Rapid change in sample temperature for assays

Abstract

Furthermore, the present invention provides apparatus and methods that enable rapid change or circulation (i.e., heating and cooling) of sample temperature with high speed, less heating energy, high energy efficiency, compact and simplified equipment (e.g., handheld equipment), easy and rapid operation, and / or low cost.

Description

[0001] Cross-referencing

[0002] This application is a divisional application of Chinese national phase application No. 201880048466.5, filed on May 23, 2018. Like the parent application, this application claims the benefits of U.S. Provisional Patent Application No. 62 / 510,063, filed May 23, 2017; International Application No. PCT / US2018 / 017307, filed February 7, 2018; International Application No. PCT / US2018 / 018108, filed February 14, 2018; International Application No. PCT / US2018 / 018405, filed February 15, 2018; and International Application No. PCT / US2018 / 028784, filed April 23, 2018, each of which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This invention relates to apparatus and methods for performing biological and chemical assays, and more particularly to apparatus and methods that feature rapid sample temperature changes, rapid assays, and ease of use. Background Technology

[0004] In some chemical, biological, or medical assays, rapid changes in sample temperature or rapid thermal cycling are required (e.g., polymerase chain reaction (PCR) or isothermal amplification for nucleic acid amplification).

[0005] To improve sample thermal cycling speed (i.e., shorter thermal cycling time), rapid heating elements (e.g., radiant heaters or electric heaters) with small sample sizes, small thermal cycling chambers, and small thermal masses have been described in the prior art. However, there remains a need for apparatuses and methods capable of improving the speed, energy efficiency, device size and weight, operating procedures and time, power consumption, and / or cost of rapid sample thermal cycling. Such improvements could offer significant economic benefits.

[0006] One object of the present invention is to meet these needs. The present invention also provides useful apparatus and methods for isothermal nucleic acid amplification. Summary of the Invention

[0007] The following brief overview is not intended to include all features and aspects of the invention.

[0008] Furthermore, the present invention provides apparatus and methods for rapidly changing or cycling (i.e., heating and cooling) sample temperatures with high speed, less heating energy, high energy efficiency, compact and simplified devices (e.g., handheld devices), easy and fast operation, and / or low cost.

[0009] The present invention has experimentally achieved the cycling of sample temperature between 95°C and 55°C within one second or less.

[0010] The present invention has six novel aspects: (1) a device and method that allows for rapid thermal cycling, (2) a device and method that allows for uniform sample thickness and mechanical stability of the sample holder for processing, (3) simple operation, (3) a device and method for performing real-time PCR, (4) biochemistry, and (5) a smartphone-based system.

[0011] To rapidly cycle the temperature of a sample or a portion thereof, the thermal mass and lateral heat must be reduced.

[0012] Radiative heating and cooling are preferred. BRIEF DESCRIPTION OF DRAWINGS

[0013] 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 invention in any way. In some cases, the drawings are not drawn to scale. In graphs presenting experimental data points, the lines connecting the data points are only to guide the observation of the data and have no other meaning.

[0014] Figure 1 A schematic showing certain components of a system for changing the temperature of a sample and for monitoring a signal from the sample, according to some embodiments, is shown.

[0015] Figure 2A An embodiment of a device having a heating layer separated from a cooling layer, according to some embodiments, is shown.

[0016] Figure 2B An embodiment of a device having a heating layer in contact with a cooling layer, according to some embodiments, is shown.

[0017] Figure 3A And 3B A perspective view and a cross-sectional view of a combined heating / cooling layer on the outer surface of a plate, according to some embodiments, are shown.

[0018] Figure 4A A perspective view and a cross-sectional view of a device in an open configuration, according to some embodiments, are shown.

[0019] Figure 4B A perspective view and a cross-sectional view of a device when the sample holder is in a closed configuration, according to some embodiments, are shown.

[0020] Figure 5 A top view of a device, according to some embodiments, is shown.

[0021] Figure 6A A perspective view of a system when the device (sample holder of the system) is in an open configuration, according to some embodiments, is shown.

[0022] Figure 6BA cross-sectional view of a system is shown, according to some embodiments, when the sample holder is in a closed configuration.

[0023] Figure 7 A cross-sectional view of a system is shown, according to some embodiments, showing additional elements that facilitate temperature change and control.

[0024] Figure 8A and 8B Perspective and cross-sectional views of a device with multiple sample contact areas, according to some embodiments, are shown.

[0025] Figure 9 A cross-sectional view of a device is shown, according to some embodiments, demonstrating how to add and squeeze a sample.

[0026] Figure 10 A cross-sectional view of a device is shown, according to some embodiments, demonstrating a PCR process.

[0027] Figure 11A and 11B Top and cross-sectional views of a heating layer on a plate of a device, according to some embodiments, are shown.

[0028] Figure 12A and 12B A cross-sectional view of a device is shown, according to some embodiments, with a first plate, a second plate, and a heating / cooling layer.

[0029] Figure 13 A cross-sectional view of a system for rapid temperature change of a sample is shown, according to some embodiments, including a heating source using a fiber.

[0030] Figure 14 A cross-sectional view of a system for rapid temperature change of a sample is shown, according to some embodiments, including a heating source using a lens.

[0031] Figure 15A and 15B Top and side views of a device with separate heating elements, according to some embodiments, are shown.

