the detection agent bind to the analyte captured on the beads or the capture agent on the bead, and some of the detection agent are unbound in the sample; e. making a washing solution to contact one side edge of the two plates, wherein the side has an over-diameter gap; wherein the surfaces of the plates are hydrophilic and the washing solution flow into the plates and a capillary force causing the sample and the washing solution flow lateral to the plate in the direction from the over-diameter gap to the sub-diameter gap; wherein the flow bring the bead in the over-diameter area to the interface between the overdiameter area and the sub-diameter area and stop at the interface; wherein the washing solution has a sufficient amount so that at least some of the beads has less unbounded detection agent than without the wash solution; and f. detection of the signal of detection agent on the at least some of the beads.

A-2. A kit for assaying an analyte in a liquid sample, comprising: a. one or more beads that comprising a capture agent immobilized on the surface of the beads, wherein the capture agent binds to the analyte in the sample that contains or is suspected of containing the analyte; b. a detection agent that binds to the analyte or the capture agent, wherein the detection agent comprises a label; c. two plates, wherein at least one of the plate surface has a different height (Fig. 1), so that the two plates can, when facing each other, form two gaps, wherein one of the gaps is a sub-diameter gap that is smaller than the diameter of the beads, and the other gap is over-diameter gap that is larger than the diameter of the beads; d. a washing solution that is capable of flowing into the plates through a capillary force.

In some embodiments, the method of separating the beads from the unbounded labels using washing with a lateral flow and a selective-barrier (WLS) can be used for analyte a cell. Under proper conditions, the device will control the cells similar to the beads.

A-3. A method of assaying a cell in a liquid sample, comprising: a. obtaining the sample that contains or is suspected of containing the cell to be analyzed; b. obtaining a detection agent that binds or stain the cell (either the cell surface, inside of the cell or both), wherein the detection agent comprises a label; c. obtaining two plates, wherein at least one of the plate surface has a different height (Fig. 1), so that the two plates can, when facing each other, form two gaps, wherein one of the gaps is a sub-diameter gap that is smaller than the diameter of the beads, and the other gap is over-diameter gap that is larger than the diameter of the beads; d. having the sample and the detection agent between the two plates and in the area of the over-diameter gap, and having at least a part of the area of the subdiameter gap without the sample, wherein some of the detection agent bind or penetrate into the cell, and some of the detection agent are unbound; e. making a washing solution to contact one side edge of the two plates, wherein the side has an over-diameter gap; wherein the surfaces of the plates are hydrophilic and the washing solution flow into the plates and a capillary force causing the sample and the washing solution flow lateral to the plate in the direction from the over-diameter gap to the sub-diameter gap; wherein the flow bring the bead in the over-diameter area to the interface between the overdiameter area and the sub-diameter area and stop at the interface; wherein the washing solution has a sufficient amount so that at least some of the beads has less unbounded detection agent than without the wash solution; and f. detection of the signal of detection agent on the at least some of the beads.

A-4. A kit for assaying an cell in a liquid sample, comprising: a. a detection agent that binds to the cell or the capture agent, wherein the detection agent comprises a label; b. two plates, wherein at least one of the plate surface has a different height (Fig. 1), so that the two plates can, when facing each other, form two gaps, wherein one of the gaps is a sub-diameter gap that is smaller than the diameter of the beads, and the other gap is over-diameter gap that is larger than the diameter of the beads; c. a washing solution that is capable of flowing into the plates through a capillary force.

In certain embodiments, the sample is flow from the edge of the two plates into the over-diameter gap area.

In certain embodiments, the sample is flow from the holes on one of the two plates into the over-diameter gap area.

In certain embodiments, the detection agent can penetrate into the cell.

In certain embodiments, the detection agent is a combination of multi agent, such as biotinylated detection antibody and dye with streptavidin.

A device for beads control and liquid sample analysis, comprising:

(a) at least two area, each area has one spacing height (spacing- 1 and spacing-2); wherein a portion of the one area is adjacent to the other area; wherein the height of spacing-1 is smaller than spacing-2;

(b) a boundary line between the two areas;

(c) an exterior liquid sample contact area on an exterior location of the device adjacent to the area with spacing-2; wherein at least some of target beads for analyzing has a size between the spacing heights of two areas; e.g. the spacing-1 is smaller than the bead size, while the bead size is smaller than the spacing-2.

