Assay method and apparatus using textured surface

Abstract

The present invention provides, inter alia, solutions to this problem, in particular, certain surfaces and certain sample holders to improve the sensitivity, speed and ease of use of optical signal based assays, such as colorimetric or fluorescence assays.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 460,088, filed on February 16, 2017 (053PRV); U.S. Provisional Application No. 62 / 460,083, filed on February 16, 2017 (035PRV3); U.S. Provisional Application No. 62 / 460,076, filed on February 16, 2017 (040PRV2); U.S. Provisional Application No. 62 / 460,075, filed on February 16, 2017 (026PRV3); U.S. Provisional Application No. 62 / 460,069, filed on February 16, 2017 (052PRV2); U.S. Provisional Application No. 62 / 460,062, filed on February 16, 2017 (045PRV2); U.S. Provisional Application No. 62 / 460,047, filed on February 16, 2017 (029PRV2); U.S. Provisional Application No. 62 / 459,972, filed on February 16, 2017 (002PRV2); U.S. Provisional Application No. 62 / 459,920, filed on February 16, 2017 (050PRV); PCT Application No. PCT / US18 / 18405, filed on February 15, 2018 (18F19); PCT Application No. PCT / US18 / 18108, filed on February 14, 2018 (18F14); PCT Application No. PCT / US18 / 18007, filed on February 13, 2018 (18F11B); PCT Application No. PCT / US18 / 17716, filed on February 9, 2018 (18F08); PCT Application No. PCT / US18 / 17713, filed on February 9, 2018 (18F07); PCT Application No. PCT / US18 / 17712, filed on February 9, 2018 (18F16); PCT Application No. PCT / US18 / 17504, filed on February 8, 2018 (18F02); PCT Application No. PCT / US18 / 17501, filed on February 8, 2018 (18F12); PCT Application No. PCT / US18 / 17499, filed on February 8, 2018 (18F17); PCT Application No. PCT / US18 / 17489, filed on February 8, 2018 (18F18); PCT Application No. PCT / US18 / 17492, filed on February 8, 2018 (18F10); PCT Application No. PCT / US18 / 17494, filed on February 8, 2018 (18F02); PCT Application No. PCT / US18 / 17502, filed on February 8, 2018 (18F09), and PCT Application No. PCT / US18 / 17307, filed on February 7, 2018 (18F15A), and each application is hereby incorporated by reference in its entirety for all purposes. TECHNICAL FIELD

[0003] In addition, the present invention relates to devices and methods for performing biological and chemical assays, and devices and methods for performing biological and chemical assays using colorimetric methods. Background Art

[0004] In biological / chemical assays, it is necessary to enhance the optical signal from a thin sample. For example, in a colorimetric assay, when the sample thickness is very thin (e.g., 100 μm (micrometers) or less), the color becomes very faint and difficult to observe, limiting the sensitivity of the colorimetric assay. Summary of the Invention

[0006] The following brief summary is not intended to include all features and aspects of the present invention. Among them, the present invention provides solutions for improving the sensitivity, speed, and ease of use of assays through optical signals such as colorimetric assays or fluorescence assays. Brief Description of the Drawings

[0007] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present invention in any way. The drawings are not drawn to scale. In the figures presenting experimental data points, the lines connecting the data points are for guiding the observation of the data only and have no other meaning.

[0008] Figure 1-A An example of an open assembled colorimetric assay sample card including a bottom plate, a top plate, and an aluminum hinge according to an embodiment of the present invention is shown.

[0009] Figure 1-B and Figure 1-C An example of the bottom plate of a colorimetric assay sample card having a textured microstructure on the top surface according to an embodiment of the present invention is shown.

[0010] Figure 1-D and Figure 1-E An example of the top plate of a colorimetric assay sample card according to an embodiment of the present invention is shown, and the top plate of the colorimetric assay sample card has an array of columns with a uniform height on the bottom surface.

[0011] Figure 1-F and Figure 1-G An example of a test colorimetric assay sample card including a bottom plate, a top plate, an aluminum hinge, and a sample liquid between the top plate and the bottom plate according to an embodiment of the present invention is shown.

[0012] Figure 2-A A test device for performing colorimetric measurement on a sample with a textured surface using side illumination with an optical fiber is shown.

[0013] Figure 2-B , Figure 2-C and Figure 2-DA test device for colorimetric measurement of a sample with a textured surface using ring illumination with an optical fiber is shown.

[0014] Figure 3 It is an illustration of an embodiment of CROF (Compression Regulated Open Flow). Figure (a) shows a first plate and a second plate, where the first plate has spacers. Figure (b) shows depositing a sample on the first plate (shown), or the second plate (not shown), or both (not shown) in an open configuration. Figure (c) shows (i) using two plates to spread the sample (the sample flows between the plates) and reduce the sample thickness, and (ii) using spacers and plates to adjust the sample thickness in a closed configuration. The inner surface of each plate has one or more binding sites and / or storage sites (not shown).

[0015] Figure 4 It is a flowchart for testing heavy metals in water. Detailed Description

[0016] The following detailed description shows some embodiments of the present invention by way of example and not limitation. The chapter headings and any subtitles used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. The content under the chapter headings and / or subtitles is not limited to the chapter headings and / or subtitles but applies to the entire description of the present invention.

[0017] Any citation of a publication is for disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of a prior invention. In addition, the provided publication date may be different from the actual publication date, which needs to be independently confirmed.

[0018] A. QMAX Colorimetric Determination with a Textured Reflective Scattering Surface

[0019] In assays involving optical signal detection (such as colorimetric or fluorescence assays), a small container that holds a liquid sample and allows a light beam to pass through the sample to measure the light or color of the sample. When the sample is very dilute, the light or color is faint and difficult to measure.

[0020] The present invention particularly provides a solution for obtaining a stronger optical signal in a thin sample.

[0021] One novelty of the present invention is to use a QMAX card (with two movable plates) to make the sample into a very uniform thin layer (less than 200 μm).

[0022] Another novelty of the present invention is to use a textured reflective surface on the surface of one of the two plates to enhance the optical signal, especially for colorimetric assays and / or fluorescence assays.

[0023] In the present invention, we have observed that the color signal for colorimetric determination can be significantly increased by using a reflective textured surface, as one of the walls of the chamber can significantly increase the color signal.

[0024] According to the present invention, a device for a plate to sandwich a sample in a thin layer, wherein one of the plates is transparent and the other plate has a textured reflective surface on its sample contact area. The probing light enters the sample from the transparent plate, passes through the sample, and is diffusely reflected back to the transparent plate by the textured surface. We have observed that even when the sample is as thin as 30 μm or less, such an arrangement can significantly increase the color signal.

[0025] Furthermore, according to the present invention, the device further comprises a dry reagent coated on one of the plates, such that a liquid sample can be dropped onto one or both of the plates, the plates are closed, and then measurement is carried out. The sample thickness can be 150 μm or less, such that the dry reagent can be mixed with the sample in a short time to accelerate the total measurement time.

[0026] The terms "CROF card (or card)", "COF card", "QMAX card", "Q card", "CROF device", "COF device", "QMAX device", "CROF plate", "COF plate", and "QMAX plate" are interchangeable, except that in some embodiments, the COF card does not include a spacer; and these terms refer to a device that comprises a first plate and a second plate that are movable relative to each other into different configurations (including an open configuration and a closed configuration), and the device comprises a spacer for adjusting the spacing between the plates (except for some embodiments of COF). The term "X plate" refers to one of the two plates in a CROF card, to which the spacer is fixed. More descriptions of the COF card, CROF card, and X plate are described in PCT applications (designating the United States) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504 filed on February 8, 2017. The entire contents of all these applications are incorporated herein by reference for all purposes.

[0027] Device_0 (conventional)

[0028] N1. In some embodiments, according to the present invention, a device for determining a sample using an optical signal, comprising:

[0029] A first plate, a second plate, a spacer, and a textured surface, wherein:

[0030] i. The first plate and the second plate are movable relative to each other into different configurations;

[0031] ii. One or both plates are flexible;

[0032] iii. The second plate has a textured structure on its inner surface for scattering light incident on the surface;

[0033] iv. The textured surface can be, but is not limited to, a raised, wavy, rough surface;

[0034] v. The textured surface is regular or irregular;

[0035] vi. The average roughness of the textured surface preferably ranges from, but is not limited to, 2 μm to 5 μm;

[0036] vii. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height;

[0037] viii. The preferred height of the spacer is greater than the average roughness of the textured surface and less than 100 μm;

[0038] Wherein one of the configurations is an open configuration, in which: the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates;

[0039] Wherein one of the configurations is a closed configuration, the closed configuration being configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the deposited sample is pressed into a continuous layer by the two plates;

[0040] Wherein the sample is in liquid form.

[0041] Device_C1 (for colorimetric signal)

[0042] A sample processing device for enhancing an optical signal (Q card), comprising:

[0043] A first plate, a second plate, a spacer, and a textured surface, wherein:

[0044] i. The plates are movable relative to each other into different configurations;

[0045] ii. One or both plates are flexible;

[0046] iii. The second plate has a textured structure on its inner surface for scattering light incident on the surface;

[0047] iv. The textured surface can be, but is not limited to, a raised, wavy, rough surface;

[0048] v. The textured surface can be regular or irregular;

[0049] vi. The average roughness of the textured surface preferably ranges from but is not limited to 2 μm - 5 μm;

[0050] vii. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height;

[0051] viii. The preferred height of the spacer is greater than the average roughness of the textured surface and less than 100 μm;

[0052] Wherein one of the configurations is an open configuration, wherein: the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates;

[0053] Wherein one of the configurations is a closed configuration, the closed configuration is configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the deposited sample is pressed into a continuous layer by the two plates;

[0054] Wherein the sample is in liquid form.

[0055] In some embodiments, the textured surface is made of an opaque white material;

[0056] Device_C2 (for colorimetric signal)

[0057] A sample processing device for enhancing an optical signal (Q card), comprising:

[0058] A first plate, a second plate, a spacer, and a textured surface, wherein:

[0059] i. The plates can be moved relative to each other into different configurations;

[0060] ii. One or both of the plates are flexible;

[0061] iii. The second plate has a textured structure on its inner surface for scattering light incident on the surface;

[0062] iv. The textured surface can be but is not limited to a raised, wavy rough surface;

[0063] v. The textured surface can be regular or irregular;

[0064] vi. The average roughness of the textured surface preferably ranges from but is not limited to 2 μm - 5 μm;

[0065] vii. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height;

[0066] viii. The preferred height of the spacer is greater than the average roughness of the textured surface and less than 100 μm;

[0067] One of the configurations is an open configuration, in which: the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates;

[0068] One of the configurations is a closed configuration, which is configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the deposited sample is pressed into a continuous layer by the two plates;

[0069] Wherein the sample is in liquid form.

[0070] In some embodiments, the textured surface is made of a semi-transparent white material and has a transmittance of 10%-30%.

[0071] Device_F (for fluorescence signal)

[0072] A sample processing device for enhancing an optical signal (Q card), comprising:

[0073] A first plate, a second plate, a spacer, and a textured surface, wherein:

[0074] i. The plates can be moved relative to each other into different configurations;

[0075] ii. One or both of the plates are flexible;

[0076] iii. The second plate has a textured structure on its inner surface for scattering light incident on the surface;

[0077] iv. The textured surface can be, but is not limited to, a raised, wavy rough surface;

[0078] v. The textured surface can be regular or irregular;

[0079] vi. The average roughness range of the textured surface is preferably, but not limited to, 2 μm - 5 μm;

[0080] vii. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height;

[0081] viii. The preferred height of the spacer is greater than the average roughness of the textured surface and less than 100 μm;

[0082] One of the configurations is an open configuration, where: two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates;

[0083] One of the configurations is a closed configuration, which is configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the deposited sample is pressed into a continuous layer by the two plates;

[0084] The sample is in liquid form.

[0085] In some embodiments, the textured surface is made of an opaque white material or coated with a reflective metal film, which may be (but is not limited to) aluminum, silver, and gold. The preferred thickness range of the metal film is preferably but not limited to 10 nm - 100 nm.

[0086] Device (for colorimetric signal).A1

[0087] A test device, comprising:

[0088] a) A sample processing device (Device_C1) for enhancing an optical signal as described in the above device claims;

[0089] b) A mobile computing device having a camera module and a light source;

[0090] c) An illumination optical device comprising angled optical fibers;

[0091] d) An external lens;

[0092] The light source emits white light;

[0093] The light source and the camera module are on the same side of the mobile computing device;

[0094] The Q card is placed directly below the camera module, and the preferred distance between the Q card and the camera module is 15 mm to 20 mm;

[0095] The external lens is placed between the Q card and the camera module such that the sample in the Q card is within the working distance of the camera module, the preferred focal length of the external lens is 12 - 18 mm, the distance between the lens and the camera module is preferably as small as possible and not more than 3 mm;

[0096] The optical fibers direct the light emitted from the light source to irradiate a sample area directly below the camera module;

[0097] One end face of the optical fiber is placed under the aperture of the light source, and the distance between them is preferably as small as possible and not greater than 3 mm;

[0098] The diameter of the optical fiber is configured to be equal to the diameter of the aperture of the light source;

[0099] The tilt angle of the optical fiber is set such that the central beam emitted from the optical fiber irradiates the sample area directly below the camera module.

[0100] Device (for colorimetric signal).A2

[0101] A test device, comprising:

[0102] a) A sample processing device (Device_C2) for enhancing optical signals as described in the above device claims;

[0103] b) A mobile computing device having a camera module and a light source;

[0104] c) An illumination optical device comprising a pair of mirrors;

[0105] d) An external lens;

[0106] - The light source emits white light;

[0107] - The light source and the camera module are on the same side of the mobile computing device;

[0108] - The Q card is placed directly below the camera module, and the preferred distance between them is 5 - 10 mm;

[0109] - The external lens is placed between the Q card and the camera module such that the sample in the Q card is within the working distance of the camera module. The preferred focal length of the external lens is 4 - 8 mm, and the preferred distance between the lens and the camera module is as small as possible and not greater than 3 mm;

[0110] - The illumination optical device turns the light emitted by the light source and irradiates the sample on the Q card. Each mirror turns the light by 90 degrees;

[0111] - The mirrors are mounted below the Q card. One mirror is in a straight line with the light source, and the other mirror is in a straight line with the camera module. The preferred distance between the Q card and the mirrors is 5 mm - 10 mm.

[0112] Device (for colorimetric signal).A3

[0113] A test device, comprising:

[0114] a) A sample processing device (Device_C2) for enhancing optical signals as described in the above device claims;

[0115] b) A mobile computing device having a camera module;

[0116] c) An independent light source;

[0117] d) An external lens;

[0118] - Wherein the light source emits white light, the light source is placed under the Q card and in a straight line with the camera module, and the preferred distance between the light source and the Q card is 5 mm - 10 mm.

[0119] - Wherein the Q card is placed directly below the camera module, and the preferred distance between them is 5 - 10 mm;

[0120] - Wherein the external lens is placed between the Q card and the camera module such that the sample in the Q card is within the working distance of the camera module, the preferred focal length of the external lens is 4 - 8 mm, the preferred distance between the lens and the camera module is as small as possible and not more than 3 mm;

[0121] Optical signal

[0122] According to the present invention, the optical signals enhanced by the textured surface of the device of any of the foregoing embodiments are selected from a group of colors, fluorescence, luminescence (electrical, chemical, optical or electro - chemical), and / or other light from emitters in the sample.

[0123] Device for fluorescence signal).A4

[0124] A test device, comprising:

[0125] a) A sample processing device (Device_F) for enhancing optical signals as described in the above device claims;

[0126] b) A mobile computing device having a camera module;

[0127] c) An independent light source;

[0128] d) Illumination optics, comprising a tilting mirror;

[0129] e) A filter, comprising a long - pass filter and a short - pass filter;

[0130] f) An external lens;

[0131] - Wherein the light source is a laser diode;

[0132] - Wherein the tilting mirror rotates the light emitted from the light source to irradiate the sample area directly below the camera module;

[0133] - wherein light is incident on the sample at an oblique angle, and the preferred angle is > 60 degrees;

[0134] - wherein the Q card is placed directly below the camera module, and the preferred distance between the Q card and the camera module is 15 mm - 20 mm;

[0135] - wherein the external lens is placed between the Q card and the camera module such that the sample in the Q card is within the working distance of the camera module, the preferred focal length of the external lens is 12 mm - 18 mm, and the preferred distance between the lens and the camera module is as small as possible and not greater than 3 mm;

[0136] - wherein the short - pass filter is placed in front of the aperture of the light source;

[0137] - wherein the long - pass filter is placed between the external lens and the camera module.

[0138] Method

[0139] A method for analyzing the optical signal of a sample, comprising the following steps:

[0140] a) Collecting the sample liquid;

[0141] b) Obtaining the device according to any of the foregoing embodiments;

[0142] c) When the plate is in the open configuration, depositing the sample on one or both of the plates in the device;

[0143] d) Placing the two plates together and pressing the plates into the closed configuration so that the sample forms a liquid layer between the two plates;

[0144] e) Inserting the device into the test equipment;

[0145] f) Turning on the light source of the test equipment;

[0146] g) Using the camera module to collect an image of the sample; and

[0147] h) The mobile computing device processes the image to analyze the colorimetric or fluorescence signal of the image, thereby obtaining some characteristics of the sample.

[0148] Application

[0149] The above - mentioned Q - card device, test equipment and method can be used to detect the presence and level of analytes of interest in the following fields:

[0150] 1) Food science and safety: Testing pH, ammonia, nitrite, nitrate, heavy metals, bacterial levels, etc. in drinking water; Testing bacteria, lactose, additives, specific protein levels, etc. in milk;

[0151] 2) Personal health monitoring: Glucose, alcohol, etc. in saliva, urine, and breath.