[0032] Figure 16A and 16B Perspective and side views of a light pipe for directing electromagnetic waves (e.g., light) from a heating source, according to some embodiments, are shown.

[0033] Figure 17 A perspective view of a light pipe, according to some embodiments, is shown.

[0034] Figure 18A and 18B Side and top views of a sample device heated with a heat source, according to some embodiments, are shown.

[0035] Figure 19 A schematic side view of a device with a lens focusing light from a heat source is shown, according to some embodiments.

[0036] Figure 20 Experimental absorption spectra of different materials are shown, according to some embodiments.

[0037] Figure 21 Experimental thermal cycling data is shown, according to some embodiments.

[0038] Figure 22 Experimental data showing the effect of the area of the heating / cooling layer on heating and cooling times, according to some embodiments.

[0039] Figure 23 Experimental data showing heating and cooling times versus the area size of the heating / cooling layer, according to some embodiments.

[0040] Figure 24A Experimental data showing the relationship between heating time and heating / cooling layer thickness, according to some embodiments.

[0041] Figure 24B Experimental data showing the relationship between cooling time and heating / cooling layer thickness, according to some embodiments.

[0042] Figure 25A Experimental data showing the relationship between heating time and the distance between the heating / cooling layer and the sample, according to some embodiments.

[0043] Figure 25B Experimental data showing the relationship between cooling time and the distance between the heating / cooling layer and the sample, according to some embodiments.

[0044] Figure 26A Experimental data showing the relationship between heating time and the sample layer thickness, according to some embodiments.

[0045] Figure 26B Experimental data showing the relationship between cooling time and the sample layer thickness, according to some embodiments.

[0046] Figure 27A Experimental data showing the relationship between heating time and heating source power, according to some embodiments.

[0047] Figure 27B Experimental data showing the relationship between cooling time and heating source power on the sample, according to some embodiments.

[0048] Figure 28AExperimental data showing the relationship of heating time to different heating / cooling layer materials according to some embodiments.

[0049] Figure 28B Experimental data showing the relationship of cooling time to different heating / cooling layer materials according to some embodiments.

[0050] Figure 29A A schematic of a device with spherical spacers according to some embodiments is shown.

[0051] Figure 29B A schematic of a device with cylindrical spacers according to some embodiments is shown.

[0052] Figure 30A And 30B A top view and a side view of a device on a support according to some embodiments are shown, respectively.

[0053] Figure 31 Experimental data showing the effect of placing a device on a device support and / or a device adapter on heating and cooling times according to some embodiments is shown. DETAILED DESCRIPTION

[0054] The following detailed description demonstrates some embodiments of the application by way of example and not by way of limitation. The section headings and any subtitles, if any, used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. The contents under a section heading and / or subtitle are not limited to the section heading and / or subtitle, but apply to the entire description of the application.

[0055] Any publication mentioned is provided solely for its disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate this publication by virtue of prior application. In addition, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0056] It should be noted that the drawings are not intended to be to scale. For the sake of clarity, some elements can be exaggerated in the drawings. The size of the elements should be determined in accordance with the description provided herein, which describes the embodiments in more detail.

[0057] DEFINITIONS

[0058] The term“sample thermal cycler” or“thermal cycler” refers to an apparatus that can raise and lower the temperature of a sample, and if desired, can repeat heating and cooling the sample between two temperatures.

[0059] The term“sample thermal cycling” or“thermal cycling” refers to repeatedly raising and lowering the temperature of a sample.

[0060] The term "sample thermal cycling" or "thermal cycling" refers to the cycling of a sample temperature to a higher temperature and then back to the original temperature.

[0061] The term "sample thermal cycling time" or "thermal cycling time" refers to the time to perform a given number of thermal cycles.

[0062] The term "sample thermal cycling speed" or "thermal cycling speed" refers to the speed at which thermal cycling is performed.

[0063] The term "thermal mass" of a material refers to the energy required to heat the temperature of the material by one degree when there are no other energy losses. Thus, the thermal mass of a material is equal to the specific heat per unit volume multiplied by the volume of the material.

[0064] The term "thermal conductivity to capacity ratio" refers to the ratio of the thermal conductivity of a material to its heat capacity. For example, at about room temperature, the thermal conductivity to capacity ratio of gold is 1.25 cm2 / sec (square centimeters per second) and the thermal conductivity to capacity ratio of water is 1.4 x 10-3cm2 / sec.

[0065] 10-3cm2 / sec.

[0066] The term "wasted energy" refers to energy provided to a sample holder that is not used to directly heat an associated sample.

[0067] The terms "a" and "an" as used herein shall be understood to mean "at least one" unless otherwise indicated.

[0068] The term "about" as used herein generally refers to a range of greater than or less than the stated value by 15% in the context of the particular usage. For example, "about 10" shall include the range of 8.5 to 11.5.

[0069] The term "sample holder support" refers to a device to which a sample holder is physically attached and mechanically supported by the device.

[0070] The term "disposable" as used herein generally refers to a device designed to be discarded after a limited use (e.g., in terms of number of reactions, thermal cycles, or time) rather than being reused indefinitely.

[0071] The term "nucleic acid amplification" refers to the production of one or more replicated copies of an existing nucleic acid.

[0072] The term "nucleic acid amplification cycle" refers to the entire set of steps used to perform a single round of nucleic acid amplification.

[0073] The term "template" refers to the nucleic acid being amplified.