A method of beads control and liquid sample analysis, comprising the steps of: (a) obtaining a device of the embodiment; (b) dropping the sample with target beads onto the exterior liquid sample contact area with spacing-2;

(c) the sample with beads is guided flow into the device from spacing-2 area to spacing-1 area due to capillary force; and the beads with size larger than spacing- 1 is trapped and enriched at the boundary line between the two areas;

(d) imaging and analyzing the beads at the boundary line;

In one embodiment, the signal measured is beads numbers.

In one embodiment, the signal measured is fluorescence intensity of beads.

In one embodiment, the signal measured is fluorescence intensity over the size of beads.

In one embodiment, the signal measured are both bright field and fluorescence images of beads.

In one embodiment, the beads for analyzing are cells.

In one embodiment, the signal measured is the number of cells at the separation interface over the total number of cells.

In one embodiment, the signal measured is the cell number in the sub-diameter gap area and over-diameter gap area.

In one embodiment, the device sorting the cell based on the cell size relationship to the gap.

In one embodiment, the parameters measured in each area is complete blood count including but not limit to white blood cell count, red blood cell count, platelet count, white blood cell differentiation and count e.g. neutrophils, lymphocytes, monocytes, eosinophils and basophils - as well as abnormal cell types if they are present.

Fig. 1 shows one embodiment of the devices and methods from (a) top-view and (b) cross-section view. The device comprises a first plate and a second plate. The sample is between the first plate and second plate. The device has at least two areas (area 1 and area 2) with two spacing heights (e.g. sub-diameter spacing 1 and over-diameter spacing 2). Liquid sample with beads is flowed into the device from the sample entrance at spacing 2 area to spacing 1 area. In one embodiment, the spacing-1 has a size smaller than bead size, and bead size is smaller than spacing-2. Once beads arriving interface or boundary line of the two areas, the beads are trapped and enriched at the line, while liquid are continuously sucked into spacing-1 area. Fig. 2 shows one method of trapping and enriching beads at the interface boundary line between the two areas with sub-diameter spacing-1 and over-diameter spacing-2, (a) Add sample with beads into spacing-2 area from sample entrance; (b) Liquid in sample are sucked into spacing-1 area, while leaving beads at the interface of the two areas; (c) In some embodiment, all the liquid is sucked into spacing-1 area, leaving all the beads at the boundary of the two areas. In some other embodiment, by controlling the total area of spacing-1 , not all the liquid is sucked into spacing-1 area.

Fig. 3 shows one embodiment of the devices for multiplexing assay with 3 samples (Sample-1 , Sample-2 and Sample-3) containing 3 beads (Bead-1 , Bead-2 and Bead-3) flowing into 3 channels (Channel-1 , Channel-2 and Channel-3), (a) Three samples are added into three spacing-2 areas separately. The spacing-2 area has 3 separated branches in this device, (b) All liquid is sucking into spacing-1 , while leaving beads in 3 separated boundary lines for analyzing.

The devices or methods of above example, the spacing heights for three samples can be different, but are all larger than the corresponding beads size.

The devices or methods of any prior embodiment, wherein the device has two areas, while each area has one spacing height.

The devices or methods of any prior embodiment, wherein the device has more than two areas, while each area has one spacing height.

The liquid sample are added into the area with higher spacing height between two areas.

The devices or methods of any prior embodiment, wherein one of the areas have a shape selected from round, polygonal, circular, square, rectangular, oval, elliptical, or any combination of the same.

The devices or methods of any prior embodiment, wherein the separation interface between the two area is a straight line or a curved line.

The device of any prior device claim, wherein the spacing of each area is 1 urn, 2 urn, 3 urn, 5 urn, 10 urn, 20 urn, 30 urn, 50 urn, 100 urn, 150 urn or in a range between any two of the values.

The device of any prior device claim, wherein the preferred spacing of one area is 2 urn, 3 urn, 5 urn, 10 urn, or in a range between any two of the values.