[0152] 3) In some embodiments, a device for enhancing an optical signal in an assay comprises:

[0154] A first plate, a second plate, a spacer, and a light-scattering layer, wherein:

[0155] i. The first plate and the second plate are movable relative to each other into different configurations and have sample contact regions on their respective inner surfaces for contacting a sample containing an analyte;

[0156] ii. One or both of the plates are flexible;

[0157] iii. The first plate is transparent to light, and

[0158] iv. The second plate substantially reflects light and comprises an inner surface light-scattering layer having a rough topology;

[0159] Wherein one of the configurations is an open configuration, wherein the average spacing between the inner surfaces of the two plates is at least 200 μm, and the sample is deposited on one or both of the plates;

[0160] Wherein the other of the configurations is a closed configuration configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the sample is between the two plates, and the average spacing between the inner surfaces of the plates is less than 200 μm; and

[0161] Wherein in the closed configuration, the light-scattering layer enhances the capture of probe photometry between the inner surfaces of the two plates.

[0162] In some embodiments, in the device, the light-scattering surface of the second plate comprises:

[0163] i. The textured surface can be, but is not limited to, a raised, wavy rough surface;

[0164] ii. The textured surface can be regular or irregular;

[0165] iii. The average roughness range of the textured surface is preferably, but not limited to, 2 μm - 5 μm; or

[0166] iv. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height; and

[0167] v. Combinations thereof.

[0168] C1. The device or system as described in any of the foregoing embodiments, wherein the light-scattering layer can be made of a highly reflective opaque white material with a reflectivity of at least 50%, 60%, 70%, 80%, 90%, 100%, or within a range between any two of these values.

[0169] C2. The device or system as described in any of the foregoing embodiments, wherein the reflection spectrum of the light-scattering surface is in the range of 300 nm to 1000 nm.

[0170] C3. The device or system as described in any of the foregoing embodiments, wherein the light-scattering layer can be made of a translucent white material, and the transmittance is 10% - 30%.

[0171] C4. The device or system as described in any of the foregoing embodiments, wherein the light-scattering layer can be made of a reflective metal film, and the light-scattering layer can be made of an opaque white dielectric film.

[0172] C5. The device or system as described in any of the foregoing embodiments, wherein the light-scattering layer has an R a (arithmetic mean roughness) of 0.5 μm - 200 μm, an R sm (mean spacing of the profile) > 0.5 μm, and an RΔ a (mean slope of the profile) > 0.1 textured surface.

[0173] C6. The device or system as described in any of the foregoing embodiments, wherein the textured surface can be regular or irregular, and the shape of a single feature on the textured surface can be, but is not limited to, square, triangular, acute angle.

[0174] C7. The device or system as described in any of the foregoing embodiments, wherein the height of the spacer is greater than the average roughness of the textured surface and less than 200 μm.

[0175] For the device or system as described in any of the foregoing embodiments, the average roughness height (R a ) of the textured reflection needs to be at least 20% of the wavelength of the illumination light and can be up to 5 times the spacing between the first plate and the second plate, or within a range between these two values;

[0176] For the device or system as described in any of the foregoing embodiments, the average lateral feature size (b a ) needs to be at least 20% of the wavelength of the illumination light and up to 10 times, or within a range between these two values;

[0177] For the device or system as described in any of the foregoing embodiments, the average period (b a ) needs to be at least 50% of the wavelength of the illumination light and up to 1000 times, or within a range between these two values.

[0178] Figure 1-A It is a schematic diagram of the colorimetric determination sample card 1 in an open state. The sample card 1 includes a top plate 12, a bottom plate 11, and an aluminum hinge 13. The hinge 13 connects the top plate 12 to the bottom plate 13.

[0179] The height of the random scattering structure is from 1 nm to 200 nm, from 1 nm to 300 nm, and from 1 nm to 5000 nm.

[0180] In some embodiments, the reflective surface can be achieved by random nanoparticles of the same size or different sizes.

[0181] In some embodiments, the reflection ranges are from 50% to 100%, from 30% to 100%, and from 50% to 80%. They are broadband or narrowband spectrally.

[0182] - Figure 1-B and Figure 1-C are schematic diagrams of the bottom plate 11 in the sample card 1, shown in isometric view and cross-sectional view respectively. The material used for the bottom plate 11 is non-absorbent and has an opaque white color. It can be, but is not limited to, white polyethylene. The bottom plate 11 has a textured surface 11S on one of its top surfaces (i.e., the surface facing the top plate 12). The textured surface 11S can be a random microstructure or a regular microstructure. For the random microstructure, it can be, but is not limited to, a raised, wavy, or rough surface. In one embodiment, the textured surface is a matte finish raised surface of a white polystyrene sheet with an average roughness of 2 - 3 μm. For the regular microstructure, it can be (but is not limited to) circular, rectangular, and triangular columns protruding from the bottom plate surface with a square, hexagonal, or other grid. A notch 11N is fabricated on one side of the bottom plate 11 to facilitate opening the top plate 12. A triangular gap 11C is fabricated at one corner of the bottom plate 11 to easily distinguish the front surface and the bottom surface of the bottom plate 11.

[0183] - Figure 1-D and Figure 1-E are schematic diagrams of the top plate 12 in the sample card 1, shown in isometric view and cross-sectional view respectively. The material used for the top plate is transparent and can be, but is not limited to, PMMA. On the bottom surface of the top plate (i.e., the surface facing the bottom plate 11), there is an array of regular micro-sized columns 12S with a uniform height. The column array can be, but is not limited to, rectangular columns with a square grid. In one embodiment, the top plate is made of 175 μm thick PMMA, and the column array has a square grid with a period of 120 μm * 110 μm. Each column is rectangular, with dimensions of 30 μm * 40 μm, and the column height is 30 μm. A triangular gap 12C is fabricated at one corner of the bottom plate 12 to easily distinguish the front surface and the bottom surface of the top plate 12.

[0184] - Figure 1-F and Figure 1-G are schematic views of the colorimetric assay sample card 1 with the sample liquid in a closed state, shown respectively from an isometric view and a cross-sectional view. The sample liquid 1L is embedded between the top plate 12 and the bottom plate 11. The textured surface 11S of the bottom plate 11 faces the bottom surface of the top plate 12 having the column array 12S. The average liquid layer thickness of the sample liquid 1L is uniform and is determined by the height of the column array 12S on the top plate 12. Thus, in the present invention, the volume of the sample liquid 1L held in the sample card 1 per unit area can be accurately determined. Under white light illumination, the textured surface 11S of the bottom plate 11 helps to deflect the light beam to increase the optical path within the sample liquid layer 1L. Thus, the light absorption of the colored compound in the sample liquid 1L increases and the color change is enhanced.

[0185] Figure 2-A 、 2-B and 2-C are schematic views showing details of the system 10 for reading the colorimetric card, particularly the device 13. Figure 2-B is a cross-sectional view showing details of the device; and Figure 2-A is a schematic view showing only the construction of the optical elements in the device. Figure 2-D shows the functions of the elements described above with reference to Figure 2-C wherein the light emitted from the light source 1L is coupled into the side-emitting fiber optic ring 135 from both end faces of the fiber optic ring 135 and propagates inside along the ring. The light beam B1 is emitted from the side wall of the fiber optic ring and passes through the diffuser film 136. The light beam B1 irradiates the sample area of the colorimetric sample card 138 directly below the camera 1C from the front side to produce uniform illumination. The irradiated sample area absorbs part of the light beam B1 and reflects the light beam B1 to the light beam B2. The light beam B2 is collected by the lens 133 and enters the camera 1C, and the lens 133 produces an image of the sample area on the image sensor plane of the camera 1C. The smartphone 1 captures and processes the image to analyze the color information in the image, thereby quantifying the color change of the colorimetric assay.

[0186] In some embodiments, no spacer is used when adjusting the sample thickness between the two plates.

[0187] In some embodiments, the textured reflective surface of the plate has one or a combination of each of these parameters: enhanced optical signal of the textured surface

[0188] The signal of colorimetric determination can be enhanced by a textured surface. In colorimetric determination, under white light illumination, light of a specific wavelength is absorbed by a colored compound, which results in a color change. Therefore, in order to obtain a stronger signal of color change, it is necessary to absorb more light of the specific absorption wavelength of the colored compound. And based on the Beer-Lambert law, which determines how much percentage of light is absorbed when light passes through an optical absorption medium, the way to increase light absorption in colorimetric determination is to increase the optical path in the sample liquid. Compared with a flat reflective surface, a textured surface can reflect small-angle incident light into large-angle outgoing light by scattering, thus increasing the optical path in the sample liquid. The textured surface can scatter the incident light in the sample liquid several times to increase the optical path before the light is emitted.

[0189] The measured fluorescence signal can also be enhanced by a textured surface. In fluorescence determination, under the illumination of excitation light of a specific wavelength, the emitted fluorescence intensity is proportional to the product of the quantum yield of the fluorescent dye and the amount of excitation light absorbed. The textured surface increases the optical path of the excitation light in the sample liquid by scattering, so more excitation light is absorbed by the fluorescent molecules.

[0190] The test device includes a device, a light source, an optical fiber, and an imager

[0191] - wherein the light source emits light in the wavelength range of 300 nm to 1000 nm;

[0192] - wherein the light source and the imager are on the same plane;

[0193] - wherein the Q card is placed directly below the imager, and the preferred distance between them is 15 mm - 20 mm;

[0194] - wherein the optical fiber guides the light emitted from the light source to irradiate the sample area directly below the camera module;

[0195] - wherein one end face of the optical fiber is placed under the aperture of the light source, and the distance between them is preferably as small as possible and not greater than 10 mm;

[0196] - wherein the diameter of the optical fiber is configured to be equal to the diameter of the light source aperture;

[0197] - wherein the tilt angle of the optical fiber is set such that the central beam emitted from the optical fiber irradiates the sample area directly below the camera module.

[0198] The test device includes a device, a light source, a loop optical fiber, and an imager,

[0199] wherein the light source emits light in the wavelength range of 300 nm to 1000 nm;

[0200] wherein the loop optical fiber is a side-emitting optical fiber capable of externally coupling light from the fiber wall;

[0201] - wherein the annular optical fiber is within a circle around the imager;

[0202] - wherein the Q card is placed directly below the imager, and the preferred distance between them is 15 mm - 20 mm;

[0203] - wherein light is emitted from the side of the annular optical fiber to irradiate the sample;

[0204] - wherein the two end faces of the annular optical fiber are placed below the aperture of the light source;

[0205] - wherein a light diffuser is placed between the annular optical fiber and the sample to diffuse the light emitted from the annular optical fiber;

[0206] B. Spacers, Hinges, and Open Notches

[0207] In biological and chemical assays (i.e., tests), devices and / or methods that simplify assay operations or accelerate assay speeds are generally of great value.

[0208] In QMAX (Q: Quantification; M: Amplification; A: Reagent addition; X: Acceleration; also known as the Compression Regulated Open Flow (CROF) assay platform), the QMAX card uses two plates to manipulate the shape of the sample into a thin layer (e.g., by pressing) (as shown in FIG. 1). In some embodiments, the plate manipulation requires multiple changes in the relative position of the two plates (referred to as: plate configuration) manually by a person or by other external forces. The QMAX card needs to be designed to make the manual operation easy and fast.

[0209] In the QMAX assay, one of the plate configurations is the open configuration, where the two plates are completely or partially separated (the spacing between the plates is not controlled by a spacer) and a sample can be deposited. Another configuration is the closed configuration, where at least a portion of the sample deposited in the open configuration is pressed by the two plates into a layer with a very uniform thickness, and the uniform thickness of this layer is defined by the inner surfaces of the plates and is adjusted by the plates and spacers.

[0210] In the QMAX measurement operation, the operator often needs to add the measurement reagent to the sample in a controlled manner. For example, in some embodiments, the reagents (e.g., the detection agent and the binding agent) are coated on the surface of the plate of the QMAX device, and some reagents (e.g., the detection agent) are released into the sample at an appropriate timing during the measurement process. Among them, in some cases, it is necessary to add the detection agent after the target analyte has been fully combined with the binding agent. In other cases, it is desirable to add the detection agent after the sample film has been formed. In other cases, it is desired to delay the addition of the detection agent for a specified period of time. The present invention aims to provide devices and methods for achieving these goals and for making biological / chemical sensing (including but not limited to immunoassays, nucleic acid assays, electrolyte analysis, etc.) faster, more sensitive, with fewer steps, easier to perform, requiring less sample volume, less or reduced (or no) professional assistance, and / or lower cost than a variety of current sensing methods and devices.

[0211] The term "compressed 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 pressing the sample between the two plates by pushing the two plates towards each other; wherein the pressing reduces the thickness of at least a portion of the sample and causes the sample to flow into the open space between the plates. The term "compressed regulated open flow" or "CROF" (or "self-calibrated compressed open flow" or "SCOF" or "SCCOF") (also known as QMAX) refers to a specific type of COF, where the final thickness of part or all of the sample after pressing is "regulated" by a spacer, where the spacer is placed between the two plates. Here, the CROF device can be used interchangeably with the QMAX device.

[0212] Unless otherwise specified, the term "spacer" or "stop" refers to a mechanical object that sets a limit on the minimum spacing between two plates when placed between the two plates, and this limit can be reached when the two plates are pressed together. That is, during the pressing process, the spacer will stop the relative movement of the two plates to prevent the plate spacing from becoming less than a preset (i.e., predetermined) value.

[0213] The terms "the spacer has a predetermined height" and "the spacer has a predetermined spacer spacing" respectively mean that the values of the spacer height and the spacer spacing are known before the QMAX process. If the values of the spacer height and the spacer spacing are not known before the QMAX process, then the values of the spacer height and the spacer spacing are not predetermined. For example, in the case where beads are sprayed on the plate as spacers and the beads land at random positions on the plate, the distance between the spacers is not predetermined. Another example of non-predetermined spacer spacing is when the spacer moves during the QMAX process.

[0214] In the QMAX process, the term "spacer fixed to its corresponding plate" means that the spacer is attached to a position on the plate and remains attached to that position during the QMAX process (i.e., the position of the spacer on the corresponding plate does not change). An example of "spacer fixed together with its corresponding plate" is that the spacer is integrally made of a piece of material of the plate, and the position of the spacer relative to the plate surface does not change during the QMAX process. An example of "spacer not fixed together with its corresponding plate" is that the spacer is adhered to the plate by an adhesive, but during the use of the plate, during the closed configuration where the other of the structures is configured after the sample is deposited in the open configuration, the adhesive cannot keep the spacer at its original position on the plate surface, and the spacer moves away from its original position on the plate surface.

[0215] In the QMAX process, the term "open configuration" of two plates refers to a configuration in which the two plates are either partially or completely separated, and the spacing between the plates is not adjusted by the spacers.

[0216] In the QMAX process, the term "closed configuration" of two plates refers to a configuration in which the plates face each other, the relevant volume of the spacers and the sample is between the plates, and the relevant spacing between the plates and thus the thickness of the relevant volume of the sample are adjusted by the plates and the spacers, where the relevant volume is at least a part of the entire volume of the sample.

[0217] In the QMAX process, the term "sample thickness adjusted by the plates and the spacers" means that for given conditions of the plates, the sample, the spacers, and the plate pressing method, the thickness of at least one port of the sample in the closed configuration of the plates can be predetermined according to the properties of the spacers and the plates.

[0218] In the QMAX device, the term "inner surface" or "sample surface" of the plate refers to the surface of the plate that contacts the sample, while the other surface of the plate (not contacting the sample) is called the "outer surface".

[0219] Unless otherwise specified, the term "height" or "thickness" of an object in the QMAX process refers to the dimension of the object in the direction perpendicular to the plate surface. For example, the spacer height is the dimension of the spacer in the direction perpendicular to the plate surface, and the spacer height and the spacer thickness refer to the same thing.

[0220] Unless otherwise specified, the term "area" of an object in the QMAX process refers to the area of the object parallel to the plate surface. For example, the spacer area is the area of the spacer parallel to the plate surface.

[0221] The term QMAX device refers to a device that performs the QMAX (e.g., CROF) process on a sample and has or does not have a hinge connecting two plates.

[0222] In some embodiments of the QMAX card, they do not use spacers to control the sample thickness in the closed configuration of the movable plate, but use other means to measure the sample thickness after reaching the closed configuration. Thickness measurement includes optical interferometry.

[0223] C. Colorimetric assay chemicals

[0224] As used herein, the term "colorimetric" and its grammatical variants refer to the physical description and quantification of chromatography, including the spectrum of human color perception (e.g., the visible spectrum). In some embodiments, colorimetric assays are particularly useful when quantification is not required and expensive detection equipment is not available. In certain embodiments, detection of a color change can be performed by visual observation by a user (e.g., the person performing the assay). Since colorimetric assays can be detected by visual observation, the user can check for a detectable change in the color of the reaction, or the assay can be performed in parallel with one or more controls (positive or negative) that replicate the color of the comparable reaction. In some embodiments, calibrated colorimetric measurements can be used to quantitatively determine the amount of a target.

[0225] Generally, colorimetric analysis involves determining the presence / absence, level, or concentration of an analyte (such as a chemical element or compound) in a sample such as a solution by means of a color reagent. It is applicable to both organic and inorganic compounds and can be used with or without an enzymatic reaction step. Generally, the equipment required is a colorimeter, one or more cuvettes, and a suitable color reagent. The process can be automated, for example, by using an autoanalyzer or by flow injection analysis. In certain embodiments, the colorimeter can be adapted to be used with a plate reader to accelerate the analysis and reduce the waste stream.