[0074] The term "amplification product" refers to the replicated copies of an existing nucleic acid produced from a template during nucleic acid amplification.

[0075] The term "black paint" means a paint that is visible to the human eye as black under daylight illumination.

[0076] The term "cooling gas" or "cooling liquid" means a gas phase or a liquid phase used to remove thermal energy, for example, from a sample, from a sample holder, from a material, or from a region.

[0077] The term "mechanical contact" as used herein generally means a contact formed between one or more materials, where the materials are in physical contact.

[0078] The term "thermal path" means a distance through which thermal energy is transferred from one location to another.

[0079] The term "relevant sample" or "relevant sample volume" means a volume of a sample that is heated and / or cooled to a desired temperature during a thermal cycle, and the relevant sample can be a portion or the entire volume of a sample on a sample holder, and there is no fluidic separation between the portion of the sample and the remainder of the sample.

[0080] The term "high K material" means a material having a thermal conductivity (K) equal to or greater than 50 W / (m-K) (e.g., gold: -314 W / (m-K) and graphite - 80 W / (m-K) are high K materials).

[0081] The term "low K material" means a material having a thermal conductivity (K) equal to or less than 1 W / (m-K) (e.g., water (-0.6 W / (m-K)) and plastic (-0.2 W / (m-K)) are low K materials).

[0082] The terms "cooling time in a thermal cycle" and "cooling cycle time" are interchangeable.

[0083] The terms "heating time in a thermal cycle" and "heating cycle time" are interchangeable.

[0084] The term "heating zone" means (a) a heating layer when the heating layer is a layer that is separate from a cooling layer; or (b) a heating region when heating and cooling use the same layer. A heating zone is directly heated by a heating source.

[0085] The term "directly heated" means energy input into the region. For example, for a heating zone heated by an LED heating source, the LED heating source projects light over the heating zone. For a heating zone heated by an electrical heating source, the electrical heating source sends an electrical current to the heating zone to generate heat in the heating zone.

[0086] The term "cooling zone" means (a) a cooling layer when the cooling layer is a layer that is separate from a heating layer; or (b) a cooling region when cooling and heating use the same layer. Unless otherwise specified, a cooling zone comprises a material having a thermal conductivity of 50 W / m- or greater.

[0087] The term "heating layer is heated by a heating source" means that "the heating zone of the heating layer or heating / cooling layer is heated by a heating source".

[0088] The term "non-sample material" refers to the material on the sample holder that is outside the relevant sample volume.

[0089] The term "wasted heating energy" refers to the energy that must be provided to non-sample materials and non-relevant samples in order to heat the relevant sample volume to the required temperature.

[0090] The term "average linear dimension" of a region is defined as the length equal to the area multiplied by 4 and then divided by the perimeter of the area. For example, if the region is a rectangle with width w and length L, then the average linear dimension of the rectangle is 4*W*L / (2*(L+W)) (where "*" represents multiplication and " / " represents division). According to this definition, for the square of width W, the average linear dimension is W, and for a circle with diameter d, the average linear dimension is d.

[0091] The term "lateral" refers to the direction of the plate parallel to the sample holder.

[0092] The term "vertical" refers to the direction perpendicular to the plate of the sample holder.

[0093] The "period" in the term "periodic array of structures" refers to the distance from the center of the structure to the center of the nearest adjacent identical structure.

[0094] The terms “smartphone” or “mobile phone” are used interchangeably to refer to a type of telephone having a camera and communication hardware and software that can use the camera to take images, manipulate images taken by the camera, and transmit data to a remote location. In some embodiments, a smartphone has a flash.

[0095] Unless otherwise stated, the terms “heating layer” or “heating zone” refer to a layer of material containing at least one layer of material with a thermal conductivity of 50 W / mK or greater.

[0096] The term "heated volume" refers to the volume of the material to be heated. "Heated sample volume" refers to the volume of a portion of the sample being heated.

[0097] The term "cooling layer" refers to a thermal radiation cooling layer with high thermal conductivity and a large surface thermal radiation capacity, which is at least 50% of the surface thermal radiation capacity of a blackbody.

[0098] The term "lateral dimension" or "lateral area" for a sample inside a sample holder used for heating and cooling refers to the lateral dimension or lateral area of ​​the portion of the sample that is heated to the desired temperature.

[0099] The term "plate" refers to a self-supporting plate, except when two plates are in a "closed configuration," where the two plates are closed together and separated by a spacer (in which case the pair of plates is self-supporting). The term "self-supporting" means that the central region of the plate has no support. For example, when two plates are in a closed configuration and a sample is placed between the plates, the central region of the plate pair has no mechanical support; only air contacts the outer surfaces of the plates.

[0100] Working principle

[0101] One aspect of the invention is to reduce thermal cycling time, reduce the heating energy used for such cycling, increase energy efficiency, and reduce total power consumption.

[0102] Thermal cycling time (rate), heating energy, energy efficiency, and power consumption are related. When raising the temperature of a given sample requires more heating energy, more energy must be removed when cooling the sample, which in turn requires more time and / or more energy for cooling.

[0103] Many existing thermal cyclers require a significant amount of heating energy to be transferred to the sample holder (e.g., the plastic chamber wall) rather than the sample; they utilize lateral heat conduction through the large thermal mass and poor thermal conductivity of the sample holder as the primary cooling channel for cooling the sample (note that the material needs to absorb and release energy for heat conduction); they use conductive cooling as the primary cooling method; and / or employ additional cooling gases or moving cooling blocks. These methods result in long thermal cycling times, high thermal energy consumption, low energy efficiency, bulky equipment, and / or high costs.