The device of any prior device claim, wherein the preferred spacing of one area is 10 urn, 30 urn, 50 urn, 100 urn, or in a range between any two of the values. The device of any prior device claim, wherein the difference between two spacing of each area is 0.5 um, 1 um, 2 um, 3 um, 5 um, 10 um, 20 um, 30 um, 50 um, 100 um, 150 um, or in a range between any two of the values.

The device of any prior device claim, wherein the beads has a size of 0.5 um, 1 um, 2 um, 3 um, 5 um, 10 um, 20 um, 30 um, 50 um, or in a range between any two of the values.

The device of any prior device claim, wherein the beads have a preferred size of 5 um, 6 um, 8 um, 10 um, 20 um, or in a range between any two of the values.

The device of any prior device claim, wherein the beads are polystyrene beads, PMMA beads, or magnetic beads.

The device of any prior device claim, wherein the difference between one spacing and beads is 0.1 um, 0.5 um, 1 um, 2 um, 3 um, 5 um, 10 um, 20 um, or in a range between any two of the values.

The device of any prior device claim, wherein the ratio of the manufacturing spacing height between two area is 1.1 fold, 1.2 fold, 1.5 fold, 2 fold, 3 fold, 5 fold, 10 fold, 30 fold, 50 fold, 100 fold, or in a range between any two of the values.

The device of any prior device claim, wherein the area of one area is 1000 um ² , 2500 um ² , 5000 um ² , 10000 um ² , 50000 um ² , 1 mm ² , or in a range between any two of the values.

The device of any prior device claim, wherein the area of one area is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the total device area, or in a range between any two of the values.

The device of any prior device claim, wherein the area of one area is 1 mm ² , 2 mm ² , 5 mm ² , 16 mm ² , 50 mm ² , 100 mm ² or in a range between any two of the values.

The device of any prior device claim, wherein one of the plates is fabricated by imprint lithography.

The device of any prior device claim, wherein one of the plates is fabricated by injection molding.

Fig. 4 shows an example of device using multi spacing device controlling beads and performing assay, (a) shows the top view and (b) shows the cross-section schematic of 2 area arrangement with spacing 1 and spacing 2 in one device, (c) shows one example microscopy photo of such device testing samples containing 10 um size beads. Spacing-1 is around 5 um while spacing-2 is around 30 um. The samples with beads are added from spacing-2 area and flowing into spacing-1 area. After flowing, the beads are trapped and enriched at the interface or boundary line of two areas.

In one example of above device,

The plate 1 has a thickness of 200 um to 1500 um. The plate 2 has a thickness of 50 um to 250 um.

The area 1 with pillar 1 array on the plate 2 has a pillar height from 3 um to 6 um with a inter pillar distance of 100 um to 200 um and a pillar size around 5 um to 40 um.

The area 2 with pillar 2 array on the plate 2 has a pillar height from 10 um to 50 um with a inter pillar distance of 100 um to 200 um and a pillar size around 10 um to 40 um.

The pillar array can also be fabricated on the plate 1 .

The size of area 1 have around 0.5 mm length and 1 mm width.

The size of area 2 have more than 2 mm length and 2 mm width.

Fig. 5 shows an example of device using multi spacing device controlling beads and performing assay, (a) shows the bright field image of device at interface line with trapped and enriched beads, (b) shows the fluorescence image of device at interface line with trapped and enriched beads. The beads have sandwich immunoassay (capture antibody, antigen, and labeled detection antibody) on it with green fluorescence label.

In one example, the beads are pre-coated with capture antibody for target antigen, the beads are then pre-mixed with antigen in sample and detection antibody with fluorescence label. After incubation, the beads are added into device and trapped at the boundary region as shown above. Then the device and beads in the device are imaged and analyzed using both bright field and fluorescence optic system. The beads are recognized, located, and marked in bright field. Then the fluorescence intensity for each bead is detected and analyzed to calculate the antigen concentration in the sample.

In some embodiment, the beads above are not pre-mixed with antigen in sample and detection antibody with fluorescence label. Instead, the beads and detection antibody are pre-coated and dried at spacing-2 region during the manufacturing of the device. During usage of device, the antigen are added into device and mixed with beads and detection antibody.

In some embodiment, another liquid can be added after adding beads sample. The liquid includes but not limited to washing buffer, enhancement buffer, detection buffer, beads separation buffer and others.