[0226] On the one hand, the colorimetric assays disclosed herein are non-enzymatic methods. For example, metal ions can react with one or more reagents to form one or more colored products. For example, calcium can react with o-cresolphthalein complexone to form a colored complex; copper can react with bathocuproine disulfonate to form a colored complex; creatinine can react with picrate to form a colored complex; iron can react with bathophenanthrolinedisulfonic acid to form a colored complex; phosphate can react with ammonium molybdate and / or ammonium metavanadate to form a colored complex.

[0227] On the other hand, the colorimetric assays disclosed herein include one or more enzyme reaction steps. Generally, the color reaction is preceded by a reaction catalyzed by an enzyme. Since enzymes are specific for one or more particular substrates, more accurate results can be obtained. For example, in a cholesterol detection assay such as the CHOD-PAP method, cholesterol in the sample first reacts with oxygen, catalyzed by cholesterol oxidase, to produce cholestanone and hydrogen peroxide. Then the hydrogen peroxide is reacted with 4-aminoantipyrine and phenol, which reaction is catalyzed by peroxidase, to produce a colored complex and water. Another example is the GOD-Perid method for detecting glucose, where glucose is the sample and first reacts with oxygen and water, catalyzed by glucose oxidase, to produce gluconate and hydrogen peroxide. The hydrogen peroxide so produced then reacts with ABTS to produce a colored complex, and this reaction can be catalyzed by peroxidase. In yet another example, the so-called GPO-PAP method detects triglycerides, which are first converted to glycerol and carboxylic acid (catalyzed by lipase); then glycerol reacts with ATP to form glycerophosphate and ADP (catalyzed by glycerol kinase); then glycerophosphate is oxidized by glycerophosphate oxidase to form dihydroxyacetone phosphate and hydrogen peroxide; and the final enzyme reaction is catalyzed by peroxidase, where the hydrogen peroxide reacts with 4-aminoantipyrine and 4-chlorophenol to form a colored complex. In some embodiments, the colorimetric assay can include non-enzyme steps and enzyme steps. For example, urea can be detected by first converting the analyte to ammonium carbonate (catalyzed by urease), and then ammonium carbonate reacts with phenol and hypochlorite in a non-enzyme reaction to form a colored complex.

[0228] In some embodiments, the colorimetric assay detects a protein target. In one aspect, the colorimetric assay involves the formation of a protein-metal chelate (e.g., protein-copper chelate), followed by a secondary detection of the reduced metal (e.g., copper). Examples of such colorimetric assays include the BCA assay and the Lowry protein assay, such as the Thermo Scientific Pierce BCA and the modified Lowry protein assay. On the other hand, the colorimetric assay involves protein-dye binding, directly detecting the color change associated with the bound dye. Examples of such colorimetric assays include the 660 nm assay and the Bradford protein assay. Other examples of colorimetric assays for detecting polypeptide or protein targets include the biuret assay, the bicinchoninic acid (Smith) assay, the Amido Black method, and the colloidal gold assay.

[0229] In certain embodiments, colorimetric assays, e.g., colorimetric screening, can be based on NAD(P)H production. The absorbance of NAD(P)H at 340 nm is commonly used to measure dehydrogenase activity. Typically, such colorimetric assays involve indirect methods that require synthetic compounds or secondary enzymes. For example, tetrazolium salts such as nitroblue tetrazolium (NBT) can be reduced to formazan dyes, which absorb light in the visible region. These reactions are essentially irreversible under biological conditions, and the increase in color can be readily monitored visually on a filter plate or a standard 96-well plate reader. The cascade of reactions leading to the formation of colored formazan links the production of NAD(P)H to the catalytic activity of dehydrogenases in the sample.

[0230] In certain embodiments, the colorimetric assay is an enzyme-linked immunosorbent assay (ELISA). Examples of colorimetric ELISA substrates include colorimetric (also known as chromogenic) substrates for alkaline phosphatase (AP) and / or horseradish peroxidase (HRP), such as p-nitrophenyl phosphate (PNPP), a substrate widely used to detect alkaline phosphatase in ELISA applications to produce a yellow water-soluble reaction product that absorbs light at 405 nm), ABTS (2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt, which is used to detect HRP and produces a water-soluble green final reaction product), o-phenylenediamine dihydrochloride (OPD), which is used to detect HRP and produces a water-soluble yellow-orange reaction product), and 3,3',5,5'-tetramethylbenzidine (TMB), which produces a blue color when detecting HRP).

[0231] Specific examples of colorimetric assays include HRP / ABTS / H2O2 assay, HRP / 4CN / H2O2 assay, D-amino acid oxidase assay, peroxidase / o-dianisidine assay, ABTS and o-dianisidine assay, TMB assay, guaiacol assay, MNBDH assay, assays based on Gibbs reagent and 4-aminoantipyrine, poly R-478 assay, horseradish peroxidase conjugate assay, MTT assay, indole assay, and p-nitrophenoxy analog (pNA) assay.

[0232] The above-described devices and methods can be used for any one or more of the following colorimetric assays. Suitable colorimetric assays include, but are not limited to, colorimetric assays for detecting proteins, nucleic acids, antibodies, or microorganisms. Colorimetric assays can be used to determine the concentration of substances in a solution. In some cases, colorimetric assays include colorimetric immunoassays. Suitable colorimetric assays can include those described in Jiang et al., Analyst (2016), 141:1196-1208; Morbioli et al., Anal. Chim. Acta. (2017), 970:1-22; Gu et al., Biotechnology Advances (2015), 33:666-690; Marin et al., Analyst. (2015), 140(1):59-70; Du et al., Small. (2013), 9(9-10):1467-81; Song et al., Adv. Mater. (2011), 23(37):4215-36; Liu et al., Nanoscale (2011), 3(4):1421-33; Martin et al., J. Animicrob Chemother. (2007) 59(2):175-83; Sapan et al., Biotechnol. Appl. Biochem. (1999), 29(pt 2):99-108.

[0233] Colorimetric immunoassays can include enzyme immunoassays, for example, enzyme-linked immunosorbent assay (ELISA) by way of example. ELISA assays can include labeling surface-bound antigens with an enzyme, for example, using a single antibody conjugate or two or more antibodies acting together to label the antigen with an enzyme. The antigen can be immobilized on a solid surface either non-specifically (e.g., by adsorption) or specifically (e.g., by antibody capture in a "sandwich" ELISA). After incubation, a washing step can be performed and a detection antibody covalently linked to the enzyme can be added. In some cases, the detection antibody is a primary antibody, which is itself detected by a secondary antibody linked to the enzyme. After labeling the enzyme and typically after one or more washing steps, the enzyme is reacted with a suitable substrate (e.g., a chromogenic substrate) in a manner that produces a signal (e.g., a chemical signal), which can be detected, for example, by spectrophotometry, fluorescence, or by visual means. This color change can indicate the presence and / or amount of antigen in the sample. Types of ELISA assays include, for example, direct ELISA, sandwich ELISA, and competitive ELISA.

[0234] Suitable enzymes for enzyme immunoassays include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Detection in these assays can be accomplished by a colorimetric method using a chromogenic substrate for the enzyme, where suitable substrates include, but are not limited to: o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine tetrahydrochloride (DAB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), etc. The fluid composition of the substrate, such as an aqueous formulation of the substrate, is typically incubated with the substrate surface for a period of time sufficient to produce a detectable product. The incubation is typically carried out at a temperature of about 0 to 37 °C, usually about 15 to 30 °C, more usually about 18 to 25 °C for about 10 seconds - 2 hours, usually about 30 seconds - 1 hour, more usually about 5 minutes - 15 minutes.

[0235] Colorimetric immunoassays can include lateral flow assays (LFA) or immunochromatographic assays. Such assays can be carried out on a series of capillary beds such as porous paper or polymers for transporting fluids. Conventional lateral flow test strips include a solid support on which a sample receiving zone and a target capture zone are supported. The material of the solid support is capable of supporting the sample receiving zone and the target capture zone and provides capillary flow of the sample from the sample receiving zone to the target capture zone when the lateral flow test strip is exposed to a suitable solvent or buffer, which serves as the carrier fluid for the sample. General classes of materials that can be used as the support include organic or inorganic polymers, as well as natural and synthetic polymers. More specific examples of suitable solid supports include, but are not limited to, glass fiber, cellulose, nylon, cross-linked dextran, various chromatographic papers, and nitrocellulose.

[0236] In the capture zone, the capture molecule can bind the complex, producing a color change in the test strip. The capture zone can include one or more components of the signal generation system. The signal generation system can vary widely depending on the specific nature of the lateral flow assay and can be any label that can be detected directly or indirectly. Detectable labels suitable for LFA include any moiety that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other methods. For example, suitable labels include biotin for staining with labeled streptavidin conjugates, fluorescent dyes (such as fluorescein, Texas Red, rhodamine, green fluorescent protein, etc.), radiolabels (such as 3 H, 125 I, 35 S, 1 C or 32) Enzymes (such as horseradish peroxidase, alkaline phosphatase, and other substances commonly used in ELISA), and colorimetric labels such as colloidal gold nanoparticles, silver nanoparticles, magnetic nanoparticles, cerium oxide nanoparticles, carbon nanotubes, graphene oxide, conjugated polymers, or colored glass or plastics (such as polystyrene, polypropylene, latex beads). Radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected by a light detector detecting the emitted light. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply observing the colored label.

[0237] In some cases, colorimetric assays can be used to measure ions in a sample. For example, chloride ions can be measured by a colorimetric assay. Chloride ions displace thiocyanate in mercury thiocyanate. Free thiocyanate reacts with iron ions to form a colored complex, iron thiocyanate, which is measured photometrically.

[0238] Similarly, magnesium can be colorimetrically measured using a calcein indicator that turns red-violet when reacting with magnesium; by a formazan dye test; emitting at 600 nm when reacting with magnesium or using methyl thymol blue, and methyl thymol blue binds to magnesium to form a blue complex.

[0239] Similarly, calcium can be detected by a colorimetric technique using O-Cresolphtalein, which turns purple when O-Cresolphtalein complexone reacts with calcium.

[0240] Similarly, due to bicarbonate (HCO3 -) and phosphoenolpyruvate (PEP) are converted to oxaloacetate and phosphate in a reaction catalyzed by phosphoenolpyruvate carboxylase (PEPC), so bicarbonate can be subjected to a two-color test. Malate dehydrogenase (MD) catalyzes the reduction of oxaloacetate to malate, accompanied by the oxidation of reduced nicotinamide adenine dinucleotide (NADH). This oxidation of NADH results in a proportional decrease in the absorbance of the reaction mixture measured at 380 / 410 nm for the two-color measurement, which is proportional to the bicarbonate content of the sample. Blood urea nitrogen can be detected in a colorimetric test, where diacetyl or fearon produces a yellow chromogen with urea and can be quantified by spectrophotometry. Similarly, creatinine can be measured colorimetrically by treating the sample with an alkaline picrate solution to produce a red complex. In addition, creatine can be measured using a non-Jaffe reaction, which measures the ammonia produced when creatinine is hydrolyzed by creatinine imino-hydrolase. Glucose can be measured in an assay where blood is exposed to a fixed amount of glucose oxidase for a limited period to estimate the concentration. After the specified time, the excess blood is removed and the color is developed for estimating the glucose concentration. For example, glucose oxidase reacts with glucose to form nascent oxygen, which converts potassium iodide (in filter paper) to iodine, forming a brown color. Glycated hemoglobin concentration serves as an indirect reading of the glucose level in the blood.

[0241] After precipitating apolipoprotein B-containing lipoproteins in whole plasma (LDL and VLDL) with heparin-manganese chloride, plasma high-density lipoprotein cholesterol (HDL-C) is determined by the same method used for total plasma cholesterol. These compounds can also be detected colorimetrically in an assay based on an enzyme-driven reaction of quantitative cholesterol esters and free cholesterol. Cholesterol esters are hydrolyzed to cholesterol by cholesterol esterase and then oxidized to cholest-4-en-3-one plus hydrogen peroxide by cholesterol oxidase. Then hydrogen peroxide is detected using a highly specific colorimetric probe. Horseradish peroxidase catalyzes the reaction between the probe and hydrogen peroxide, and the two combine in a 1:1 ratio. The sample can be compared with a cholesterol standard of known concentration.

[0242] Reagent examples

[0243] A. Glucose colorimetric (fluorometric) assay

[0244] Samples: Whole blood, plasma, serum, saliva

[0245] Reagent formulation 1:

[0246] 100 units / ml glucose oxidase, 100 units / ml horseradish peroxidase, 20 mM 4-aminoantipyrine, 20 mM TOOS

[0247] Reagent formulation 2:

[0248] 100 units / ml glucose oxidase, 100 units / ml horseradish peroxidase, 20 mM 3,5,3',5'-tetramethylbenzidine (TMB)

[0249] Reagent formula 3:

[0250] 100 units / ml glucose oxidase, 100 units / ml horseradish peroxidase, 20 mM Amplex Red

[0251] Reagent formula 4:

[0252] 1 unit / ml hexokinase, 220 mg / ml ATP, 400 mg / ml NAD

[0253] B. Colorimetric determination of calcium

[0254] Samples: whole blood, plasma, serum, saliva

[0255] Reagent formula 1:

[0256] 17 mg / ml arsenazo III

[0257] C. Colorimetric determination of albumin

[0258] Samples: whole blood, plasma, serum, saliva

[0259] Reagent formula 1:

[0260] 22 mg / ml bromocresol purple

[0261] D. Colorimetric determination of total protein

[0262] Samples: whole blood, plasma, serum, saliva

[0263] Reagent formula 1:

[0264] 1.34 mg / ml copper sulfate, 3.43 mg / ml potassium sodium tartrate, 0.28 mg / ml potassium iodide

[0265] E. Colorimetric determination method of sodium

[0266] Samples: whole blood, plasma, serum, saliva

[0267] Reagent formula 1:

[0268] 220 mg / ml ONPG, 0.05 unit / ml β-galactosidase

[0269] F. Colorimetric determination method of potassium

[0270] Samples: whole blood, plasma, serum, saliva

[0271] Reagent formula 1:

[0272] 220 mg / ml ADP, 0.05 units / ml phosphoenolpyruvate, 0.1 units / ml pyruvate kinase, 480 mg / ml NADH, 13.6 mg / ml potassium phosphate, 95 mg / ml magnesium sulfate, 7.85 mg / ml FAD, 130 mg / ml 4 - aminophenazone, 10 units / ml horseradish peroxidase, 1.88 mg / ml TBHBA

[0273] G. Colorimetric determination of chloride

[0274] Samples: whole blood, plasma, serum, saliva

[0275] Reagent formulation 1:

[0276] 530 mg / ml CNPG3, 0.36 units / ml α - amylase, 250 mg / ml calcium acetate

[0277] H. Colorimetric determination of blood urea nitrogen

[0278] Samples: whole blood, plasma, serum, saliva

[0279] Reagent formulation 1:

[0280] 0.5 U / ml urease aminohydrolase, 570 μg / ml PEP, 220 μg / ml ATP, 1 U / ml pyruvate kinase

[0281] 10 U / ml pyruvate oxidase, 13.6 mg / ml potassium phosphate, 95 μg / ml MgCl2, 7.85 μg / ml FAD

[0282] 1.88 mg / ml TBHBA, 130 μg / ml 4 - AAP, 10 U / ml peroxidase

[0283] I. Colorimetric determination of creatinine

[0284] Samples: whole blood, plasma, serum, saliva

[0285] Reagent formulation 1:

[0286] 10 U / ml creatinine amidohydrolase, 30 U / ml creatinine amidinohydrolase, 10 U / ml sarcosine oxidase, 1.88 mg / ml TBHBA, 130 μg / ml 4 - AAP, 10 U / ml peroxidase

[0287] J. Colorimetric determination of alkaline phosphatase

[0288] Samples: whole blood, plasma, serum, saliva

[0289] Reagent formulation 1:

[0290] 560 μg / ml p-Nitrophenyl phosphate, 0.5 U / ml Zinc sulfate, 330 μg / ml Magnesium sulfate

[0291] K. Colorimetric determination of alanine aminotransferase

[0292] Samples: Whole blood, plasma, serum, saliva

[0293] Reagent formula 1:

[0294] 8.74 mg / ml L-Alanine, 1.01 mg / ml α-Ketoglutaric acid

[0295] 10 U / ml Pyruvate oxidase, 13.6 mg / ml Potassium phosphate, 95 μg / ml MgCl2, 7.85 μg / ml FAD, 1.88 mg / ml TBHBA, 130 μg / ml 4-AAP, 10 U / ml Peroxidase

[0296] L. Colorimetric determination of aspartate aminotransferase

[0297] Samples: Whole blood, plasma, serum, saliva

[0298] Reagent formula 1:

[0299] 4.26 mg / ml L-Aspartic acid, 1.01 mg / ml α-Ketoglutaric acid, 10 U / ml Oxaloacetate decarboxylase, 1.88 mg / ml TBHBA, 130 μg / ml 4-AAP, 10 U / ml Peroxidase

[0300] M. Colorimetric determination of bilirubin

[0301] Samples: Whole blood, plasma, serum, saliva

[0302] Reagent formula 1:

[0303] 1 U / ml Bilirubin oxidase

[0304] N. Colorimetric (fluorescence) determination of cholesterol

[0305] Samples: Whole blood, plasma, serum, saliva

[0306] Reagent formula 1:

[0307] 100 units / ml Cholesterol oxidase, 100 units / ml Horseradish peroxidase, 20 mM 4-Aminoantipyrine, 20 mM TOOS

[0308] Reagent formula 2:

[0309] 100 units / ml cholesterol oxidase, 100 units / ml horseradish peroxidase, 20 mM 3,5,3',5'-tetramethylbenzidine (TMB)

[0310] Reagent formulation 3:

[0311] 100 units / ml cholesterol oxidase, 100 units / ml horseradish peroxidase, 20 mM Amplex Red

[0312] O. Colorimetric (fluorometric) determination of triglyceride

[0313] Samples: whole blood, plasma, serum, saliva

[0314] Reagent formulation 1:

[0315] 100 units / ml lipase, 100 units / ml glycerol kinase, 100 units / ml glycerol phosphate oxidase, 20 mM 4-aminoantipyrine, 20 mM TOOS

[0316] Reagent formulation 2:

[0317] 100 units / ml lipase, 100 units / ml glycerol kinase, 100 units / ml glycerol phosphate oxidase, 20 mM 3,5,3',5'-tetramethylbenzidine (TMB)

[0318] Reagent formulation 3:

[0319] 100 units / ml lipase, 100 units / ml glycerol kinase, 100 units / ml glycerol phosphate oxidase, 20 mM Amplex Red

[0320] P. Colorimetric (fluorometric) determination of ethanol

[0321] Samples: whole blood, plasma, serum, saliva

[0322] Reagent formulation 1:

[0323] 100 units / ml alcohol oxidase, 100 units / ml horseradish peroxidase, 20 mM 4-aminoantipyrine, 20 mM TOOS

[0324] Reagent formulation 2:

[0325] 100 units / ml alcohol oxidase, 100 units / ml horseradish peroxidase, 20 mM 3,5,3',5'-tetramethylbenzidine (TMB)

[0326] Reagent formulation 3:

[0327] 100 units / ml ethanol oxidase, 100 units / ml horseradish peroxidase, 20 mM Amplex Red

[0328] Q. Hydrogen peroxide (fluorescence) assay

[0329] Samples: whole blood, plasma, serum, saliva

[0330] Reagent formulation 1:

[0331] 100 units / ml horseradish peroxidase, 20 mM 4 - aminophenazone, 20 mM TOOS

[0332] Reagent formulation 2:

[0333] 100 units / ml horseradish peroxidase, 20 mM 3,5,3',5'-tetramethylbenzidine (TMB)

[0334] Reagent formulation 3:

[0335] 100 units / ml horseradish peroxidase, 20 mM Amplex Red

[0336] R. Gram staining

[0337] Samples: blood smear, vaginal sample, genital sample

[0338] Gram crystal violet

[0339] 20 g crystal violet, 8 g ammonium oxalate, 200 mL methanol

[0340] Gram iodine

[0341] 3.33 g iodine crystals, 6.67 g potassium iodide

[0342] Gram decolorizer

[0343] 500.0 mL ethanol, 500.0 mL acetone

[0344] Gram safranin

[0345] 0.25 g safranin O, 10 mL ethanol

[0346] Gram basic fuchsin

[0347] 0.7 g fuchsin, 3.5 mL phenol, 14 mM ethanol

[0348] S. Leishman staining

[0349] Samples: smear samples

[0350] Formulation 1

[0351] 0.2 g Leishman dye, 100 mL acetone-free methanol

[0352] T. Giemsa staining

[0353] Sample: smear sample

[0354] Formulation 1

[0355] 0.15 g Giemsa powder, 12.5 mL glycerol, 12.5 mL methanol

[0356] U. Wright staining

[0357] Sample: smear sample

[0358] Formulation 1

[0359] 1.5 g Wright stain, 500 mL methanol

[0360] V. Field staining

[0361] Sample: smear sample

[0362] Field solution A

[0363] 1.6 g methylene blue, 10 g sodium dihydrogen phosphate, 12.5 g potassium dihydrogen phosphate, 1 g Azur, 1000 mL distilled water Field solution B

[0364] 2 g eosin Y, 10 g sodium dihydrogen phosphate, 12.5 g potassium dihydrogen phosphate, 1000 mL distilled water

[0365] W. Jenner staining

[0366] Sample: smear sample

[0367] Formulation 1

[0368] 0.5 g Jenner stain, 100 mL methanol

[0369] X. JSB staining

[0370] Sample: smear sample

[0371] Formulation 1

[0372] 0.5 g Atine orange dye, 3 mL 1% sulfuric acid, 0.5 g potassium dichromate, 3.5 g dehydrated disodium hydrogen phosphate, 500 mL distilled water

[0373] JSB staining II

[0374] 1 g eosin Y, 500 ml distilled water

[0375] Y. Leukocyte staining for counting and classification

[0376] Samples: blood, urine, other body fluids

[0377] Formulation 1

[0378] 1 μg / mL to 1 mg / mL acridine orange (detecting agent)

[0379] Formulation 2

[0380] 150 μM propidium iodide (PI) (detecting agent), 100 μM fluorescein isothiocyanate (FITC), 250 μM basic orange 21 (BO21) dye

[0381] Z. Platelet staining and counting

[0382] Samples: blood, urine, other body fluids

[0383] Formulation 1

[0384] 1 μg / mL to 1 mg / mL acridine orange (detecting agent)

[0385] Formulation 2

[0386] 150 μM propidium iodide (PI) (detecting agent), 100 μM fluorescein isothiocyanate (FITC), 250 μM basic orange 21 (BO21) dye

[0387] Testing system using a QMAX device

[0388] One aspect of the present invention provides a system and method for analyzing biological / chemical samples using a QMAX device.

[0389] AA1. A system for analyzing a sample, comprising:

[0390] a) depositing the sample on a Q card and closing the Q card;

[0391] b) inserting the closed Q card into an adapter connected to a camera of a handheld mobile communication device;

[0392] c) using the camera of the handheld mobile communication device to take an image of the closed Q card;

[0393] d) transmitting the image from the handheld mobile communication device and / or the analysis result of the image to a remote location;

[0394] e) analyzing the image transmitted from the handheld mobile communication device and / or the analysis result of the image at the remote location; and

[0395] f) if an abnormality is detected, notifying a third party and / or the handheld mobile communication device;

[0396] wherein the Q card comprises two plates that are movable relative to each other and have an open configuration and a closed configuration;

[0397] wherein the sample is deposited on one or both plates of the Q - card in the open configuration, and at least a portion of the sample is between the two plates in the closed configuration.

[0398] wherein the mobile communication device is configured to generate an image of the Q - card in the adapter and transmit the image and / or the analysis result of the image to a remote location.

[0399] AA2. The method according to any of the preceding embodiments, wherein the sample deposited on the Q - card is from a subject, and the subject performs step a).

[0400] AA3. The method according to any of the preceding embodiments, wherein an anomaly is identified if the analysis result of the sample is not within the normal range.

[0401] AA4. The method according to any of the preceding embodiments, wherein an anomaly is identified if the analysis results generated by the remote device and the mobile handheld communication device differ by a predetermined value.

[0402] AA5. The method according to any of the preceding embodiments, wherein the sample comprises a body fluid selected from the group consisting of amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), earwax (cerumen), chyle, chyme, endolymph, perilymph, feces, gastric acid, gastric juice, lymph fluid, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled condensate.

[0403] AA6. The method according to any of the preceding embodiments, wherein the sample comprises an environmental sample obtained from rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rock, concrete, wood, brick, sewage; air, radiator, industrial waste gas, or vehicle exhaust gas.

[0404] AA7. The method according to any of the preceding embodiments, wherein the sample comprises a food sample, and the food sample includes: raw food ingredients, cooked food or processed food, plant and animal food sources, pre - processed food, or fully processed food.

[0405] AA8. The method according to any of the preceding embodiments, wherein in step (a), the Q - card is pressed by hand.

[0406] AA9. The method according to any of the preceding embodiments, wherein step e) comprises comparing the result with a threshold or a normal range to identify samples containing anomalies.

[0407] AA10. The method as described in any of the foregoing embodiments, wherein the method further comprises: updating the handheld mobile communication device if the analysis at the remote location yields significantly different results.

[0408] AA11. The method as described in any of the foregoing embodiments, wherein the sample deposited on the Q card is from a subject, and the analysis result is not transmitted to the subject.

[0409] AA12. The method as described in any of the foregoing embodiments, wherein the third party is a medical professional.

[0410] AA13. The method of embodiment AA12, wherein the medical professional is a doctor or a nurse practitioner.

[0411] AA14. The method as described in any of embodiments AA1 - AA12, wherein the third party is an insurance company.

[0412] AA15. The method as described in any of the foregoing embodiments, wherein the results from the mobile communication device and / or the results from the remote location are sent to the emergency room.

[0413] AA16. The method of embodiment AA1, wherein based on the results, the handheld mobile communication device or the remote location transmits follow-up information to the subject.

[0414] AA17. The method of embodiment AA16, wherein the follow-up information includes an explanation of the results, education about the disease or condition, information related to possible treatments, information about the location of appropriate physicians, information related to changes in diet and / or exercise, or advertisements.

[0415] AA18. The method as described in any of the foregoing embodiments, wherein the Q card includes a spacer having a substantially uniform height and a predetermined constant spacer pitch, and in the closed configuration, at least a portion of the sample is pressed by two plates of the Q card to form a layer with a very uniform thickness and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is defined by the inner surfaces of the two plates and is adjusted by the plates and the spacer.

[0416] AA19. The method of embodiment AA18, wherein at least one of the plates is flexible.

[0417] AA20. The method of embodiment AA19, wherein for the flexible plate, the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is in the range of 60 to 750 GPa-μm.

[0418] AA21. The method according to embodiment AA19, wherein for the flexible plate, the fourth power of the spacer pitch (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate, ISD 4 / (hE), is equal to or less than 106 μm 3 / GPa.

[0419] AA22. The method according to embodiment AA18, wherein the spacer for adjusting the layer of uniform thickness has a filling factor of at least 1%, where the filling factor is the ratio of the spacer area in contact with the layer of uniform thickness to the total plate area in contact with the layer of uniform thickness.

[0420] AA23. The method according to embodiment AA18, wherein for the spacer for adjusting the layer of uniform thickness, the Young's modulus of the spacer multiplied by the filling factor of the spacer is equal to or greater than 10 MPa, where the filling factor is the ratio of the spacer area in contact with the layer of uniform thickness to the total plate area in contact with the layer of uniform thickness.

[0421] AA24. The method according to any one of the preceding embodiments, wherein one or both of the plates comprise position marks located on or within the surface of the plate, the position marks providing information about the position of the plate.

[0422] AA25. The method according to any one of the preceding embodiments, wherein one or both of the plates comprise scale marks on or within the surface of the plate, the scale marks providing information about the lateral dimensions of the sample and / or the structure of the plate.

[0423] AA26. The method according to any one of the preceding embodiments, wherein one or both of the plates comprise imaging marks located on or within the surface of the plate, the imaging marks assisting in the imaging of the sample.

[0424] AA27. The method according to embodiment AA18, wherein the spacer acts as a position mark, a scale mark, an imaging mark, or any combination thereof.

[0425] AA28. The method according to embodiment AA18, wherein the average thickness of the layer of uniform thickness is in the range of 0.2 μm to 3.8 μm and the sample is blood.

[0426] AA29. The method according to embodiment AA18, wherein the spacer pitch is in the range of 7 μm to 50 μm.

[0427] AA30. The method according to embodiment AA18, wherein the spacer pitch is in the range of 50 μm to 120 μm.

[0428] AA31. The method as described in embodiment AA18, wherein the spacer pitch is in the range of 120 μm to 200 μm.

[0429] AA32. The method as described in embodiment AA18, wherein the spacer pitch is substantially fixed.

[0430] AA33. The method as described in embodiment AA18, wherein the spacer is a pillar having a cross-sectional shape selected from circular, polygonal, annular, square, rectangular, oval, elliptical, or any combination thereof.

[0431] AA34. The method as described in embodiment AA18, wherein the spacer has a columnar shape and has a substantially flat top surface, and for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1.

[0432] AA35. The method as described in embodiment AA18, wherein for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1.

[0433] AA36. The method as described in embodiment AA18, wherein the minimum lateral dimension of the spacer is less than or substantially equal to the minimum size of the analyte in the sample.

[0434] AA37. The method as described in embodiment AA18, wherein the minimum lateral dimension of the spacer is in the range of 0.5 μm to 100 μm.

[0435] AA38. The method as described in embodiment AA18, wherein the spacer has a columnar shape, and the sidewall corners of the spacer have a rounded shape with a radius of curvature of at least 1 μm.

[0436] AA39. The method as described in embodiment AA18, wherein the spacer has a density of at least 1000 / mm 2 of.

[0437] AA40. The method as described in any of the preceding embodiments, wherein at least one of the plates is transparent.

[0438] AA41. The method as described in any of the preceding embodiments, wherein at least one of the plates is made of a flexible polymer.

[0439] AA42. The method as described in embodiment AA18, wherein for the pressure on the pressing plate, the spacer is non-pressible and / or independently, only one of the plates is flexible.

[0440] AA43. The method as described in any of the preceding embodiments, wherein the thickness of the flexible plate is in the range of 10 μm to 200 μm.

[0441] AA44. The method as described in embodiment AA18, wherein the variation in the uniform thickness is less than 30%.

[0442] AA45. The method as described in embodiment AA18, wherein the variation in the uniform thickness is less than 10%.

[0443] AA46. The method as described in embodiment AA18, wherein the variation in the uniform thickness is less than 5%.

[0444] AA47. The method as described in any of the preceding embodiments, wherein the plates are connected by a hinge and are configured to change from an open configuration to a closed configuration by folding the plates along the hinge.

[0445] AA48. The method as described in any of the preceding embodiments, wherein the uniform thickness sample layer is uniform over a lateral area of at least 1 mm 2 .

[0446] AB1. A system for analyzing a sample, comprising:

[0447] a) A Q card for manipulating a sample for analysis, the Q card comprising two plates that are movable relative to each other and have an open configuration and a closed configuration;

[0448] b) A handheld mobile communication device that comprises a camera;

[0449] c) An adapter having a slot configured to hold the closed Q card, wherein the adapter is connected to the handheld mobile communication device and allows the camera to take an image of the closed Q card; and

[0450] d) A remote device capable of storing information and communicating with the mobile communication device;

[0451] wherein the sample is deposited on one or both plates of the Q card in the open configuration, and at least a portion of the sample is between the two plates in the closed configuration,

[0452] wherein the system is configured to generate an image of the Q card in the adapter and transmit the image and / or the analysis result of the image to a remote location.

[0453] AB2. The system as described in embodiment AB1, wherein the Q card can be placed in the closed configuration by folding.

[0454] AB3. The system as described in embodiment AB1, wherein the remote device is configured to analyze the image and / or the analysis result of the image.

[0455] AB4. The system as described in embodiment AB1, wherein the remote device is configured to communicate with other remote devices.

[0456] AB5. The system as described in embodiment AB1, wherein the remote device is configured to notify a third party in the event of an anomaly detected in a sample placed in the Q card.

[0457] AC1. A method for providing health advice to a subject, comprising:

[0458] a) using a Q card and an associated mobile communication device to analyze one or more analytes in a sample from the subject;

[0459] b) transmitting the analysis result of the analyte from the mobile communication device to a remote location;

[0460] c) storing the analysis result in a data set;

[0461] d) at the remote location, generating a series of health advice based on the cumulative analysis results in the data set; and

[0462] e) providing health advice to the subject by sending a message to the mobile communication device;

[0463] wherein the health advice includes advice on medicine, nutrition / diet, exercise, and / or treatment for the subject.

[0464] AC2. The method as described in paragraph AC1, further comprising identifying the needs of the subject before providing health advice to the subject.

[0465] B. Testing cholesterol with a QMAX device

[0466] Another aspect of the present invention provides a device and a method for cholesterol testing using a QMAX device.

[0467] BA1. A method for analyzing a liquid sample, comprising:

[0468] (a) obtaining the liquid sample;

[0469] (b) obtaining a device, the device comprising a first plate, a second plate, and a spacer fixed to one or both of the plates; wherein:

[0470] i. the plates are movable relative to each other into different configurations, which include an open configuration and a closed configuration;

[0471] ii. each plate respectively comprises an inner surface having a sample contact area, and

[0472] iii. the spacer has a predetermined substantially uniform height, and at least one of the spacers is located within the sample contact area;

[0473] (c) depositing the sample on one or both of the plates when the plates are in the open configuration,

[0474] In the open configuration, the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers; and

[0475] (d) After (c), the two plates are joined together and the plates are pressed into a closed configuration,

[0476] wherein in the closed configuration, at least a portion of the sample is pressed by the two plates into a layer of very uniform thickness, the layer being defined by the inner surfaces of the two plates and adjusted by spacers;

[0477] wherein one or both of the sample contact surfaces include one or more storage sites that store one or more reagents, the reagents being configured to dissolve and diffuse in the sample in the closed configuration and react with cholesterol in the sample to produce or alter a luminescent signal;

[0478] (e) Reading the luminescent signal from the layer of very uniform thickness to obtain a measurement of total cholesterol in the sample.

[0479] BA2. The method according to paragraph BA1, wherein the one or more reagents are configured to react with cholesterol to produce or alter a colorimetric luminescent signal,

[0480] wherein the reading step (e) includes detecting and quantifying the colorimetric luminescent signal from the analyte in the layer of very uniform thickness.

[0481] BA3. The method according to paragraph BA1, wherein the one or more reagents include cholesterol ester hydrolase and cholesterol oxidase.

[0482] BA4. The method according to paragraph BA3, wherein the one or more reagents further include peroxidase and a color probe.

[0483] BA5. The method according to paragraph BA4, wherein the color probe includes 4 - aminophenazone and phenol.

[0484] BA6. The method according to paragraph BA1, wherein the one or more storage sites include a first storage site on the first plate and a second storage site on the second plate.

[0485] BA7. The method according to paragraph BA6, wherein:

[0486] i. The first storage site includes cholesterol ester hydrolase and cholesterol oxidase; and

[0487] ii. The second storage site includes 4 - aminophenazone, phenol, and peroxidase.

[0488] C. Heavy metal testing

[0489] Another aspect of the present invention provides an apparatus and method for testing heavy metals in biological / chemical samples. More specifically, the present invention provides a method for detecting heavy metal ions in an aqueous system, an apparatus comprising a heavy metal ion test strip, and a sensor. The apparatus provided by the present invention provides a portable test method for detecting heavy metal ions in a convenient, efficient, and rapid manner.