[0104] Based on theoretical and experimental research, this invention provides a solution to certain shortcomings of sample thermal cycling in existing technologies.

[0105] To demonstrate the working principle of this invention, let's examine the energy components in the heating and cooling of the sample using a thermal cycler. Heating and cooling share three energy components: (i) one relating to thermal mass (i.e., the material's ability to absorb and store energy); the greater the thermal mass, the more energy is required for heating and the more energy needs to be removed during cooling; (ii) heat loss due to thermal radiation; and (iii) heat loss due to thermal conduction / convection. For rapid heating, all three energy components need to be small. However, for rapid cooling, the first energy component needs to be small, but at least one of the last two needs to be large.

[0106] Through theoretical and experimental studies, this invention is based on certain designs that can balance and / or optimize the three energy components used for rapid heating and cooling. In particular, in some embodiments, this invention reduces the thermal mass that must be heated in the thermal cycle, limits lateral heat conduction, and uses radiative heat loss as the primary means of removing energy from the heated sample.

[0107] According to the present invention, the sample is cooled primarily by thermal radiation, rather than by thermal conduction. Therefore, during thermal cycling, most or all of the non-sample material on the sample holder does not absorb and release as much energy as thermal conduction dominates the system.

[0108] One aspect of the present invention provides an apparatus and method for reducing heating of non-sample materials on a sample holder.

[0109] Another aspect of the invention provides apparatus and method for reducing lateral thermal conduction through large thermal mass and poor thermal conductivity material on a sample holder.

[0110] Another aspect of the invention provides an apparatus and method for cooling a sample using thermal radiation cooling as the primary cooling channel.

[0111] Another aspect of the invention provides an apparatus and method for placing spacers between plates (i.e., walls) that clamp a sample. The spacers provide good sample uniformity over a large area, even when the plates are thin (e.g., 25 μm thick) and flexible. Without spacers, it is difficult to obtain uniform sample thickness when the two plates defining the sample become very thin.

[0112] Another aspect of the invention provides apparatus and methods for simplifying device operation.

[0113] According to the present invention, thermal radiation cooling uses a material layer (in terms of material and shape) that has good thermal radiation cooling properties during cooling and low thermal mass (and therefore low heating energy) during heating.

[0114] According to the present invention, the sample holder is configured to limit / minimize thermal conduction cooling.

[0115] According to the present invention, the sample thickness of the sample chamber wall, the first plate and the second plate (facing each other) are configured to reduce lateral heat conduction (i.e., in the direction of the plates).

[0116] According to the present invention, in some embodiments, the thermal radiation cooling layer is the same heating / cooling layer as the heating layer, but the ratio of the cooling zone to the heating zone, the material properties, and the material thickness and geometry are configured such that the heating / cooling layer has a low thermal mass and a high thermal radiation cooling rate during heating.

[0117] Another object of the present invention is to perform a cycle of sample temperature change (e.g., from 95°C to 55°C) within a few seconds or even a few seconds (e.g., 0.7 seconds).

[0118] Another aspect of the invention is to provide a useful apparatus and method for isothermal nucleic acid amplification, wherein the sample temperature needs to be raised from ambient temperature to an elevated temperature (i.e., 65°C) and maintained for a period of time (i.e., 5-10 minutes). One aspect of the invention is rapid heating, using less energy, and making the apparatus compact, lightweight, and portable.

[0119] The thermal mass of the card and the sample is minimized to reduce the energy required for heating and cooling.

[0120] Another aspect of the invention is that, in some embodiments, only a small portion of the sample is heated and / or cooled.

[0121] Another aspect of the invention is that it uses a thin, high thermal conductivity layer with an area larger than the area of ​​the relevant sample region.

[0122] Another aspect of the invention is the use of a thin, highly thermally conductive layer with an area larger than the area of ​​the heating zone.

[0123] Another aspect of the invention provides apparatus and method for reducing heating of non-sample materials on a sample holder.

[0124] Another aspect of the invention provides apparatus and method for reducing large thermal mass on sample holders and lateral thermal conduction in poorly thermally conductive materials.

[0125] Another aspect of the invention provides an apparatus and method for cooling a sample using thermal radiation cooling as the primary cooling channel.

[0126] Another aspect of the invention is that it can achieve rapid thermal cycling without the use of cooling gas.

[0127] The thermal mass of the card and the sample is minimized to reduce the energy required for heating and cooling.

[0128] Another aspect of the invention is to adjust radiative cooling and convection cooling for rapid cooling.

[0129] Radiators used for radiative and / or convective cooling are used for rapid cooling.

[0130] An embodiment of the sample thermal cycling device of the present invention (e.g.) Figure 1The device shown includes (i) a sample holder, referred to as an “RHC (Rapid Heating and Cooling) Card” or “sample card,” which allows for rapid heating and cooling of the sample on the card; (ii) a heat source; (iii) an additional heat sink (optional); (iv) a temperature control system; and (v) a signal monitoring system (optional). The temperature control system and signal monitoring system are not included. Figure 1 The invention is explicitly shown, but can be used to control the output of the heating source. In some embodiments, a signal sensor is included to detect optical signals from the sample on the sample holder. Note that some embodiments of the invention may only have... Figure 1 One or more components are displayed.