Fig. 6 shows an example of device added at least two kinds of liquid into it. Liquid-1 has beads, (a-c) Same as Fig.2, after flowing liquid-1 into the device from the sample entrance at spacing-2 area, the beads are trapped and enriched at the boundary of two area, (d) Add liquid-2 as washing buffer solution into the device, (e) After liquid-2 reaching the boundary, the liquid is further sucked into spacing-1 passing through the beads, (f) Finally, all liquid is sucked into spacing-1 area. In some other embodiment, by controlling the total area of spacing-1 , not all the liquid is sucked into spacing-1 area. With above process, the beads are washed by the liquid-2.

Fig. 7 shows another example of device added at least two kinds of liquids into it. Liquid-1 has beads, (a) After flowing liquid-1 into the device from the sample entrance at spacing-2 area, by controlling the liquid volume, the liquid does not touch the interface line of two areas, thus the beads are not yet trapped and enriched at the boundary of two area, (b) Add liquid-2 as washing buffer solution into the device, (c) After liquid 1 reaching the boundary, the liquid is further sucked into spacing-1 passing through the beads, until all liquid is sucked into spacing-1 area, leaving beads on the boundary of two areas. With above process, the beads are washed by the liquid-2.

In some embodiment, a coating is on at least one interior opposing surface of at least one of the plates, or both. The coating uses hydrophilic treatment, including but not limit to dielectric material coating, silicon oxide coating, plasma treatment, ozone treatment, polymer coating, acid-base treatment, surfactant chemical coating.

In some embodiments, the wetting angle at one interior surface is 10 °, 20 °, 30 °, 45°, 60°, 75° or in a range between any of these values.

The assay performed locally at beads or cells includes but not limit to colorimetric assay, immunoassay, cell counting, cell staining, and others.

In some embodiments, different assay is performed on different beads in the device.

In some embodiments, there is another spacing-3 area adjacent to spacing-1 area which has spacing height smaller than spacing-1 to further sucking out the liquid in spacing- 1.

In some embodiments, the two plates are QMAX device with spacers.

In some embodiments, the analyte comprises protein, peptide, nucleic acids, virus, bacterial, cell, nanoparticle, molecule, synthetic compounds, inorganic compounds, or any combination thereof.

In some embodiments, the detection of the signal of the detection agent comprises taking images, wherein the image comprises bright field image, dark field image, fluorescence image, or phosphorescence image.

In some embodiments, the detection of the signal of the detection agent comprises taking images, wherein one image is a bright field image and another image is fluorescence image, wherein the both bright filed image the fluorescence image are used in detection.

One advantage of using different types of images to analyze the signal of the detection agent is to distinguish the real signal from the detection agent from the false signal generated by others (scattering, dust, air bubble, etc). Machine Learning

Details of the Network are described in detail in a variety of publications including International Application (IA) No. PCT/US2018/017504 filed February 8, 2018, and PCT/US2018/057877 filed October 26, 2018, each of which are hereby incorporated by reference herein for all purposes.

One aspect of the present invention provides a framework of machine learning and deep learning for analyte detection and localization. A machine learning algorithm is an algorithm that is able to learn from data. A more rigorous definition of machine learning is “A computer program is said to learn from experience E with respect to some class of tasks T and performance measure P, if its performance at tasks in T, as measured by P, improves with experience E.” It explores the study and construction of algorithms that can learn from and make predictions on data - such algorithms overcome the static program instructions by making data driven predictions or decisions, through building a model from sample inputs.

Deep learning is a specific kind of machine learning based on a set of algorithms that attempt to model high level abstractions in data. In a simple case, there might be two sets of neurons: ones that receive an input signal and ones that send an output signal. When the input layer receives an input, it passes on a modified version of the input to the next layer. In a deep network, there are many layers between the input and output (and the layers are not made of neurons but it can help to think of it that way), allowing the algorithm to use multiple processing layers, composed of multiple linear and non-linear transformations.

One aspect of the present invention is to provide two analyte detection and localization approaches. The first approach is a deep learning approach and the second approach is a combination of deep learning and computer vision approaches.