[0490] Heavy metal (ion) pollution refers to environmental pollution caused by heavy metals or their compounds. The increase in the content of heavy metals in the environment, especially in the case of heavy metal pollution in water systems, is mainly due to human factors, such as mining, exhaust gas emissions, sewage irrigation, and the use of heavy metal-containing products, which leads to the deterioration of environmental quality. There is still a need for a heavy metal ion test strip that can be used to detect small amounts or even trace amounts of heavy metal ions in water systems in a simple, low-cost, highly sensitive, highly reliable, and stable manner. At the same time, it is required that the test piece can be detected in-situ and can detect heavy metal ions with high sensitivity. In addition, it is expected that heavy metal ions can be detected not only qualitatively but also quantitatively or semi-quantitatively. The present invention provides an apparatus and method for achieving these purposes.

[0491] C-1. Apparatus and Method for Heavy Metal Testing

[0492] Figure C1 shows that the present invention comprises two parts: 1. Testing, comprising a test card having a dry reagent in a sample chamber with a controlled volume and can be inserted into a smartphone-based reader for measurement; 2. Calculation, comprising a method for converting a photo taken by a smartphone into a signal for calculating the analyte concentration.

[0493] As shown in Figure C1, the present invention is an apparatus and method for obtaining a collection point of an analyte on a test platform and selecting a quantitative label, comprising:

[0494] 1. Providing a modular colorimetric reactive test platform having a test area and a calibration area;

[0495] 2. Providing an analyte to be tested on the test area of the modular colorimetric test platform, wherein the test area is adapted to enable a colorimetric reaction with the analyte;

[0496] 3. Obtaining a color image of the test area and the calibration area containing the analyte;

[0497] 4. Selecting a pixel array in each color image of the test area and the calibration area containing the analyte;

[0498] 5. Determining the median RGB color value of each pixel array;

[0499] 6. Converting the median RGB color value of each pixel array into a characteristic value;

[0500] 7. Provide calibration marks, the calibration marks being related to selected quantitative marks of the eigenvalue;

[0501] 8. Correlate the eigenvalues to determine the selected quantitative marks of the analyte

[0502] As shown in FIG. C2, a first plate is uniformly printed with a color indicator and a pH regulator. The first plate is a forced white substrate. The color indicator is a biological / chemical reagent that exhibits a specific reaction to heavy metals in a liquid sample. The liquid sample includes but is not limited to water, soil samples, oils, body fluids, and foods. In some embodiments, the sample is drinking water. In some embodiments, the sample is food. In some embodiments, the second plate is a forced white polystyrene plate.. In some embodiments, the color indicator is dried on the first plate. In some embodiments, the pH regulator is dried on the first plate. In some embodiments, the concentration of the dried color indicator is from 1 μM to 10 mM. In some embodiments, the concentration of the dried pH regulator is from 1 μM to 10 mM.

[0503] As shown in FIG. C2, the surface of the first plate facing the second plate is defined as the inner surface of the first plate; the surface of the second plate facing the first plate is also defined as the inner surface of the second plate. In some embodiments, the inner surface of each plate includes a sample contact area for contacting a sample containing the analyte. The sample contact area may occupy part or all of the corresponding inner surface.

[0504] As shown in FIG. C2, in order to use a colorimetric test to test for heavy metals in water, a pH regulator must be added to the sample to adjust the pH level to an optimal condition. This is because the chemical reaction rate of the color indicator with heavy metal ions varies significantly at different pH levels, which results in large color changes in the test if the pH is not adjusted. For heavy metal testing, combinations of pH regulators or multiple combinations thereof dried on the plate to adjust the sample pH level include but are not limited to: formic acid (methanoic acid), oxalic acid (ethanedioic acid), lactic acid (2-hydroxypropanoic acid), malic acid (2-hydroxybutanedioic acid), citric acid (2-hydroxypropane-1,2,3-tricarboxylic acid), carbonic acid (hydroxyformic acid, not an IUPAC name), aminomethylphosphonic acid.

[0505] As shown in FIG. C2, the second plate includes spacers fixed on the inner surface of the second plate. However, it should be noted that in some embodiments, the spacers are fixed on the inner surface of the first plate, while in other embodiments, the spacers are fixed on the inner surfaces of both the second plate and the first plate.

[0506] As shown in Figure C2, the spacer is in the range between 1μm, 2μm, 5μm, 10μm, 20μm, 50μm, 100μm, 200μm, 500μm, 1000μm or between any two values. The diameter of the holes in the spacer is about 0.5mm, 1mm, 2mm, 3mm, 4mm, 5mm, or in the range between any two values. The center-to-center spacing between the holes is 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 20mm, 50mm or in the range between any two values. The second plate is a transparent flat film with a thickness of about 1μm, 2μm, 5μm, 10μm, 20μm, 50μm, 100μm, 200μm, 500μm, 1000μm or in the range between any two values.

[0507] As shown in Figure C2, the first and second plates can be moved relative to each other into different configurations; one of the configurations is an open configuration where the two plates are partially or fully separated and the spacing between the plates is not adjusted by the spacer. Figure C1 shows the plates in the open configuration where a sample (such as but not limited to blood) can be added to the first plate, the second plate, or both plates. In some embodiments, the inner surface of the corresponding plate contains a sample contact area that occupies a portion of the entire inner surface. In certain embodiments, the spacer is positioned within the sample contact area. In some embodiments, the spacer is not fixed to either plate but is mixed in the sample.

[0508] As shown in Figure C2. The second plate is a transparent thin film with a smooth surface. The absorption of the second plate must not interfere with the absorption of the color indicator. Depending on the flexibility of the material, a thickness of 10μm to 300μm can be used for the second plate as long as there is no deformation of the sample chamber after pressing the second plate onto the sample.

[0509] Figure 3 A schematic diagram of the test procedure is shown. 1. First, a small sample is added to each well printed with the color indicator and pH regulator. 2. Then the transparent second plate is pressed onto the top of the spacer to form a closed sample chamber. 3. Incubate for 1 minute to allow each individual sample to develop color. In this method, the color indicator and pH regulator are completely dissolved and mixed.

[0510] As shown in Figure C3, a white polystyrene (PS) substrate printed with a self-made color indicator and a pH regulator. The amounts of the color indicator and the pH regulator on the sensing area are carefully controlled according to the size of the pores such that when each pore is filled with the sample, a desired pH level and color indicator concentration can be obtained. Different chemicals are used as color indicators according to the type of heavy metals or their combination. The color indicator can be: (1) for lead detection, the color indicator is 0.01%-0.2% sodium rhodizonate (preferably 0.2% after being dissolved in the sample), or (2) for copper, cadmium, chromium, mercury, 10 μM-1 mM dithizone (preferably 30 μM after being dissolved in the sample).

[0511] As shown in Figure C3, the printing parameters of the color indicator can vary as long as uniform drying is achieved on the first plate. The printing conditions, i.e., droplet volume, speed, depend on the surface wettability of the first plate, which is well known to those skilled in the art and thus need not be described. In the present invention, the printing conditions are a droplet diameter of 500-600 μm, a pitch of ~1 mm, and a printing speed of ~10 mm / second.

[0512] As shown in Figure C3, the pore size is determined by the size of the pore array on the spacer. The thickness of the spacer, the diameter of the pores, and their spacing determine the sample volume. Their structure is flexible, but it is crucial to avoid deformation of the sample chamber in certain structures, i.e., small aspect ratios. Here, the thickness of the spacer can be 2 μm-1 mm (preferably, 100 μm), and the pore diameter can be 100 μm-10 mm (preferably, 3 mm), and the center-to-center spacing can be 100 μm-10 mm (preferably, 6 mm).

[0513] As shown in Figure C3, in some embodiments, the method of the present invention further comprises incubating a layer of uniform thickness for a predetermined period of time after step (2) and before step (3). In certain embodiments, the predetermined period of time is equal to or longer than the time required for the detection antibody to diffuse through the layer of uniform thickness into the sample. In certain embodiments, the predetermined period of time is less than 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, or 60 minutes, or within a range between any two values.

[0514] Figure C4 is a chemical reaction diagram for testing lead in water. Lead ions react with sodium rhodizonate (dark yellow) dissolved in the sample to form insoluble lead rhodizonate with a red-dark red color. The color absorption can be analyzed to calculate the lead concentration in water.

[0515] Figure C5 is a chemical reaction diagram for testing heavy metals in water. The heavy metals can be Cd, Cu, Cr, Hg. The heavy metal ions react with dithizone dissolved in the sample to form dithizone-metal complexes, which produce different colors for different heavy metals. The colors can be used to identify the types of heavy metals, and the color absorption can be analyzed to calculate the heavy metal concentration in water.

[0516] Figure C6 is a schematic diagram of converting the colorimetric water lead test standard curves of each R, G, B channel into a single standard curve. For each sample containing different concentrations of heavy metals, the R, G, B signals are different. The combination of the R, G, B channel signals of different lead concentrations is used for this conversion. In some embodiments, the combination method is a linear combination. In some embodiments, the coefficients for combining the RGB channel signals are constants. In some embodiments, the coefficients for combining the RGB channel signals are matrices. In some embodiments, the coefficients for combining the RGB channel signals are functions of the lead concentration in water.

[0517] As shown in Figure C7, the algorithm for converting the standard curves of individual R, G, B channels into a single standard curve is a process of finding the optimal coefficients for combining the R, G, B signals to achieve the best measurement sensitivity. In some embodiments, the linear combination of the R, G, B channel signals of different lead concentrations is used for this conversion. In some embodiments, the generalized reduced gradient algorithm is used to train the linear coefficients. This algorithm is open source and known to those skilled in the art and does not need to be elaborated. Here, the process of this algorithm is simply shown in the figure:

[0518] 1. First, we define 4 constants: C1, C2, C3, and C4 such that

[0519] Signal = C1 * R + C2 * G + C3 * B + C4

[0520] 2. Make a small change to the linear coefficients by a predetermined amount

[0521] 3. Calculate the limit of detection (LOD),

[0522] 4. Keep changing the linear coefficients until the minimum LOD can be achieved

[0523] In the present invention, we use 48 different tests to train the data. It is expected that further improving the accuracy can be achieved with more training data. This is well known to those skilled in the art and does not need further explanation.

[0524] C-2. Example: Testing the lead concentration in tap water

[0525] For example, we prepared a chip for testing lead in water. On a white rigid PS substrate, we printed a self-made color indicator. The color indicator is 0.2% sodium rhodizonate (this is the saturated concentration), and by adding citric acid, the pH regulator is pH - 3.0 (this pH was optimized through our own experiments). We printed the reagent mixture with parameters of a droplet diameter of 500 - 600 μm, a spacing of ∼1 mm, and a printing speed of ∼10 mm / sec.

[0526] For this example, we fabricated a plate with 48 holes per plate, and the hole diameter is 3 mm.

[0527] The center - to - center distance is 6 mm, and the hole height is ∼100 μm (controlled using double - sided tape from adhesive research). Then, 0.7 μL of the sample was dropped into each hole. Then, the holes were covered with a 175 - μm - thick PET film and waited for 1 minute. Immediately after incubating for 1 minute, each hole was measured. For the test, the light source used was the smartphone camera flash. Images were taken using the smartphone camera.

[0528] As assay verification, we calculated 4 key performances: 1. The limit of detection (LOD) per plate; 2. The within - assay CV% per plate, 3. The between - assay CV% per test day, and 4. The inter - day CV%. For this example, we prepared a total of 8 plates, and each plate was prepared with different batches of reagents at different times. We conducted the tests on 2 different days, and each day we tested on 4 different plates. On each plate, we performed assays at 8 different concentrations: 417 ppb, 213 ppb, 106 ppb, 53.4 ppb, 26.7 ppb, 13.3 ppb, 6.7 ppb, and 0 ppb. For each concentration, we repeated the measurement 6 times.

[0529] Figure C8 shows the water lead test standard curves for each R, G, B channel. The RGB channel signals vary with the Pb 2+ concentration curve, and using the conversion equation signal = - 0.88 * R + G – 0.27 * B + 56.12 is converted into a single standard curve. The converted data was fitted using 5PL logistic fitting. The error bars are the standard deviations of 6 replicate holes. After conversion, the LOD is 8.5 ppb.

[0530] Figure C9 shows the sensitivities of all 8 different test plates in this example of the present invention. Each test plate was prepared with different reagents and tested at different times. The average LOD obtained is 8 ppb, which is lower than the EPA action level at 15 ppb.

[0531] Figure C10 is a table of within-run, between-run, and day-to-day CV% for the determination of lead in water. Near the LOD, the within-run CV% for each test was approximately 4%, the between-run CV% was approximately 4%, and the day-to-day CV% was approximately 1.1%.

[0532] In summary, this example shows the testing of lead concentration in tap water, which shows (1) sensitivity: average LOD - 8 ppb. All test plates showed an LOD meeting the EPA standard (15 ppb), with the best LOD obtained being 3.9 ppb. (2) Repeatability: within-run CV% approximately 4% at LOD, between-run CV% approximately 4% at LOD, and day-to-day CV% approximately 1.1% at LOD. D. Food safety and allergen testing using the QMAX device

[0533] Another aspect of the present invention provides an apparatus and method for safety and allergen testing in food samples.

[0534] As described above, the apparatus, system, and method of the present invention can be used to analyze food samples, such as samples from raw food, processed food, cooked food, drinking water, etc., for the presence of food markers. A food marker can be any suitable marker, such as those shown in Table B9 below, which can be captured by a capture agent that specifically binds to the food marker in the CROF device configured with the capture agent. Environmental samples can be obtained from any suitable source, such as tap water, drinking water, prepared food, processed food, or raw food, etc. In some embodiments, the presence, absence, or quantitative level of a food marker in a sample can indicate the safety or hazard to a subject if the food is consumed. In some embodiments, the food marker is a substance derived from a pathogen or microorganism that indicates the presence of the organism in the food from which the sample was obtained. In some embodiments, the food marker is a toxic or harmful substance if consumed by the subject. In some embodiments, the food marker is a bioactive compound that may inadvertently or accidentally alter physiology if consumed by the subject. In some embodiments, the food marker is an indication of the way the food was obtained (grown, obtained, captured, harvested, processed, cooked, etc.). In some embodiments, the food marker indicates the nutritional content of the food. In some embodiments, the food marker is an allergen that can induce an allergic reaction if the food from which the sample was obtained is consumed by the subject.

[0535] In some embodiments, the apparatus, system, and method of the present invention further include receiving or providing a report that indicates the safety or hazard to a subject of consuming the food from which the sample was obtained, based on information including the measured level of the food marker. Information for evaluating the safety of food for consumption can include data of types and measured amounts different from the food marker. These other data can include any health conditions related to the consumer (allergies, pregnancy, chronic or acute diseases, current prescription medications, etc.).

[0536] The report can be generated by a device configured to read the CROF device or can be generated at a remote location when sending data including the measured amount of the food marker. In some cases, a food safety expert can be at a remote location or can access the data sent to the remote location and can analyze or view the data to generate a report. The food safety expert can be a scientist or administrator of a government agency (e.g., the U.S. Food and Drug Administration (FDA) or the CDC), a research institution (e.g., a university), or a private company. In certain embodiments, the food safety expert can send instructions or recommendations to a user based on the data sent by the device and / or analyzed at the remote location.

[0537] The food markers are listed in Table D1. In some embodiments of the present invention, the QMAX device is used to detect the presence and / or amount of an analyte, including but not limited to the food markers listed in Table D1.

[0538] Table D1: Food Markers

[0539]

[0540]

[0541]

[0542]

[0543] E. Uniform sample thickness with imprecise force pressing.

[0544] Some embodiments of the device or method for forming a uniform sample thickness by pressing with an imprecise force as described herein and in the provisional 62 / 456504 filed on February 8, 2017, are hereby incorporated by reference in their entirety for all purposes.

[0545] In some embodiments, the imprecise force is about 0.01 kg, 0.05 kg, 0.1 kg, 0.25 kg, 0.5 kg, 1 kg, 2.5 kg, 5 kg, 7.5 kg, 10 kg, 20 kg, 25 kg, 30 kg, 40 kg, 50 kg, 60 kg, 70 kg, 80 kg, 100 kg, 200 kg, or in the range between any two of these values; and the preferred ranges of 0.5 - 2 kg, 2 - 5 kg, 5 - 7.5 kg, 7.5 - 10 kg, 10 - 20 kg, 20 - 40 kg, 40 - 60 kg, or 60 - 100 kg.

[0546] In some embodiments, the imprecise force is applied by hand, such as by clamping an object between the thumb and forefinger, or by clamping and rubbing an object between the thumb and forefinger.

[0547] In some embodiments, the hand pressing force is about 0.05 kg, 0.1 kg, 0.25 kg, 0.5 kg, 1 kg, 2.5 kg, 5 kg, 7.5 kg, 10 kg, 20 kg, 25 kg, 30 kg, 40 kg, 50 kg, 60 kg, or within a range between any two values; and preferred ranges of 0.5 - 1 kg, 1 - 2 kg, 2 - 4 kg, 4 - 6 kg, 6 - 10 kg, 10 - 20 kg, 20 - 40 kg, or 40 - 60 kg.

[0548] In some embodiments, the pressure of hand pressing is 0.01 kg / cm 2 , 0.1 kg / cm 2 , 0.5 kg / cm 2 , 1 kg / cm 2 , 2 kg / cm 2 , 2.5 kg / cm 2 , 5 kg / cm 2 , 10 kg / cm 2 , 20 kg / cm 2 , 30 kg / cm 2 , 40 kg / cm 2 , 50 kg / cm 2 , 60 kg / cm 2 , 100 kg / cm 2 , 150 kg / cm 2 , 200 kg / cm 2 , or a range between any two values; and preferred ranges of 0.1 kg / cm 2 to 0.5 kg / cm 2 , 0.5 kg / cm 2 to 1 kg / cm 2 , 1 kg / cm 2 to 5 kg / cm 2 or 5 kg / cm 2 to 10 kg / cm 2 .