[0131] Figure 2A and 2B Cross-sectional views of two embodiments of the device of the present invention are shown. Figure 2A An embodiment is shown comprising a separate heating layer (112-1) and a separate cooling layer (112-2), wherein the heating layer (112-1) is on the outer surface of one plate and the cooling layer (112-2) is on the outer surface of another plate. Figure 2B An embodiment comprising a heating layer (112-1) and a cooling layer (112-2) is shown, wherein the heating layer (112-1) and the cooling layer (112-2) are structurally different but in contact with each other, and both layers are on the outer surface of one of the plates.

[0132] SH-1 provides a detailed description of an embodiment of the RHC card of the present invention, which is a device for rapidly changing the temperature of a fluid sample, comprising:

[0133] The structure comprises a first plate (10), a second plate (20), a heating layer (112-1), and a cooling layer (112-2), wherein:

[0134] Each of the first and second plates has a sample contact area on its respective inner surface for contacting the fluid sample; wherein the sample contact areas face each other, are separated by an average spacing of 200 μm or less, and are capable of clamping the sample between them;

[0135] Heating layer:

[0136] It is positioned on the inner surface, outer surface, or inner side of one of the plates.

[0137] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0138] Cooling layer:

[0139] Located on the inner surface, outer surface, or inner side of one of the plates;

[0140] Configured for cooling-related sample volumes; and

[0141] It includes a material layer with a thermal conductivity and heat capacity ratio of 0.6 cm² / sec or greater;

[0142] Wherein the distance between the cooling layer and the surface of the relevant sample volume is zero or less than a distance configured such that the thermal conductivity per unit area between the cooling layer and the surface of the relevant sample volume is equal to 70 W / (m2·K) or greater; and

[0143] In some embodiments, the heating layer and the cooling layer are layers of the same material having heating and cooling zones, and the heating and cooling zones may have the same area or different areas.

[0144] SH-2 provides another detailed description of an embodiment of the RHC card (sample holder) of the present invention, which is a device for rapidly changing the temperature of a fluid sample, comprising:

[0145] The structure comprises a first plate (10), a second plate (20), a heating layer (112-1), and a cooling layer (112-2), wherein:

[0146] Each of the first and second plates has a sample contact area on its respective inner surface for contacting the fluid sample; wherein the sample contact areas face each other, are separated by an average separation distance of 200 μm or less, and are capable of clamping the sample between them;

[0147] Heating layer:

[0148] It is positioned on the inner surface, outer surface, or inner side of one of the plates.

[0149] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0150] Cooling layer:

[0151] Located on the inner surface, outer surface, or inner side of one of the plates;

[0152] Configured for cooling-related sample volumes; and

[0153] Includes a material layer with a thermal conductivity to heat capacity ratio of 0.6 cm² / sec or greater, wherein the layer with a high thermal conductivity to heat capacity ratio has an area greater than the lateral area of ​​the sample volume;

[0154] Wherein the distance between the cooling layer and the surface of the relevant sample volume is zero or less than a distance configured such that the thermal conductivity per unit area between the cooling layer and the surface of the relevant sample volume is equal to 150 W / (m²·K) or greater; and

[0155] In some embodiments, the heating layer and the cooling layer are layers of the same material having heating and cooling zones, and the heating and cooling zones may have the same area or different areas.

[0156] like Figure 3A and 3B As shown, in some embodiments of the invention, the heating layer and cooling layer are combined into one layer (heating / cooling layer), creating a heating zone and a cooling zone, wherein the cooling zone is larger than the heating zone. The sample card 100 (also referred to as an "RHC card") may include two thin plates (10, 20) that sandwich a fluid sample (90) between them, and the heating / cooling layer (112) is located below the sample and is heated by a heating source located away from the card's positioning. According to one embodiment, there are no walls at the edge of the sample to accommodate it, but the edge of the sample does not flow due to capillary forces that maintain the shape of the fluid sample edge.

[0157] like Figure 4A As shown, according to one embodiment, plates 10 and 20 may have inner surfaces 11 and 21 separated by a spacer 102. The spacer 102 may be large when the device is ready to receive a sample (e.g., in the open position). Figure 4B A closed configuration of the device 100 is shown, wherein the spacing 102 is made small (e.g., less than about 200 μm) to clamp the sample 90 between plates 10 and 20. In this embodiment, a heating / cooling layer 112 is located on the outer surface 22 of plate 20.

[0158] SH-3 provides another detailed description of an embodiment of the RHC card of the present invention, which is a device for rapidly changing the temperature of a fluid sample, comprising:

[0159] The first plate (10), the second plate (20), and the heating / cooling layer (112), wherein:

[0160] The first board (10) and the second board (20) face each other and are separated by a certain distance;

[0161] Each plate has a sample contact area on its respective inner surface (11, 21) for contacting a fluid sample; wherein these sample contact areas face each other, contact the sample, confine the sample between them, and have an average spacing (102) between them, as well as the sample;

[0162] The heating / cooling layer (112) is located on the outer surface (22) of the second plate (20); and

[0163] The heating / cooling layer is configured to include a heating zone and a cooling zone; wherein the heating zone is configured to heat the fluid sample, and the cooling zone is configured to cool the sample by thermal radiation.

[0164] The heating zone is configured to receive heating energy from a heating source and has an area smaller than the total area of ​​the heating / cooling layer; and

[0165] At least a portion of the heating zone of the heating layer overlaps with the sample area.