In some embodiments, the beads and/or the analytes are recognized, measured or detected using machine learning. In some embodiments, the machine learning is assisted by the spacers which provides optical, location, and dimensional signals. For example, the spacers serves as imaging calibration marks, focus marks, location marks, and dimension (scale marks).

(i) Deep Learning Approach. In the first approach, the disclosed analyte detection and localization workflow consists of two stages, training and prediction. We describe training and prediction stages in the following paragraphs.

(a)Training Stage

In the training stage, training data with annotation is fed into a convolutional neural network. Convolutional neural network is a specialized neural network for processing data that has a grid-like, feed forward and layered network topology. Examples of the data include timeseries data, which can be thought of as a 1 D grid taking samples at regular time intervals, and image data, which can be thought of as a 2D grid of pixels. Convolutional networks have been successful in practical applications. The name “convolutional neural network” indicates that the network employs a mathematical operation called convolution. Convolution is a specialized kind of linear operation. Convolutional networks are simply neural networks that use convolution in place of general matrix multiplication in at least one of their layers.

The machine learning model receives one or multiple images of samples that contain the analytes taken by the imager over the sample holding QMAX device as training data. Training data are annotated for analytes to be assayed, wherein the annotations indicate whether or not analytes are in the training data and where they locate in the image. Annotation can be done in the form of tight bounding boxes which fully contains the analyte, or center locations of analytes. In the latter case, center locations are further converted into circles covering analytes or a Gaussian kernel in a point map.

When the size of training data is large, training machine learning model presents two challenges: annotation (usually done by human) is time consuming, and the training is computationally expensive. To overcome these challenges, one can partition the training data into patches of small size, then annotate and train on these patches, or a portion of these patches. The term “machine learning” can refer to algorithms, systems and apparatus in the field of artificial intelligence that often use statistical techniques and artificial neural network trained from data without being explicitly programmed.

The annotated images are fed to the machine learning (ML) training module, and the model trainer in the machine learning module will train a ML model from the training data (annotated sample images). The input data will be fed to the model trainer in multiple iterations until certain stopping criterion is satisfied. The output of the ML training module is a ML model - a computational model that is built from a training process in the machine learning from the data that gives computer the capability to perform certain tasks (e.g. detect and classify the objects) on its own.

The trained machine learning model is applied during the predication (or inference) stage by the computer. Examples of machine learning models include ResNet, DenseNet, etc. which are also named as “deep learning models” because of the depth of the connected layers in their network structure. In certain embodiments, the Caffe library with fully convolutional network (FCN) was used for model training and predication, and other convolutional neural network architecture and library can also be used, such as TensorFlow.

The training stage generates a model that will be used in the prediction stage. The model can be repeatedly used in the prediction stage for assaying the input. Thus, the computing unit only needs access to the generated model. It does not need access to the training data, nor requiring the training stage to be run again on the computing unit. (b) Prediction Stage

In the predication/inference stage, a detection component is applied to the input image, and an input image is fed into the predication (inference) module preloaded with a trained model generated from the training stage. The output of the prediction stage can be bounding boxes that contain the detected analytes with their center locations or a point map indicating the location of each analyte, or a heatmap that contains the information of the detected analytes.

When the output of the prediction stage is a list of bounding boxes, the number of analytes in the image of the sample for assaying is characterized by the number of detected bounding boxes. When the output of the prediction stage is a point map, the number of analytes in the image of the sample for assaying is characterized by the integration of the point map. When the output of the prediction is a heatmap, a localization component is used to identify the location and the number of detected analytes is characterized by the entries of the heatmap.

One embodiment of the localization algorithm is to sort the heatmap values into a onedimensional ordered list, from the highest value to the lowest value. Then pick the pixel with the highest value, remove the pixel from the list, along with its neighbors. Iterate the process to pick the pixel with the highest value in the list, until all pixels are removed from the list.

In the detection component using heatmap, an input image, along with the model generated from the training stage, is fed into a convolutional neural network, and the output of the detection stage is a pixel-level prediction, in the form of a heatmap. The heatmap can have the same size as the input image, or it can be a scaled down version of the input image, and it is the input to the localization component. We disclose an algorithm to localize the analyte center. The main idea is to iteratively detect local peaks from the heatmap. After the peak is localized, we calculate the local area surrounding the peak but with smaller value. We remove this region from the heatmap and find the next peak from the remaining pixels. The process is repeated only all pixels are removed from the heatmap.