[0549] As used herein, in the context of a force (e.g., "imprecise pressing force"), the term "imprecise" means a force

[0550] (a) having a magnitude that is not precisely known or precisely predictable when the force is applied;

[0551] (b) whose magnitude varies from one application of the force to the next; and

[0552] (c) the imprecision (i.e., variation) of the force in (a) and (c) is at least 20% of the total force actually applied.

[0553] Imprecise forces can be applied by hand, for example, by clamping an object between the thumb and forefinger, or by clamping and rubbing an object between the thumb and forefinger.

[0554] EA. Imprecise force, specific IGS^4 / hE

[0555] EA1. An apparatus for forming a thin fluid sample layer of a uniform predetermined thickness by pressing with an imprecise pressure, comprising:

[0556] A first plate, a second plate, and a spacer, wherein:

[0557] i. The plates are movable relative to each other into different configurations;

[0558] ii. One or both of the plates are flexible;

[0559] iii. Each plate comprises an inner surface having a sample contact area for contacting a blood sample;

[0560] iv. Each plate comprises a force area on its respective outer surface for applying an imprecise pressure that forces the plates together;

[0561] v. One or both of the plates comprise spacers permanently fixed to the inner surfaces of the respective plates;

[0562] vi. The spacers have a predetermined substantially uniform height equal to or less than 200 microns and a predetermined fixed spacer spacing;

[0563] vii. The fourth power of the spacer spacing (IDS) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5×10 6 μm 3 / GPa or less; and

[0564] viii. At least one of the spacers is located inside the sample contact area;

[0565] One of the configurations is an open configuration, in which: 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;

[0566] The other of the configurations is a closed configuration, which is configured after the sample is deposited in the open configuration and the plates are forced into the closed configuration by applying an imprecise pressure on the force areas; and in the closed configuration: at least a portion of the sample is pressed by the two plates into a layer of very uniform thickness and is substantially stationary relative to the plates, wherein the uniform thickness of the layer is defined by the sample contact areas of the two plates and is adjusted by the plates and the spacers.

[0567] EA2. A method of forming a thin fluid sample layer having a uniform predetermined thickness by pressing with an imprecise pressure, comprising the following steps:

[0568] (a) Obtaining a first plate, a second plate, and a spacer, wherein:

[0569] i. The plates are movable relative to each other into different configurations;

[0570] ii. One or both of the plates are flexible;

[0571] iii. Each plate includes an inner surface having a sample contact area for contacting a blood sample;

[0572] iv. Each plate includes a force area on its respective outer surface for applying an imprecise pressure that forces the plates together;

[0573] v. One or both of the plates include a spacer permanently fixed to the inner surface of the respective plate;

[0574] vi. The spacer has a predetermined substantially uniform height equal to or less than 200 microns and a predetermined fixed spacer spacing;

[0575] vii. The fourth power of the spacer spacing (IDS) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5×10 6 μm 3 / GPa or less; and

[0576] viii. At least one of the spacers is located inside the sample contact area;

[0577] (b) Obtaining a fluid sample;

[0578] (c) Depositing the sample on one or both of the plates; when the plates are configured in an open configuration, wherein the open configuration is a configuration in which the two plates are partially or fully separated and the spacing between the plates is not regulated by the spacer;

[0579] (d) After (c), using the two plates to press at least a portion of the sample into a layer of substantially uniform thickness defined by the sample contact surfaces of the plates, wherein the uniform thickness of the layer is regulated by the spacer and the plates, and wherein the pressing includes:

[0580] Placing the two plates together; and

[0581] conformally pressing an area of ​​at least one of the plates in parallel or sequentially to press the plates together into a closed configuration, wherein the conformal pressing produces a substantially uniform pressure on the at least a portion of the sample on the plates, and the pressing causes at least a portion of the sample to spread laterally between the sample contacting surfaces of the plates, and wherein the closed configuration is a configuration in which the spacing between the plates in the uniform thickness region layer is regulated by the spacers; and wherein the reduced thickness of the sample reduces the time for mixing the reagents on the storage site with the sample, and

[0582] The force in which the two panels are pressed into the closed configuration is an imprecise pressing force provided by the human hand.

[0583] EB. Manual press, specific spacer hardness - contact area product

[0584] EB1. A device for forming a thin layer of a fluid sample having a uniform predetermined thickness by pressing with an imprecise force, comprising:

[0585] A first plate, a second plate and a spacer, wherein:

[0586] i. The plates can be moved relative to each other into different configurations;

[0587] ii. One or both plates are flexible;

[0588] iii. Each plate comprises a sample contact area on its respective inner surface for contacting and / or pressing a fluid sample;

[0589] iv. Each plate comprises an area on its respective outer surface for applying a force forcing the plates together;

[0590] v. One or both panels contain spacers permanently fixed to the inner surface of the respective panel;

[0591] vi. The spacers have a predetermined substantially uniform height equal to or less than 200 microns, a predetermined width, and a predetermined spacer pitch;

[0592] vii. The ratio of the spacing between the spacers to the width of the spacers is 1.5 or greater; and

[0593] viii. At least one of the spacers is located inside the sample contact area;

[0594] One of the configurations is an open configuration in which: the two plates are partially or completely separated, the spacing between the plates is not regulated by a spacer, and the sample is deposited on one or both of the plates;

[0595] Another of the configurations is a closed configuration configured after the sample is deposited in the open configuration; and in the closed configuration: at least a portion of the sample is pressed by two plates into a layer with a very uniform thickness and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is defined by the sample contact regions of the two plates and is adjusted by the plates and spacers; and

[0596] wherein the force pressing the two plates into the closed configuration is an imprecise pressing force provided by a human hand.

[0597] EB2. A method of forming a thin fluid sample layer with a uniform predetermined thickness by pressing with imprecise pressure, comprising the steps of:

[0598] (a) Obtaining a first plate, a second plate, and spacers, wherein:

[0599] i. The plates are movable relative to each other into different configurations;

[0600] ii. One or both of the plates are flexible;

[0601] iii. Each plate includes a sample contact region on its respective inner surface for contacting and / or pressing the fluid sample;

[0602] iv. Each plate includes a region on its respective outer surface for applying a force that forces the plates together;

[0603] v. One or both of the plates include spacers permanently fixed to the inner surface of the respective plate;

[0604] vi. The spacers have a predetermined substantially uniform height, a predetermined width, and a predetermined spacer pitch that is equal to or less than 200 microns;

[0605] vii. The ratio of the spacer pitch to the spacer width is 1.5 or greater; and

[0606] viii. At least one of the spacers is located inside the sample contact region;

[0607] (b) Obtaining a fluid sample;

[0608] (c) Depositing the sample on one or both of the plates; when the plates are configured in an open configuration, wherein the open configuration is a configuration in which the two plates are partially or fully separated and the spacing between the plates is not adjusted by the spacers;

[0609] (d) After (c), using the two plates to press at least a portion of the sample into a layer of substantially uniform thickness, the layer being defined by the sample contact surfaces of the plates, wherein the uniform thickness of the layer is adjusted by the spacers and the plates, wherein the pressing includes:

[0610] Place two plates together; and

[0611] Conformably press at least one region of at least one of the plates either parallelly or sequentially to press the plates together into a closed configuration, wherein the conformable pressing creates a substantially uniform pressure on the plates over at least a portion of the sample, and the pressing causes at least a portion of the sample to laterally spread between the sample contact surfaces of the plates, and wherein the closed configuration is a configuration in which the spacing between the plates in the uniformly thick region layer is adjusted by the spacers; and wherein the reduced thickness of the sample reduces the time for mixing the reagent at the storage site with the sample, and

[0612] wherein the force for pressing the two plates into the closed configuration is an imprecise pressing force provided by a human hand.

[0613] EC. Manual pressing, specifying IDS / hE& spacer hardness - contact area product

[0614] EC1. An apparatus for forming a thin fluid sample layer having a uniform predetermined thickness by pressing with an imprecise force, comprising:

[0615] A first plate, a second plate, and spacers, wherein:

[0616] i. The plates are movable relative to each other into different configurations;

[0617] ii. One or both of the plates are flexible;

[0618] iii. Each plate includes a sample contact area on its respective inner surface for contacting and / or pressing the fluid sample;

[0619] iv. Each plate includes a region on its respective outer surface for applying a force to force the plates together;

[0620] v. One or both of the plates include spacers permanently fixed to the inner surface of the respective plate;

[0621] vi. The spacers have a predetermined substantially uniform height, a predetermined width, and a predetermined spacer pitch that is equal to or less than 200 microns;

[0622] vii. The ratio of the spacer pitch to the spacer width is 1.5 or greater; and

[0623] viii. At least one of the spacers is located inside the sample contact area;

[0624] wherein one configuration is an open configuration, in which: 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;

[0625] Another one of the structures is a closed structure configured after the sample is deposited on the open structure; and in the closed structure: at least a portion of the sample is pressed by two plates into a layer with a very uniform thickness and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is defined by the sample contact areas of the two plates and is adjusted by the plates and the spacers;

[0626] The force pressing the two plates into the closed structure is imprecise and is provided by a human hand.

[0627] EC2. A method of forming a thin fluid sample layer having a uniform predetermined thickness by pressing with imprecise pressure, comprising the steps of:

[0628] (a) Obtaining a first plate, a second plate, and spacers, wherein:

[0629] i. The plates are movable relative to each other into different structures;

[0630] ii. One or both of the plates are flexible;

[0631] iii. Each plate includes a sample contact area on its respective inner surface for contacting and / or pressing the fluid sample;

[0632] iv. Each plate includes an area on its respective outer surface for applying a force that forces the plates together;

[0633] v. One or both of the plates include spacers permanently fixed to the inner surface of the corresponding plate;

[0634] vi. The spacers have a predetermined substantially uniform height, a predetermined width, and a predetermined spacer pitch that is equal to or less than 200 microns;

[0635] vii. The ratio of the spacer pitch to the spacer width is 1.5 or greater; and

[0636] viii. At least one of the spacers is located inside the sample contact area;

[0637] (b) Obtaining a fluid sample;

[0638] (c) Depositing the sample on one or both of the plates; when the plates are configured in an open structure, wherein the open structure is a structure in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacers;

[0639] (d) After (c), use the two plates to press at least a portion of the sample into a layer of substantially uniform thickness, the layer being defined by the sample contact surfaces of the plates, wherein the uniform thickness of the layer is adjusted by the spacer and the plates, and wherein the pressing comprises:

[0640] Place the two plates together; and

[0641] Conformably press at least one region of at least one of the plates either parallelly or sequentially to press the plates together into a closed configuration, wherein the conformable pressing creates a substantially uniform pressure on the plates over at least a portion of the sample, and the pressing causes at least a portion of the sample to laterally spread between the sample contact surfaces of the plates, and wherein the closed configuration is a configuration in which the spacing between the plates in the region of the uniform thickness layer is adjusted by the spacers; and wherein the reduced thickness of the sample reduces the time for mixing the reagent on the storage site with the sample, and

[0642] wherein the force for pressing the two plates into the closed configuration is an imprecise pressing force provided by a human hand.

[0643] ED. Manual pressing, specified strut spacing, and IDS / W ratio

[0644] ED1. An apparatus for forming a thin fluid sample layer of uniform predetermined thickness by pressing with an imprecise force, comprising:

[0645] A first plate, a second plate, and a spacer, wherein:

[0646] i. The plates are movable relative to each other into different configurations;

[0647] ii. One or both of the plates are flexible;

[0648] iii. Each plate includes a sample contact area on its respective inner surface for contacting and / or pressing a fluid sample;

[0649] iv. Each plate includes a region on its respective outer surface for applying a force that forces the plates together;

[0650] v. One or both of the plates include spacers permanently fixed to the inner surfaces of the respective plates;

[0651] vi. The spacers have a predetermined substantially uniform height, a predetermined width, and a predetermined spacer spacing that is equal to or less than 200 microns;

[0652] vii. The ratio of the spacer spacing to the spacer width is 1.5 or greater;

[0653] viii. At least one of the spacers is located inside the sample contact area; and

[0654] One of the configurations is an open configuration, in which: the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers, and the sample is deposited on one or both of the plates;

[0655] Another of the configurations is a closed configuration configured after the sample is deposited in the open configuration; and in the closed configuration: at least a portion of the sample is pressed by the two plates into a layer of very uniform thickness and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is defined by the sample contact areas of the two plates and is adjusted by the plates and spacers;

[0656] The force pressing the two plates into the closed configuration is imprecise and is provided by hand.

[0657] ED2. A method of forming a thin fluid sample layer of uniform predetermined thickness by pressing with imprecise pressure, comprising the steps of:

[0658] (a) obtaining a first plate, a second plate, and spacers, wherein:

[0659] i. the plates are movable relative to each other into different configurations;

[0660] ii. one or both of the plates are flexible;

[0661] iii. each plate includes a sample contact area on its respective inner surface for contacting and / or pressing the fluid sample;

[0662] iv. each plate includes a region on its respective outer surface for applying a force that forces the plates together;

[0663] v. one or both of the plates include spacers permanently fixed to the inner surface of the respective plate;

[0664] vi. the spacers have a predetermined substantially uniform height, a predetermined width, and a predetermined spacer spacing that is equal to or less than 200 microns;

[0665] vii. the ratio of the spacer spacing to the spacer width is 1.5 or greater;

[0666] viii. at least one of the spacers is located inside the sample contact area; and

[0667] (b) obtaining a fluid sample;

[0668] (c) depositing the sample on one or both of the plates; when the plates are configured in an open configuration, wherein the open configuration is a configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacers;

[0669] (d) After (c), use the two plates to press at least a portion of the sample into a layer of substantially uniform thickness, the layer being defined by the sample contact surfaces of the plates, wherein the uniform thickness of the layer is adjusted by the spacer and the plates, and wherein the pressing comprises:

[0670] Place the two plates together; and

[0671] Conformably press at least one region of at least one of the plates either parallelly or sequentially to press the plates together into a closed configuration, wherein the conformable pressing creates a substantially uniform pressure on the plates over at least a portion of the sample, and the pressing causes at least a portion of the sample to laterally spread between the sample contact surfaces of the plates, and wherein the closed configuration is a configuration in which the spacing between the plates in the region of the uniform thickness layer is adjusted by the spacers; and wherein the reduced thickness of the sample reduces the time for mixing a reagent at a storage site with the sample, and

[0672] wherein the force for pressing the two plates into the closed configuration is an imprecise pressing force provided by a human hand.

[0673] EE. Volume determination, specified IGS4 / hE

[0674] EE1. An apparatus for determining the volume of a relevant sample by pressing with an imprecise force provided by a human hand, comprising:

[0675] A first plate, a second plate, a spacer, and a region determination device, wherein:

[0676] i. The plates are movable relative to each other into different configurations;

[0677] ii. One or both of the plates are flexible;

[0678] iii. Each plate includes on its respective inner surface a sample contact region for contacting and / or pressing a fluid sample having a relevant volume to be measured;

[0679] iv. Each plate includes on its respective outer surface a region for applying a force to urge the plates together;

[0680] v. One or both of the plates include spacers permanently fixed to the inner surfaces of the respective plates;

[0681] vi. The spacers have a predetermined substantially uniform height equal to or less than 200 microns and a predetermined constant spacer spacing;

[0682] vii. The fourth power of the spacer spacing (IDS) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5×106 μm 3 / GPa or less;

[0683] viii. At least one of the spacers is located inside the sample contact area; and

[0684] ix. The area determination device is configured to determine the lateral area of the relevant volume;

[0685] One of the configurations is an open configuration, in which: the two plates are partially or completely separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates;

[0686] The other of the configurations is a closed configuration configured after the sample is deposited in the open configuration; and in the closed configuration: at least a portion of the sample is pressed by the two plates into a layer with a very uniform thickness and is substantially stationary relative to the plates, wherein the uniform thickness of the layer is defined by the sample contact areas of the two plates and is adjusted by the plates and the spacers;

[0687] Wherein, the relevant volume of the sample is part or all of the volume of the layer with a uniform thickness, and the value of the relevant volume is determined by the uniform thickness and the determined lateral area;

[0688] Wherein the force pressing the two plates into the closed configuration is imprecise and is provided by a human hand.

[0689] The device of any of the preceding embodiments, wherein the area determination device is a camera.

[0690] + The area determination device includes an area in the sample contact area of the plate, wherein the area is less than 1 / 100, 1 / 20, 1 / 10, 1 / 6, 1 / 5, 1 / 4, 1 / 3, 1 / 2, 2 / 3 of the sample contact area, or within a range between any two values.

[0691] + The area determination device includes a camera and an area in the sample contact area of the plate, wherein the area contacts the sample.