[0166] SH-4. A device for rapidly changing the temperature of a fluid sample, comprising:

[0167] The structure comprises a first plate (10), a second plate (20), a heating layer (112-1), and a cooling layer (112-2), wherein:

[0168] The first and second plates can be moved relative to each other to form different configurations;

[0169] Each of the first and second plates has a sample contact area on its respective inner surface for contacting the fluid sample; wherein the sample contact areas face each other, are separated by an average spacing of 200 μm or less, and are capable of clamping the sample between them;

[0170] Heating layer:

[0171] It is positioned on the inner surface, outer surface, or inner side of one of the plates.

[0172] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0173] Cooling layer:

[0174] Located on the inner surface, outer surface, or inner side of one of the plates;

[0175] Configured for cooling-related sample volumes; and

[0176] It includes a material layer with a thermal conductivity and heat capacity ratio of 0.6 cm² / sec or greater;

[0177] One type of structure is an open structure, wherein the two plates are partially or completely separated and the average spacing between the plates is at least 300 μm;

[0178] Another type of these structures is a closed structure, which is configured after a fluid sample is deposited on one or two sample contact areas of an open structure; and in the closed structure: at least a portion of the sample is defined as a layer by two plates, wherein the average sample thickness is 200 μm or less.

[0179] SH-5. A device for rapidly changing the temperature of a fluid sample, comprising:

[0180] The components include a first plate (10), a second plate (20), a spacer, a heating layer (112-1), and a cooling layer (112-2), wherein:

[0181] The first and second plates can be moved relative to each other to form different configurations;

[0182] Each of the first and second plates has a sample contact area on its respective inner surface for contacting the fluid sample; wherein the sample contact areas face each other, are separated by an average spacing of 200 μm or less, and are capable of clamping the sample between them;

[0183] One or two plates include spacers, and the spacers are fixed to the inner surface of the respective plates;

[0184] The spacers have a predetermined substantially uniform height of 200 micrometers or less, and a predetermined spacer spacing.

[0185] Heating layer:

[0186] It is positioned on the inner surface, outer surface, or inner side of one of the plates.

[0187] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0188] Cooling layer:

[0189] Located on the inner surface, outer surface, or inner side of one of the plates;

[0190] Configured for cooling-related sample volumes; and

[0191] It includes a material layer with a thermal conductivity and heat capacity ratio of 0.6 cm² / sec or greater;

[0192] 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 plates; and

[0193] Another configuration of these structures involves depositing the sample in an open structure followed by configuring the closed structure; and in the closed structure: at least a portion of the sample is compressed by two plates into a layer of very uniform thickness, wherein the uniform thickness of the layer is defined by the sample contact surface of the plates and adjusted by the plates and spacers.

[0194] In some embodiments, the heating / cooling layer (112) may be on or inside the inner surface (21) of the second plate (20), rather than on the outer surface (22) of the second plate (20).

[0195] In some embodiments of the device, the RHC card further includes a spacer positioned between the first and second plates to adjust the distance between the two plates (i.e., the plate spacing), and thus the sample thickness. The spacer can allow the sample thickness between the two plates to be uniform over a large area, even when the plates are thin and flexible.

[0196] In some embodiments, there is more than one heating / cooling layer.

[0197] A. Smaller relevant sample volume (RE ratio)

[0198] Reducing the volume of the sample that should be heated or cooled to the desired temperature can shorten the heating and cooling times and heating power during thermal cycling. This reduction in sample volume during thermal cycling can be achieved by (a) reducing the entire sample volume or (b) heating only a portion of the sample in the sample holder. The terms “relevant sample” or “relevant sample volume” refer to the volume of the sample that is heated and / or cooled to the desired temperature during thermal cycling, and the relevant sample can be a portion or the entire volume of the sample in the sample holder, with no fluid separation between the portion and the remainder of the sample.

[0199] In some embodiments, the relevant sample volume is 0.001 μl, 0.005 μl, 0.01 μl, 0.02 μl, 0.05 μl, 0.1 μl, 0.2 μl, 0.5 μl, 1 μl, 2 μl, 5 μl, 10 μl, 20 μl, 30 μl, 50 μl, 100 μl, 200 μl, 500 μl, 1 ml, 2 ml, 5 ml, or within any two values.

[0200] In some preferred embodiments, the relevant sample volume is 0.001 μL to 0.1 μL, 0.1 μL to 2 μL, 2 μL to 10 μL, 10 μL to 30 μL, 30 μL to 100 μL, 100 μL to 200 μL, or 200 μL to 1 mL.

[0201] In some preferred embodiments, the relevant sample volume is 0.001 μL to 0.1 μL, 0.1 μL to 1 μL, 0.1 μL to 5 μL, or 0.1 μL to 10 μL.

[0202] In some embodiments, the ratio of the relevant sample to the total sample volume (RE ratio) is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or within any two values.

[0203] In some preferred embodiments, the RE ratios are 0.01% and 0.1%, 0.1% and 1%, 1% and 10%, 10% and 30%, 30% and 60%, 60% and 90%, or 90% and 100%.

[0204] In order to heat only a portion of the sample, in some embodiments, the area of ​​the heating zone is only a portion of the transverse area of ​​the sample, and this portion (i.e., the ratio of the heating zone to the transverse area of ​​the sample) is 0.01%, 0.05%, 0.1%, 0.5%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or within any two values.