In certain embodiments, the present invention provides the localization algorithm to sort the heatmap values into a one-dimensional ordered list, from the highest value to the lowest value. Then pick the pixel with the highest value, remove the pixel from the list, along with its neighbors. Iterate the process to pick the pixel with the highest value in the list, until all pixels are removed from the list.

Algorithm GlobalSearch (heatmap)

Input: heatmap

Output: loci loci «— {} sort(heatmap) while (heatmap is not empty) { s <— pop(heatmap)

D <— {disk center as s with radius R} heatmap = heatmap \ D // remove D from the heatmap add s to loci

}

After sorting, heatmap is a one-dimensional ordered list, where the heatmap value is ordered from the highest to the lowest. Each heatmap value is associated with its corresponding pixel coordinates. The first item in the heatmap is the one with the highest value, which is the output of the pop(heatmap) function. One disk is created, where the center is the pixel coordinate of the one with highest heatmap value. Then all heatmap values whose pixel coordinates resides inside the disk is removed from the heatmap. The algorithm repeatedly pops up the highest value in the current heatmap, removes the disk around it, till the items are removed from the heatmap.

In the ordered list heatmap, each item has the knowledge of the proceeding item, and the following item. When removing an item from the ordered list, we make the following changes:

• Assume the removing item is x _r , its proceeding item is x _p , and its following item is Xf.

• For the proceeding item x _p , re-define its following item to the following item of the removing item. Thus, the following item of x _p is now Xf.

• For the removing item x _r , un-define its proceeding item and following item, which removes it from the ordered list.

• For the following item Xf, re-define its proceeding item to the proceeding item of the removed item. Thus, the proceeding item of Xf is now x _p .

After all items are removed from the ordered list, the localization algorithm is complete. The number of elements in the set loci will be the count of analytes, and location information is the pixel coordinate for each s in the set loci.

Another embodiment searches local peak, which is not necessary the one with the highest heatmap value. To detect each local peak, we start from a random starting point, and search for the local maximal value. After we find the peak, we calculate the local area surrounding the peak but with smaller value. We remove this region from the heatmap and find the next peak from the remaining pixels. The process is repeated only all pixels are removed from the heatmap.

Algorithm LocalSearch (s, heatmap)

Input: s: starting location (x, y) heatmap

Output: s: location of local peak.

We only consider pixels of value > 0.

Algorithm Cover (s, heatmap)

Input: s: location of local peak. heatmap:

Output: cover: a set of pixels covered by peak:

This is a breadth-first-search algorithm starting from s, with one altered condition of visiting points: a neighbor p of the current location q is only added to cover if heatmap[p]> 0 and heatmap[p] <= heatmap[q]. Therefore, each pixel in cover has a non-descending path leading to the local peak s.

Algorithm Localization (heatmap)

Input: heatmap

Output: loci loci «— {} pixels <— {all pixels from heatmap} while pixels is not empty { s <— any pixel from pixels s <— LocalSearch(s, heatmap) // s is now local peak probe local region of radius R surrounding s for better local peak r <— Cover(s, heatmap) pixels <— pixels \ r // remove all pixels in cover add s to loci

(ii) Mixture of Deep Learning and Computer Vision Approaches. In the second approach, the detection and localization are realized by computer vision algorithms, and a classification is realized by deep learning algorithms, wherein the computer vision algorithms detect and locate possible candidates of analytes, and the deep learning algorithm classifies each possible candidate as a true analyte and false analyte. The location of all true analyte (along with the total count of true analytes) will be recorded as the output.

(a) Detection. The computer vision algorithm detects possible candidate based on the characteristics of analytes, including but not limited to intensity, color, size, shape, distribution, etc. A pre-processing scheme can improve the detection. Pre-processing schemes include contrast enhancement, histogram adjustment, color enhancement, de-nosing, smoothing, defocus, etc. After pre-processing, the input image is sent to a detector. The detector tells the existing of possible candidate of analyte and gives an estimate of its location. The detection can be based on the analyte structure (such as edge detection, line detection, circle detection, etc.), the connectivity (such as blob detection, connect components, contour detection, etc.), intensity, color, shape using schemes such as adaptive thresholding, etc.