[0692] EE2. A method of forming a thin fluid sample layer with a uniform predetermined thickness by pressing with imprecise pressure, comprising the following steps:

[0693] (a) Obtaining a first plate, a second plate, and a spacer, wherein:

[0694] i. The plates are movable relative to each other into different configurations;

[0695] ii. One or both of the plates are flexible;

[0696] iii. Each plate includes a sample contact area on its respective inner surface for contacting and / or pressing a fluid sample having a relevant volume to be measured;

[0697] iv. Each plate includes an area on its respective outer surface for applying a force that forces the plates together;

[0698] v. One or both plates include spacers permanently fixed to the inner surface of the respective plate;

[0699] vi. The spacers have a predetermined substantially uniform height equal to or less than 200 microns and a predetermined constant spacer pitch;

[0700] vii. The fourth power of the spacer pitch (IDS) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5×10 6 μm 3 / GPa or less;

[0701] viii. At least one of the spacers is located inside the sample contact area; and

[0702] ix. The area determination device is configured to determine the lateral area of the relevant volume;

[0703] (b) Obtain a fluid sample;

[0704] (c) Deposit the sample on one or both plates; when the plates are configured in an open configuration, where the open configuration is a configuration in which the two plates are partially or fully separated and the spacing between the plates is not regulated by the spacers;

[0705] (d) After (c), use the two plates to press at least a portion of the sample into a layer of substantially uniform thickness, which layer is defined by the sample contact surfaces of the plates, where the uniform thickness of the layer is regulated by the spacers and the plates, and where the pressing includes:

[0706] Place the two plates together; and

[0707] Conformally press at least one area of at least one of the plates parallelly or sequentially to press the plates together into a closed configuration, where the conformal pressing produces a substantially uniform pressure on the plates over at least a portion of the sample, and the pressing causes at least a portion of the sample to spread laterally between the sample contact surfaces of the plates, and where the closed configuration is a configuration in which the spacing between the plates in the layer of uniform thickness region is regulated by the spacers; and where the reduced thickness of the sample reduces the time for mixing the reagent on the storage site with the sample, and

[0708] The force for pressing the two plates into a closed configuration is an imprecise pressing force provided by human hands.

[0709] EF. Volume determination, specifying IGS^4 / hE

[0710] EF1. An apparatus for determining the volume of a relevant sample by pressing with an imprecise force provided by human hands, comprising:

[0711] A first plate, a second plate, a spacer, and an area determination device, wherein:

[0712] i. The plates are movable relative to each other into different configurations;

[0713] ii. One or both of the plates are flexible;

[0714] iii. Each plate includes, on its respective inner surface, a sample contact area for contacting and / or pressing a fluid sample having a relevant volume to be measured;

[0715] iv. Each plate includes, on its respective outer surface, an area for applying a force that forces the plates together;

[0716] v. One or both of the plates include a spacer permanently fixed to the inner surface of the corresponding plate;

[0717] vi. The spacer has a predetermined substantially uniform height equal to or less than 200 microns and a predetermined constant spacer pitch;

[0718] vii. The fourth power of the spacer pitch (IDS) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5×10 6 μm 3 / GPa or less;

[0719] viii. At least one of the spacers is located inside the sample contact area; and

[0720] ix. The area determination device is configured to determine the lateral area of the relevant volume;

[0721] One of the configurations is an open configuration, in which: the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates;

[0722] The other of the configurations is a closed configuration configured after the sample is deposited in the open configuration; and in the closed configuration: at least a portion of the sample is pressed by the two plates into a layer with a very uniform thickness and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is defined by the sample contact areas of the two plates and is adjusted by the plates and the spacer;

[0723] Wherein, the relevant volume of the sample is part or all of the volume of a layer of uniform thickness, and the value of the relevant volume is determined by the uniform thickness and a defined lateral area;

[0724] Wherein the force pressing the two plates into a closed configuration is imprecise and is provided by human hands.

[0725] EF2. A method of forming a thin fluid sample layer having a uniform predetermined thickness by pressing with imprecise pressure, comprising the following steps:

[0726] (a) Obtaining a first plate, a second plate, and spacers, wherein:

[0727] i. The plates are movable relative to each other into different configurations;

[0728] ii. One or both of the plates are flexible;

[0729] iii. Each plate includes, on its respective inner surface, a sample contact area for contacting and / or pressing a fluid sample having a relevant volume to be measured;

[0730] iv. Each plate includes, on its respective outer surface, an area for applying a force that forces the plates together;

[0731] v. One or both of the plates include spacers permanently fixed to the inner surface of the respective plate;

[0732] vi. The spacers have a predetermined substantially uniform height equal to or less than 200 microns and a predetermined constant spacer spacing;

[0733] vii. The fourth power of the spacer spacing (IDS) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5×10 6 μm 3 / GPa or less;

[0734] viii. At least one of the spacers is located inside the sample contact area; and

[0735] ix. The area determination means is configured to determine the lateral area of the relevant volume;

[0736] (b) Obtaining a fluid sample;

[0737] (c) Depositing the sample on one or both of the plates; when the plates are configured in an open configuration, wherein the open configuration is a configuration in which the two plates are partially or fully separated and the spacing between the plates is not adjusted by the spacers;

[0738] (d) After (c), use the two plates to press at least a portion of the sample into a layer of substantially uniform thickness, the layer being defined by the sample contact surfaces of the plates, wherein the uniform thickness of the layer is adjusted by the spacer and the plates, and wherein the pressing comprises:

[0739] Place the two plates together; and

[0740] Conformably press at least one region of at least one of the plates, either in parallel or sequentially, to press the plates together into a closed configuration, wherein the conformable pressing creates a substantially uniform pressure on the plates over at least a portion of the sample, and the pressing causes at least a portion of the sample to spread laterally between the sample contact surfaces of the plates, and wherein the closed configuration is one in which the spacing between the plates in the region of the uniform thickness layer is adjusted by the spacers; and wherein the reduced thickness of the sample reduces the time for mixing the reagent on the storage site with the sample, and

[0741] wherein the force for pressing the two plates into the closed configuration is an imprecise pressing force provided by a human hand.

[0742] EG. More embodiments

[0743] The term "imprecise force" refers to a force having a completely unknown magnitude, only known within a range of magnitudes but not known at a specific magnitude (the magnitude range varies by at least 20% from the minimum to the maximum of the range), or a force that is unpredictable when applied. Examples of imprecise forces include: the magnitude of the imprecise force can vary from one application of the force to the next, can be non-uniform over the area where the force is applied, and can vary over the time of application of the force. It is not necessary to measure an imprecise force when applied.

[0744] The apparatus or method as described in any of the preceding embodiments, wherein the deformable sample is a fluid sample.

[0745] The apparatus or method as described in any of the preceding embodiments, wherein the deformable sample is a liquid sample.

[0746] The apparatus or method as described in any of the preceding embodiments, wherein the variation of the imprecise force is at least 30% of the total force actually applied.

[0747] The apparatus or method as in any of the preceding embodiments, wherein the variation of the imprecise force is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 500% of the total force actually applied, or within a range between any two values.

[0748] 1. The apparatus as described in any of the preceding embodiments, wherein the spacer has a flat top.

[0749] 2. The apparatus according to any one of the preceding embodiments, wherein the apparatus is further configured to have a sample thickness after removal of the pressure that is substantially the same as the thickness and uniformity when the force is applied.

[0750] 3. The apparatus according to any one of the preceding embodiments, wherein the imprecise force is provided by a human hand.

[0751] 4. The apparatus according to any one of the preceding embodiments, wherein the spacer spacing is substantially constant.

[0752] 5. The apparatus according to any one of the preceding embodiments, wherein the spacer spacing is substantially fixed in the region of the uniform sample thickness region.

[0753] 6. The apparatus according to any one of the preceding embodiments, wherein the product of the packing factor and the Young's modulus of the spacer is 2 MPa or greater.

[0754] 7. The apparatus according to any one of the preceding embodiments, wherein the force is applied directly or indirectly by hand.

[0755] 8. The apparatus according to any one of the preceding embodiments, wherein the applied force is in the range of 5 N to 20 N.

[0756] 9. The apparatus according to any one of the preceding embodiments, wherein the thickness variation of the very uniform layer is less than 15%, 10%, or 5% of the average thickness.

[0757] 10. The apparatus according to any one of the preceding embodiments, wherein the imprecise force is applied by clamping the apparatus between the thumb and forefinger.

[0758] 11. The apparatus according to any one of the preceding embodiments, wherein the predetermined sample thickness is greater than the spacer height.

[0759] 12. The apparatus according to any one of the preceding embodiments, wherein the apparatus holds itself in a closed configuration after removal of the pressure.

[0760] 13. The apparatus according to any one of the preceding embodiments, wherein the region of the uniform thickness sample layer is greater than the region where the pressure is applied.

[0761] 14. The apparatus according to any one of the preceding embodiments, wherein the spacer does not deform significantly during the application of the pressure.

[0762] 15. The apparatus according to any one of the preceding embodiments, wherein the pressure is not predetermined and not measured.

[0763] F. Binding sites and storage sites on the same plate

[0764] Another aspect of the present invention provides an apparatus and method for performing a biological / chemical assay using a QMAX device, wherein the binding site and the storage site are on the same plate, meaning that both the capture agent and the second reagent are coated on the same plate.

[0765] FA1. A method for assaying a sample, comprising

[0766] (a) obtaining a first plate having on its inner surface a sample contact area for contacting a sample containing a target analyte;

[0767] (b) obtaining a second plate having a sample contact area, the sample contact area including an assay area, wherein the assay area includes

[0768] (i) an immobilized capture agent that binds to the target analyte in the sample, and

[0769] (ii) a second reagent that is capable of diffusing in the sample upon contact with the sample;

[0770] wherein the first and second plates are movable relative to each other into different configurations, including an open configuration and a closed configuration;

[0771] (c) depositing the sample on one or both of the sample contact areas of the plates in the open configuration, wherein in the open configuration, the sample contact areas of the plates are spaced more than 200 μm apart;

[0772] (d) after (c), bringing the two plates into the closed configuration, wherein in the closed configuration, at least a portion of the sample deposited in (c) is confined between the sample contact areas of the two plates and has an average thickness in the range of 0.01 μm to 200 μm; and

[0773] (e) detecting a signal associated with the analyte captured at the binding site.

[0774] FB1. An apparatus for performing a competitive assay, comprising:

[0775] a first plate having on its inner surface a sample contact area for contacting a sample containing a target analyte;

[0776] a second plate having a sample contact area, the sample contact area including an assay area, wherein the assay area includes

[0777] (i) an immobilized capture agent that binds to the target analyte in the sample, and

[0778] (ii) a second reagent that is capable of diffusing in the sample upon contact with the sample;

[0779] wherein the first and second plates are movable relative to each other into different configurations;

[0780] One of the configurations is an open configuration, in which the plates are partially or completely separated, and the average spacing between the sample contact regions of the plates is greater than 300 μm; and

[0781] The other of the configurations is a closed configuration, in which the average spacing between the sample contact regions of the plates is 200 μm or less.

[0782] The method or apparatus as described in any of the foregoing embodiments, wherein the capture agent is spaced from the second agent by a distance that is at least 2 times less than the average spacing between the sample contact regions of the two plates.

[0783] The method or apparatus as described in any of the foregoing embodiments, wherein the capture agent and the second reagent are separated by a distance that is at least 2 times, 3 times, 5 times, 10 times, 20 times, 30 times, 50 times, 100 times, 200 times, 300 times, 500 times, 1000 times, 2000 times, 5000 times, 10000 times, 5000 times less than the average spacing between the sample contact regions of the two plates, or within the range of any two values.

[0784] The method or apparatus as described in any of the foregoing embodiments, wherein the signal associated with the analyte captured by the capture agent is a signal from (i) the analyte captured by the capture agent, (ii) a label attached to the analyte captured by the binding site, or (iii) both (i) and (ii).

[0785] The method or apparatus as described in any of the foregoing embodiments, wherein one or both of the sample contact regions contain spacers, and the spacers adjust the spacing between the sample contact regions of the plates when the plates are in the closed configuration.

[0786] The method as described in any of the foregoing embodiments, wherein when the plates are in the closed configuration, the spacing between the sample contact regions is adjusted by the spacers. The apparatus as described in any of the foregoing embodiments, wherein the apparatus further comprises spacers that adjust the spacing between the sample contact regions when the plates are in the closed configuration.

[0787] The method or apparatus as described in any of the foregoing embodiments, wherein the storage location further contains another reagent.

[0788] The method or apparatus as described in any of the foregoing embodiments, wherein in addition to the immobilized capture agent, the binding site further contains another reagent that is capable of diffusing in the sample when in contact with the sample.

[0789] The method or apparatus as described in any of the foregoing embodiments, wherein the detection of the signal is electrical, optical, or both. (Detection will be further introduced later. Fluorescence, SPR, etc.).

[0790] The method or device as described in any of the foregoing embodiments, wherein the sample is a blood sample (whole blood, plasma or serum).

[0791] The method or device as described in any of the foregoing embodiments, wherein the material of the fluorescent microspheres is dielectric (e.g., SiO2, polystyrene) or a combination of dielectric materials thereof.

[0792] The method or device as described in any of the foregoing embodiments, which comprises the step of adding a fluorescently labeled detection agent to a first plate to bind to a competitor.

[0793] The method or device as described in any of the foregoing embodiments, which comprises the step of washing after adding the detection agent.

[0794] The embodiments in these applications incorporated herein by reference may be considered to be combined with each other or as a single invention, rather than as discrete and independent documents.

[0795] In addition, the exemplary assay recipes disclosed herein can be applied to embodiments including but not limited to the following: bio / chemical assays, QMAX cards and systems, QMAX with hinges, notches, grooved edges and sliders, assays and devices with uniform sample thickness, smartphone detection systems, cloud computing designs, various detection methods, labels, capture agents and detection agents, analytes, diseases, applications and samples; various embodiments are disclosed, described and / or referenced in the foregoing applications, all of which are incorporated herein by reference in their entirety.

[0796] Other embodiments

[0797] The present invention includes various embodiments that can be combined in various ways as long as the various components are not contradictory to each other. The embodiments should be considered as a single invention document: each application has other applications as references and is also incorporated by reference in its entirety for all purposes, rather than as discrete and independent documents. These embodiments include not only the disclosures in the current document but also the documents cited, incorporated or claimed priority herein.

[0798] (1) Definitions

[0799] The terms used to describe the devices, systems and methods disclosed herein are defined in this application or in PCT applications (designating the United States) No. PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504 filed on February 8, 2017. The entire contents of all these applications are incorporated herein by reference for all purposes.

[0800] The terms "CROF card (or card)", "COF card", "QMAX card", "Q card", "CROF device", "COF device", "QMAX device", "CROF plate", "COF plate", and "QMAX plate" are interchangeable, except that in some embodiments, the COF card does not include a spacer; and these terms refer to a device that includes a first plate and a second plate that are movable relative to each other into different configurations (including an open configuration and a closed configuration), and the device includes a spacer (except for some embodiments of COF) that adjusts the spacing between the plates. The term "X plate" refers to one of the two plates in a CROF card to which the spacer is fixed. Further descriptions of the COF card, CROF card, and X plate are described in the provisional application serial number 62 / 456065 filed on February 7, 2017, all of which are incorporated herein by reference in their entirety for all purposes.

[0801] (2) Q-Card, Spacer, and Uniform Sample Thickness

[0802] The devices, systems, and methods disclosed herein may include or use a Q card, a spacer, and embodiments of uniform sample thickness for sample detection, analysis, and quantification. In some embodiments, the Q card includes a spacer that helps to make at least a portion of the sample a very uniform layer. The structure, materials, functions, variations, and dimensions of the spacer and the uniformity of the spacer and the sample layer are listed, described, and summarized in the PCT applications (designating the United States) numbered PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016, and September 14, 2016, respectively, the U.S. provisional application number 62 / 456065 filed on February 7, 2017, the U.S. provisional application number 62 / 456287 filed on February 8, 2017, and the U.S. provisional application number 62 / 456504 filed on February 8, 2017, all of the entire contents of which are incorporated herein by reference for all purposes.

[0803] (3) Hinge, Open Notch, Groove Edge, and Slide

[0804] The devices, systems, and methods disclosed herein may include or use a Q card for sample detection, analysis, and quantification. In some embodiments, the Q card includes a hinge, notch, groove, and slider that facilitate the operation of the Q card and the measurement of the sample. The structures, materials, functions, variations, and dimensions of the hinge, notch, groove, and slider are listed, described, and summarized herein or in PCT applications (designating the United States) No. PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504 filed on February 8, 2017, the entire contents of all of which are incorporated herein by reference for all purposes.

[0805] In some embodiments of QMAX, the sample contact region of one or both plates includes a press-open flow monitoring surface structure (MSS) configured to monitor how much flow has occurred after COF. For example, in some embodiments, the MSS includes a shallow square array that will cause friction on components in the sample (such as blood cells in blood). By examining the distribution of some components of the sample, information related to the flow of the sample and its components under COF can be obtained.

[0806] The depth of the MSS can be 1 / 1000, 1 / 100, 1 / 100, 1 / 5, 1 / 2 of the spacer height or within the range of any two values, and is in a protruding or hole shape.

[0807] (4) Q-Card, Slide, and Smartphone Detection System

[0808] The devices, systems, and methods disclosed herein may include or use a Q card for sample detection, analysis, and quantification. In some embodiments, the Q card is used with a slider of the card that allows a smartphone detection system to read. The structures, materials, functions, variations, dimensions, and connections of the Q card, slider, and mobile phone detection system are listed, described, and summarized herein or in PCT applications (designating the United States) No. PCT / US2016 / 045437 and PCT / US0216 / 051775 filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065 filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287 filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504 filed on February 8, 2017, the entire contents of all of which are incorporated herein by reference for all purposes.

[0809] (5)Detection Method

[0810] The devices, systems, and methods disclosed herein may include or be used in a variety of types of detection methods. Detection methods are listed, described, and summarized herein or in PCT applications (designating the United States) No. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017. The entire content of all these applications is incorporated herein by reference for all purposes.

[0811] (6) Label, Capture Agent, and Detection Agent

[0812] The devices, systems, and methods disclosed herein may use a variety of types of labels, capture agents, and detection agents for analyte detection. Labels are listed, described, and summarized herein or in PCT applications (designating the United States) No. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017. The entire content of all these applications is incorporated herein by reference for all purposes.

[0813] (7) Analyte

[0814] The devices, systems, and methods disclosed herein may be used to operate and detect various types of analytes (including biomarkers). Analytes are listed, described, and summarized herein or in PCT applications (designating the United States) No. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016 and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017. The entire content of all these applications is incorporated herein by reference for all purposes.