[0205] In some preferred embodiments, the ratio of the heating zone area to the sample transverse area is in the range of 0.01% and 0.1%, 0.1% and 1%, 1% and 10%, 10% and 30%, 30% and 60%, 60% and 90%, or 90% and 99%.

[0206] B. Localized heating, with heat transfer perpendicular to the lateral direction.

[0207] When a high-k (high thermal conductivity) layer (e.g., a metal layer) is located on the inner, outer, or inner surface of a plate of a sample holder (RHC card), several conditions must be met to heat only a portion of the high-k layer and a portion of the sample volume beyond that portion to the desired temperature, while keeping the remainder of the high-k layer and the remainder of the sample volume at a much lower temperature during thermal cycling. The key conditions are: (1) the heat source must directly heat a portion of the high-k layer (this portion is referred to as the “heated zone”, for example, only this portion is directly heated by an LED lamp or has a local electric heater, while the rest is not); (2) the vertical heat transfer between the heated zone and a portion of the sample should be much greater than the lateral heat transfer within the high-k material (i.e., in the lateral direction of the high-k material); (3) the relevant sample should have a large lateral to vertical dimension ratio; and (4) the heating power of the heated zone must be sufficient to heat the relevant sample volume within a time range where lateral heat transfer (i.e., thermal conduction) is relatively negligible.

[0208] To describe the requirement of satisfying condition (2) above, the scaled thermal conductivity (STC ratio) of the vertical heat transferred from the high-K heating zone to the sample through the intermediate layer between the high-K and the sample and the lateral heat transfer within the high-K layer is defined as:

[0209] STC ratio=η=0.025·(K_m K_s D^2) / (K_k(K_m t_s+K_s t_m)t_k)

[0210] Where Kk, K_s, and Km are the thermal conductivity of the high-K layer, the relevant sample, and the intermediate layer (i.e., the layer between the high-K layer and the sample), respectively; tk, t_s, and tm are the thicknesses of the high-K layer, the sample, and the intermediate layer, respectively; D is the average lateral dimension of the relevant sample, and 0.025 is the scaling factor.

[0211] In some embodiments, in order to locally heat a portion of the high-K layer and a portion of the sample volume above that portion of the high-K layer to a desired temperature while keeping the remainder of the high-K layer and the remainder of the sample volume at a much lower temperature during thermal cycling, the scaled thermal conductivity (STM ratio) is 2 or greater, 5 or greater, 10 or greater, 20 or greater, 30 or greater, 40 or greater, 50 or greater, 100 or greater, 1000 or greater, 10000 or greater, 10000 or greater, or within any two values.

[0212] In some preferred embodiments, the scaled thermal conductivity (STM ratio) is in the range of 10 to 20, 30 to 50, 100 to 1000, 1000 to 10000, or 10000 to 1000000.

[0213] To satisfy conditions (2) and (3) above, in some embodiments, the lateral to vertical dimension (LVS) ratio of the relevant sample is 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, or within any two values.

[0214] In some preferred embodiments, the LVS ratio of the relevant samples is in the range of 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000 to 10,000, or 10,000 to 100,000.

[0215] In some embodiments, the thickness of the relevant sample is reduced (which also helps the sample heating rate), and the thickness of the relevant sample is 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, or within any two values.

[0216] In some preferred embodiments, the thickness of the relevant sample is 0.05 μm and 0.5 μm, 0.5 μm and 1 μm, 1 μm and 5 μm, 5 μm and 10 μm, 10 μm and 30 μm, 30 μm and 50 μm, 50 μm and 70 μm, 70 μm and 100 μm, 100 μm and 200 μm, or 200 μm and 300 μm.

[0217] C. Larger sample-to-non-sample thermal mass ratio (NSTM ratio)

[0218] Increasing the thermal mass ratio of the sample to the non-sample can shorten heating time, reduce heating energy, and increase energy efficiency. In embodiments where the sample is sandwiched between two plates, assuming no heat loss in these volumes, the thermal mass ratio can be estimated by considering only the relevant sample volume and the portions of the two plates sandwiched between the relevant sample. Therefore, one parameter for measuring the thermal mass ratio is the ratio of the "specific surface area thermal mass" of the relevant sample to the non-sample (the plate portion sandwiching the relevant sample and portions of the plate portion with heating / cooling layers in between). The material term "specific surface area thermal mass" refers to the material's volumetric specific heat multiplied by its thickness.

[0219] Therefore, assuming that heat loss due to heat conduction and radiation is negligible, the sample to non-sample thermal mass ratio is the ratio of useful thermal energy (which directly heats the relevant sample) to “wasted thermal energy” (which heats the non-sample material).

[0220] For example, water has a volumetric specific heat of 4.2 J / (cm³⁻¹C), therefore the area specific heat of a 30 μm thick water layer is 1.26 × 10⁻² J / (cm²⁻¹C). PMMA has a volumetric specific heat of 1.77 J / (cm³⁻¹C), therefore the area specific heat of a 25 μm thick PMMA layer is 4.43 × 10⁻³ J / (cm²⁻¹C), which is about 2.8 times smaller than the area specific heat of a 30 μm water layer. Gold has a volumetric specific heat of 2.5 J / (cm³⁻¹C), therefore the area specific heat of a 0.5 μm thick gold layer is 1.25 × 10⁻⁴ J / (cm²⁻¹C), which is 100 times smaller than the area specific heat of a 30 μm water layer and can be ignored. The negligible area specific heat of Au is due to its thin thickness.