(b) Localization. After detection, the computer vision algorithm locates each possible candidate of analytes by providing its boundary or a tight bounding box containing it. This can be achieved through object segmentation algorithms, such as adaptive thresholding, background subtraction, floodfill, mean shift, watershed, etc. Very often, the localization can be combined with detection to produce the detection results along with the location of each possible candidates of analytes.

(c) Classification. The deep learning algorithms, such as convolutional neural networks, achieve start-of-the-art visual classification. We employ deep learning algorithms for classification on each possible candidate of analytes. Various convolutional neural network can be utilized for analyte classification, such as VGGNet, ResNet, MobileNet, DenseNet, etc.

Given each possible candidate of analyte, the deep learning algorithm computes through layers of neurons via convolution filters and non-linear filters to extract high-level features that differentiate analyte against non-analytes. A layer of fully convolutional network will combine high-level features into classification results, which tells whether it is a true analyte or not, or the probability of being a analyte.

A) Applications, Bio/Chemical Biomarkers, and Health Conditions

The applications of the present invention include, but not limited to, (a) the detection, purification and quantification of chemical compounds or biomolecules that correlates with the stage of certain diseases, e.g., infectious and parasitic disease, injuries, cardiovascular disease, cancer, mental disorders, neuropsychiatric disorders and organic diseases, e.g., pulmonary diseases, renal diseases, (b) the detection, purification and quantification of microorganism, e.g.,, e.g., tissues, bodily fluids, (c) the detection, quantification of chemical compounds or biological samples that pose hazard to food safety or national security, e.g. toxic waste, anthrax, (d) quantification of vital parameters in medical or physiological monitor, e.g., glucose, blood oxygen level, total blood count, (e) the detection and quantification of specific DNA or RNA from biosamples, e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing of genetic sequences in DNA in the chromosomes and mitochondria for genome analysis or (g) to detect reaction products, e.g.,

The detection can be carried out in various sample matrix, such as cells, tissues, bodily fluids, and stool. Bodily fluids of interest include but are not limited to, amniotic fluid, aqueous humour, vitreous humour, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaled condensate. In some embodiments, the sample comprises a human body fluid. In some embodiments, the sample comprises at least one of cells, tissues, bodily fluids, stool, amniotic fluid, aqueous humour, vitreous humour, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, cerumen, chyle, chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, phlegm, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled condensate.

In embodiments, the sample is at least one of a biological sample, an environmental sample, and a biochemical sample.

The devices, systems and the methods in the present invention find use in a variety of different applications in various fields, where determination of the presence or absence, and/or quantification of one or more analytes in a sample are desired. For example, the subject method finds use in the detection of proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, and the like. The various fields include, but not limited to, human, veterinary, agriculture, foods, environments, drug testing, and others.

In certain embodiments, the subject method finds use in the detection of nucleic acids, proteins, or other biomolecules in a sample. The methods can include the detection of a set of biomarkers, e.g., two or more distinct protein or nucleic acid biomarkers, in a sample. For example, the methods can be used in the rapid, clinical detection of two or more disease biomarkers in a biological sample, e.g., as can be employed in the diagnosis of a disease condition in a subject, or in the ongoing management or treatment of a disease condition in a subject, etc. As described above, communication to a physician or other health-care provider can better ensure that the physician or other health-care provider is made aware of, and cognizant of, possible concerns and can thus be more likely to take appropriate action.

The applications of the devices, systems and methods in the present inventions of employing a CROF device include, but are not limited to, (a) the detection, purification and quantification of chemical compounds or biomolecules that correlates with the stage of certain diseases, e.g., infectious and parasitic disease, injuries, cardiovascular disease, cancer, mental disorders, neuropsychiatric disorders and organic diseases, e.g., pulmonary diseases, renal diseases, (b) the detection, purification and quantification of microorganism, e.g., virus, fungus and bacteria from environment, e.g., water, soil, or biological samples, e.g., tissues, bodily fluids, (c) the detection, quantification of chemical compounds or biological samples that pose hazard to food safety or national security, e.g. toxic waste, anthrax, (d) quantification of vital parameters in medical or physiological monitor, e.g., glucose, blood oxygen level, total blood count, (e) the detection and quantification of specific DNA or RNA from biosamples, e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing of genetic sequences in DNA in the chromosomes and mitochondria for genome analysis or (g) to detect reaction products, e.g., during synthesis or purification of pharmaceuticals. Some of the specific applications of the devices, systems and methods in the present invention are described now in further detail.