[0815] (8) Applications (Fields and Samples)

[0816] The devices, systems, and methods disclosed herein can be used for a variety of applications (fields and samples). Applications are listed, described, and summarized herein or in PCT applications (designating the United States) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016, and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, the entire contents of all of which are incorporated herein by reference for all purposes.

[0817] (9) Cloud

[0818] The devices, systems, and methods disclosed herein can utilize cloud technology for data transfer, storage, and / or analysis. Related cloud technologies are listed, described, and summarized herein or in PCT applications (designating the United States) Nos. PCT / US2016 / 045437 and PCT / US0216 / 051775, filed on August 10, 2016, and September 14, 2016, respectively, U.S. Provisional Application No. 62 / 456065, filed on February 7, 2017, U.S. Provisional Application No. 62 / 456287, filed on February 8, 2017, and U.S. Provisional Application No. 62 / 456504, filed on February 8, 2017, the entire contents of all of which are incorporated herein by reference for all purposes.

[0819] Other notes

[0820] Other embodiments of the inventive subject matter according to the present disclosure are described in the paragraphs listed below.

[0821] 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, such as when the word "single" is used. For example, reference to "analyte" includes a single analyte and multiple analytes, reference to "capture agent" includes a single capture agent and multiple capture agents, reference to "detection agent" includes a single detection agent and multiple detection agents, and reference to "reagent" includes a single reagent and multiple reagents.

[0822] As used herein, the terms "adapted" and "configured" mean that an element, component, or other subject 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 simply "can" perform a given function. Similarly, a subject stated to be configured to perform a particular function may additionally or alternatively be described as operable to perform that function.

[0823] As used herein, when referring to one or more components, features, details, structures, embodiments, and / or methods in accordance with the present disclosure, the phrases “for example,” the phrase “as an example,” and / or the terms “example” and “exemplary” in short are intended to convey that the described components, features, details, structures, embodiments, and / or methods are illustrative, non-exclusive examples of the 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 restrictive, required, or exclusive / exhaustive; and other components, features, details, structures, embodiments, and / or methods, including components, features, details, structures, embodiments, and / or methods that are structurally and / or functionally similar and / or equivalent, are also within the scope of the present disclosure.

[0824] As used herein, the phrases “at least one” and “one or more” with respect 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 at least one of each and every 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.

[0825] As used herein, the term “and / or” placed between a first entity and a second entity refers to (1) the first entity, (2) the second entity, and (3) one of the first entity and the second entity. Multiple entities listed using “and / or” shall be construed in the same manner, i.e., “one or more” of the entities so combined. Optionally, there may be other entities in addition to those specifically identified by the “and / or” clause, whether or not they are related to those specifically identified.

[0826] When a numerical range is recited herein, the present invention includes embodiments that include the endpoints, embodiments that exclude both endpoints, and embodiments that include one endpoint and exclude the other. It should be assumed that both endpoints are included unless otherwise stated. Further, unless otherwise stated or apparent to one of ordinary skill in the art from the context and understanding.

[0827] If any patent, patent application, or other reference is incorporated herein by reference and (1) defines a term in a manner inconsistent with the unincorporated portion of the present disclosure or other incorporated references and / or (2) is otherwise inconsistent with the unincorporated portion of the present disclosure or other incorporated references, the unincorporated portion of the present disclosure shall govern, and the term or the incorporated disclosure therein shall be construed only in accordance with the reference in which the term was first defined and / or the incorporated disclosure first appeared.

Claims

1. An apparatus for enhancing an optical signal when measuring a sample, comprising: A first plate, a second plate, a spacer, and a textured surface, wherein: i. The first plate and the second plate are movable relative to each other into different configurations; ii. One or both of the plates are flexible; iii. The first plate is transparent; iv. The second plate has a textured structure on its inner surface for scattering light incident on the inner surface; v. The textured surface may be an uneven, wavy, rough surface; vi. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height, and the height is greater than the average roughness of the textured surface and less than 200 μm; Wherein one of the configurations is an open configuration, wherein: the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; Wherein one of the configurations is a closed configuration, the closed configuration is configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the deposited sample is pressed by the two plates into a continuous layer.

2. A sample processing apparatus for enhancing an optical signal (Q card), comprising: A first plate, a second plate, a spacer, and a textured surface, wherein: i. The plates are movable relative to each other into different configurations; ii. One or both of the plates are flexible; iii. The second plate has a textured structure on its inner surface for scattering light incident on the surface; iv. The textured surface may be, but is not limited to, an uneven, wavy, rough surface; v. The textured surface may be regular or irregular; vi. The average roughness range of the textured surface is preferably, but not limited to, 2 μm to 5 μm; vii. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height; viii. The preferred height of the spacer is greater than the average roughness of the textured surface and less than 100 μm; Wherein one of the configurations is an open configuration, wherein: the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; Wherein one of the configurations is a closed configuration, the closed configuration is configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the deposited sample is pressed by the two plates into a continuous layer; Wherein the sample is liquid.

3. A sample processing apparatus for enhancing an optical signal (Q card), comprising: A first plate, a second plate, a spacer, and a textured surface, wherein: i. The plates are movable relative to each other into different configurations; ii. One or both of the plates are flexible; iii. The second plate has a textured structure on its inner surface for scattering light incident on the surface; iv. The textured surface may be, but is not limited to, an uneven, wavy, rough surface; v. The textured surface may be regular or irregular; vi. The average roughness of the textured surface preferably ranges from, but is not limited to, 2 μm to 5 μm; vii. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height; viii. The preferred height of the spacer is greater than the average roughness of the textured surface and less than 100 μm; wherein one of the configurations is an open configuration, in which: the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; wherein one of the configurations is a closed configuration, the closed configuration being configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the deposited sample is pressed into a continuous layer by the two plates; wherein the sample is in a liquid state.

4. The sample processing device according to any one of the preceding claims, wherein the textured surface is made of a semi-transparent white material and has a transmittance of 10% to 30%.

5. A sample processing device for enhancing an optical signal (Q card), comprising: a first plate, a second plate, a spacer, and a textured surface, wherein: i. The plates are movable relative to each other into different configurations; ii. One or both of the plates are flexible; iii. The second plate has a textured structure on its inner surface for scattering light incident on the surface; iv. The textured surface can be, but is not limited to, an uneven, wavy rough surface; v. The textured surface can be regular or irregular; vi. The average roughness of the textured surface preferably ranges from, but is not limited to, 2 μm to 5 μm; vii. The spacer is fixed to the inner surface of the first plate and has a predetermined uniform height; viii. The preferred height of the spacer is greater than the average roughness of the textured surface and less than 100 μm; wherein one of the configurations is an open configuration, in which: the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; wherein one of the configurations is a closed configuration, the closed configuration being configured after the sample is deposited in the open configuration, and in the closed configuration, at least a portion of the deposited sample is pressed into a continuous layer by the two plates; wherein the sample is in a liquid state.

6. A testing device, comprising: a) A sample processing device for enhancing an optical signal (Device_C1) according to the above device claims; b) A mobile computing device having a camera module and a light source; c) An illumination optical device comprising angled optical fibers; d) An external lens; - wherein the light source emits white light; - wherein the light source and the camera module are on the same side of the mobile computing device; - wherein the Q card is placed directly below the camera module, and the preferred distance between the Q card and the camera module is 15 mm to 20 mm; - Wherein the external lens is placed between the Q card and the camera module such that the sample in the Q card is within the working distance of the camera module. The preferred focal length of the external lens is 12 - 18 mm. The distance between the lens and the camera module is preferably as small as possible and not greater than 3 mm; Wherein the optical fiber guides the light emitted from the light source to irradiate the sample area directly below the camera module; Wherein one end face of the optical fiber is placed under the aperture of the light source, and the distance between them is preferably as small as possible and not greater than 3 mm; Wherein the diameter of the optical fiber is configured to be equal to the diameter of the light source aperture; Wherein the inclination angle of the installed optical fiber is set such that the central beam emitted from the optical fiber irradiates the sample area directly below the camera module.

7. A testing device, comprising: a) A sample processing device for enhancing optical signals as described in the above device claims; b) A mobile computing device having a camera module; c) An independent light source; d) An external lens; - Wherein the light source emits white light, the light source is placed under the Q card and in a straight line with the camera module. The preferred distance between the light source and the Q card is 5 mm - 10 mm. Wherein the Q card is placed directly below the camera module, and the preferred distance between them is 5 - 10 mm; Wherein the external lens is placed between the Q card and the camera module such that the sample in the Q card is within the working distance of the camera module. The preferred focal length of the external lens is 4 - 8 mm. The preferred distance between the lens and the camera module is as small as possible and not greater than 3 mm.

8. A method for analyzing the optical signal of a sample, comprising the following steps: a) Collecting a sample liquid; b) Obtaining the device as described in any of the foregoing embodiments; c) When the plate is in an open configuration, depositing the sample on one or both of the plates in the device; d) Placing the two plates together, pressing the plates into the closed configuration so that the sample forms a liquid layer between the two plates; e) Inserting the device into the testing device; f) Turning on the light source of the testing device; g) Using the camera module to collect an image of the sample; And h) The mobile computing device processes the image to analyze the colorimetric or fluorescence signal of the image, thereby obtaining some characteristics of the sample.

9. The device, system or method as claimed in any of the foregoing claims, wherein the textured surface is made of a semi - transparent white material and has a transmittance of 10% - 30%.

10. The device, system or method as claimed in any of the foregoing claims, the textured surface is made of an opaque white material or coated with a reflective metal film, and the metal film can be, but is not limited to, aluminum, silver and gold. The preferred thickness range of the metal film is preferably, but not limited to, 10 nm - 100 nm.

11. The device, system or method as claimed in any one of the preceding claims, wherein the textured surface is made of an opaque white material or coated with a reflective metal film, which may be, but is not limited to, aluminum, silver and gold. The preferred thickness range of the metal film is preferably, but is not limited to, 10 nm to 100 nm.

12. The device, system or method as claimed in any one of the preceding claims, wherein the light-scattering layer may be made of a highly reflective opaque white material with a reflectivity of at least 50%, 60%, 70%, 80%, 90%, 100% or within a range between any two values.

13. The device, system or method as claimed in any one of the preceding claims, wherein the reflection spectrum of the light-scattering surface is in the range of 300 nm to 1000 nm.

14. The device, system or method as claimed in any one of the preceding claims, wherein the light-scattering layer may be made of a semi-opaque white material, and the transmittance is 10% to 30%.

15. The device, system or method as claimed in any one of the preceding claims, wherein the light-scattering layer may be made of a reflective metal film, and the light-scattering layer may be made of an opaque white dielectric film.

16. The device, system or method as claimed in any one of the preceding claims, wherein the light-scattering layer has a textured surface with Ra (arithmetic mean roughness) of 0.5 μm to 200 μm, Rsm (average spacing of profile elements) > 0.5 μm, and RΔa (average slope of the profile) > 0.

1.

17. The device, system or method as claimed in any one of the preceding claims, wherein the textured surface is made of an opaque white material.

18. The device, system or method as claimed in any one of the preceding claims, wherein the textured surface may be regular or irregular, and the shape of a single feature on the textured surface may be, but is not limited to, square, triangle, acute angle.

19. The device, system or method as claimed in any one of the preceding claims, wherein the height of the spacer is greater than the average roughness of the textured surface and less than 200 μm.

20. The device, system or method as claimed in any one of the preceding claims, wherein the average roughness height (Ra) of the textured reflection needs to be at least 20% of the wavelength of the illumination light, and can be up to 5 times the spacing between the first plate and the second plate, or within a range between these two values.

21. The device or system as claimed in any one of the preceding claims, wherein the average lateral feature size (ba) needs to be at least 20% of the wavelength of the illumination light and can be up to 10 times, or within a range between these two values.

22. The device or system as claimed in any one of the preceding claims, wherein the average period (ba) needs to be at least 50% of the wavelength of the illumination light and can be up to 1000 times, or within a range between these two values.

23. The device, system or method as claimed in any one of the preceding claims, wherein the sample deposited on the Q card is from a subject, and the subject performs step a).

24. The device, system or method as claimed in any one of the preceding claims, wherein if the analysis result of the sample is not within the normal range, an abnormality is identified.

25. The apparatus, system or method according to any of the preceding claims, wherein if the analysis results generated by the remote device and the mobile handheld communication device differ by a predetermined value, the anomaly is identified.

26. The apparatus, system or method according to any of the preceding claims, wherein the sample comprises a body fluid selected from the group consisting of amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (ear wax), chyle, chyme, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled condensate.

27. The apparatus, system or method according to any of the preceding claims, wherein the sample comprises an environmental sample obtained from: rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rock, concrete, wood, brick, sewage; air, radiator, industrial exhaust gas or vehicle exhaust gas.

28. The device, system or method according to any one of the preceding claims, wherein the sample comprises a food sample, the food sample comprising: Raw food ingredients, cooked food or processed food, plant and animal food sources, pre-processed food or fully processed food.

29. The apparatus, system or method according to any of the preceding claims, wherein in step (a), the Q-card is pressed by hand.

30. The apparatus, system or method according to any of the preceding claims, wherein step (e) comprises comparing the result with a threshold or normal range to identify a sample containing an anomaly.

31. The apparatus, system or method according to any of the preceding claims, wherein the method further comprises: updating the handheld mobile communication device if the analysis at the remote location produces significantly different results.

32. The apparatus, system or method according to any of the preceding claims, wherein the sample deposited on the Q-card is from a subject, and the analysis result is not transmitted to the subject.

33. The apparatus, system or method according to any of the preceding claims, wherein the third party is a medical professional.

34. The apparatus, system or method according to any of the preceding claims, wherein the medical professional is a doctor or a nurse practitioner.

35. The method according to any one of claims 22 - 34, wherein the third party is an insurance company.

36. The apparatus, system or method according to any of the preceding claims, wherein the result from the mobile communication device and / or the result from the remote location is sent to the emergency room.

37. The apparatus, system or method according to any of the preceding claims, wherein based on the result, the handheld mobile communication device or the remote location transmits follow-up information to the subject.

38. The apparatus, system or method according to any of the preceding claims, wherein the follow-up information includes an explanation of the result, education about the disease or medical condition, information related to possible treatments, information about the location of a suitable physician, information related to changes in diet and / or exercise, or advertising.

39. The apparatus, system or method according to any of the preceding claims, wherein the Q card includes spacers having a substantially uniform height and a predetermined constant spacer pitch, and in the closed configuration, at least a portion of the sample is pressed by two plates of the Q card to form a layer having a very uniform thickness and being substantially stagnant relative to the plates, wherein the uniform thickness of the layer is defined by the inner surfaces of the two plates and is adjusted by the plates and the spacers.

40. The apparatus, system or method according to any of the preceding claims, wherein at least one of the plates is flexible.

41. The apparatus, system or method according to any of the preceding claims, wherein for a flexible plate, the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is in the range of 60 to 750 GPa-μm.

42. The apparatus, system or method according to any one of the preceding claims, wherein for the flexible plate, the fourth power of the spacer pitch (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible plate, ISD 4 / (hE), is equal to or less than 10 6 μm 3 / GPa.

43. The apparatus, system or method according to any of the preceding claims, wherein the spacers that adjust the layer of uniform thickness have a filling factor of at least 1%, where the filling factor is the ratio of the spacer area in contact with the layer of uniform thickness to the total plate area in contact with the layer of uniform thickness.

44. The apparatus, system or method according to any of the preceding claims, wherein for the spacers that adjust the layer of uniform thickness, the Young's modulus of the spacers multiplied by the filling factor of the spacers is equal to or greater than 10 MPa, where the filling factor is the ratio of the spacer area in contact with the layer of uniform thickness to the total plate area in contact with the layer of uniform thickness.

45. The apparatus, system or method according to any of the preceding claims, wherein one or both of the plates include position markers on or within the surface of the plate, which provide position information of the plate.

46. The apparatus, system or method according to any of the preceding claims, wherein one or both of the plates include scale markers on or within the surface of the plate, which provide information about the lateral dimensions of the sample and / or the structure of the plate.

47. The apparatus, system or method according to any of the preceding claims, wherein one or both of the plates include imaging markers on or within the surface of the plate, which assist in imaging the sample.

48. The apparatus, system or method according to any of the preceding claims, wherein the spacers act as position markers, scale markers, imaging markers, or any combination thereof.

49. The apparatus, system or method according to any of the preceding claims, wherein the average thickness of the layer of uniform thickness is in the range of 0.2 μm to 3.8 μm and the sample is blood.

50. The apparatus, system or method according to any of the preceding claims, wherein the spacer pitch is in the range of 7 μm to 50 μm.

51. The apparatus, system or method according to any of the preceding claims, wherein the spacer pitch is in the range of 50 μm to 120 μm.

52. The device, system or method according to any one of the preceding claims, wherein the spacer pitch is in the range of 120 μm to 200 μm.

53. The device, system or method according to any one of the preceding claims, wherein the spacer pitch is substantially a fixed interval.

54. The device, system or method according to any one of the preceding claims, wherein the spacer is a column having a cross-sectional shape selected from circular, polygonal, annular, square, rectangular, oval, elliptical or any combination thereof.

55. The device, system or method according to any one of the preceding claims, wherein the spacer has a columnar shape and has a substantially flat top surface, and for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1.

56. The device, system or method according to any one of the preceding claims, wherein each spacer has a ratio of the lateral dimension of the spacer to its height of at least 1.

57. The device, system or method according to any one of the preceding claims, wherein the minimum lateral dimension of the spacer is less than or substantially equal to the minimum size of the analyte in the sample.

58. The device, system or method according to any one of the preceding claims, wherein the minimum lateral dimension of the spacer is in the range of 0.5 μm to 100 μm.

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