The applications of the present invention include, but not limited to, (a) the detection, purification and quantification of chemical compounds or biomolecules that correlates with the stage of certain diseases, e.g., infectious and parasitic disease, injuries, cardiovascular disease, cancer, mental disorders, neuropsychiatric disorders and organic diseases, e.g., pulmonary diseases, renal diseases, (b) the detection, purification and quantification of microorganism, e.g., virus, fungus and bacteria from environment, e.g., water, soil, or biological samples, e.g., tissues, bodily fluids, (c) the detection, quantification of chemical compounds or biological samples that pose hazard to food safety or national security, e.g. toxic waste, anthrax, (d) quantification of vital parameters in medical or physiological monitor, e.g., glucose, blood oxygen level, total blood count, (e) the detection and quantification of specific DNA or RNA from biosamples, e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing of genetic sequences in DNA in the chromosomes and mitochondria for genome analysis or (g) to detect reaction products, e.g., during synthesis or purification of pharmaceuticals.

An implementation of the devices, systems and methods in the present invention can include a) obtaining a sample, b) applying the sample to CROF device containing a capture agent that binds to an analyte of interest, under conditions suitable for binding of the analyte in a sample to the capture agent, c) washing the CROF device, and d) reading the CROF device, thereby obtaining a measurement of the amount of the analyte in the sample. In some embodiments, the analyte can be a biomarker, an environmental marker, or a foodstuff marker. The sample in some instances is a liquid sample, and can be a diagnostic sample (such as saliva, serum, blood, sputum, urine, sweat, lacrima, semen, or mucus); an environmental sample obtained from a river, ocean, lake, rain, snow, sewage, sewage processing runoff, agricultural runoff, industrial runoff, tap water or drinking water; or a foodstuff sample obtained from tap water, drinking water, prepared food, processed food or raw food.

Additional Notes

Further examples of inventive subject matter according to the present disclosure are described in the following enumerated paragraphs.

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 multiple analytes, reference to “a capture agent” includes a single capture agent and multiple capture agents, reference to “a detection agent” includes a single detection agent and multiple detection agents, and reference to “an agent” includes a single agent and multiple agents.

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

As used herein, the phrase, “for example,” the phrase, “as an example,” and/or simply the terms “example” and “exemplary” when used with reference to one or more components, features, details, structures, embodiments, and/or methods according to the present disclosure, are intended to convey that the described component, feature, detail, structure, embodiment, and/or method is an illustrative, non-exclusive example of components, features, details, structures, embodiments, and/or methods according to the present disclosure. Thus, the described component, feature, detail, structure, embodiment, and/or method is not intended to be limiting, required, or exclusive/exhaustive; and 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 of” and “one or more of,” in reference to a list of more than one entity, means any one or more of the entity in the list of entity, and is not limited to at least one of each and every entity specifically listed within the list of entity. 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”) may refer to A alone, B alone, or the combination of A and B.

As used herein, the term “and/or” placed between a first entity and a second entity means one of (1) the first entity, (2) the second entity, and (3) the first entity and the second entity. Multiple entity listed with “and/or” should be construed in the same manner, i.e. , “one or more” of the entity so conjoined. Other entity may optionally be present other than the entity specifically identified by the “and/or” clause, whether related or unrelated to those entities specifically identified.

Where numerical ranges are mentioned herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art.

In the event that any patents, patent applications, or other references are incorporated by reference herein and (1) define a term in a manner that is inconsistent with and/or (2) are otherwise inconsistent with, either the non-incorporated portion of the present disclosure or any of the other incorporated references, the non-incorporated portion of the present disclosure shall control, and the term or incorporated disclosure therein shall only control with respect to the reference in which the term is defined and/or the incorporated disclosure was present originally.