[0221] In the RHC card embodiment, if the relevant sample is sandwiched between two plates, each 25 μm thick, and the heating / cooling layer is 0.5 μm thick, then the thermal mass ratio of the sample to the non-sample is 1.4. That is, when heat loss due to thermal conduction and radiation is ignored, the ratio of useful energy to wasted energy is 1.4, and the ratio of useful energy to total heating energy is 58%.

[0222] In some embodiments, the sample to non-sample thermal mass ratio (NSTM ratio) is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 100, 200, 300, 1000, 4000, or within any two values.

[0223] In a preferred embodiment, the thermal mass ratio (NSTM ratio) of the sample to the non-sample is in the range of 0.1 to 0.2, 0.2 to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30 to 50, 50 to 100, 100 to 300, 300 to 1000, or 1000 to 4000.

[0224] To achieve a high thermal mass ratio between the sample and the non-sample, it is necessary to maintain a low thermal mass in the non-sample area, which in turn requires thin plates and heating / cooling layers, and / or low volumetric specific heat.

[0225] To achieve a high thermal-to-mass ratio, one embodiment uses a thin material having multiple layers or a mixture of materials. For example, the thickness of the carbon fiber layer having a plastic sheet or carbon mixed with plastic can be 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 25 μm, 50 μm, or within any two values.

[0226] D. The thickness of the relevant samples and the ratio of their largest horizontal to vertical dimensions (LVS ratio).

[0227] The term "sample lateral to vertical dimension ratio" or "sample LVS ratio" refers to the ratio of the average lateral dimension of the relevant sample volume to its average vertical dimension. In embodiments where heating and / or cooling are primarily derived from the vertical direction, a larger LVS ratio of the sample can reduce wasted heating energy and increase heating and / or cooling rates, and can also reduce lateral heat conduction losses at the edges of the relevant sample relative to the total heat energy. All of these can increase and / or may increase cooling time.

[0228] In some embodiments, the LVS ratio of the relevant samples is 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, or within any two values.

[0229] In some preferred embodiments, the LVS ratio of the relevant samples is in the range of 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000 to 10,000, or 10,000 to 100,000.

[0230] For example, if the sample has a lateral dimension of 15 mm and a thickness of 30 μm, then the LVS of the sample is 500.

[0231] In some embodiments, the thickness of the relevant sample is reduced (which also helps the sample heating rate), and the thickness of the relevant sample is 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, or within any two values.

[0232] In some preferred embodiments, the thickness of the relevant sample is 0.05 μm and 0.5 μm, 0.5 μm and 1 μm, 1 μm and 5 μm, 5 μm and 10 μm, 10 μm and 30 μm, 30 μm and 50 μm, 50 μm and 70 μm, 70 μm and 100 μm, 100 μm and 200 μm, or 200 μm and 300 μm.

[0233] E. Thinness of non-sample components and large lateral / vertical dimension ratio (LVS ratio)

[0234] The term "non-sample lateral to vertical dimension ratio" or "non-sample LVS ratio" refers to the ratio of the average lateral dimension (which is the same as the average lateral dimension of the relevant sample volume) of the portion of the two plates sandwiching the relevant sample to its thickness. A large non-sample LVS ratio can reduce lateral heat conduction losses at the edges of the non-sample relative to the total heat energy.

[0235] In some embodiments, the LVS ratio of non-samples is 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, or within any two values.

[0236] In a preferred embodiment, the LVS ratio of non-samples is in the range of 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000 to 10,000, or 10,000 to 100,000.

[0237] For example, two 25 μm thick plates sandwich a sample with a lateral dimension of 5 mm or more between them, so the non-sample LVS of each plate is 200 or higher.

[0238] To shorten heating time, reduce heating energy, and improve energy efficiency, lateral heat conduction through non-sample materials (on the sample holder) should be reduced.

[0239] In particular, when the first and second plates are made of materials that are not good heat-dissipating materials, the thickness of the plates should be minimized.

[0240] In some embodiments, the thickness of the first plate or the second plate or each of the two plates is 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2.5 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 200 μm or 500 μm, 1000 μm, or within any two values.

[0241] In some preferred embodiments, the thickness of the first plate or the second plate or each of the two plates is 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2.5 μm, 5 μm, 10 μm, 25 μm, 50 μm, 75 μm, or within any two values.

[0242] The first and second plates can have the same thickness or different thicknesses, and can be made of the same material or different materials.

[0243] In some preferred embodiments, the thickness of each of the first plate or the second plate or both plates is 10 nm and 500 nm, 500 nm and 1 μm, 1 μm and 2.5 μm, 2.5 μm and 5 μm, 5 μm and 10 μm, 10 μm and 25 μm, 25 μm and 50 μm, 50 μm and 100 μm, 100 μm and 200 μm, or 200 μm and 500 μm, or 500 μm and 1000 μm.

[0244] In some preferred embodiments, the first plate and the second plate are plastic, thin glass, or materials with similar physical properties. The thickness of the first plate or the second plate is 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 175 μm, 250 μm, or within any two values.

[0245] In some preferred embodiments, the first plate and the second plate are plastic, thin glass, or materials with similar physical properties. The thickness of the first plate is 5 μm, 10 μm, 25 μm, 50 μm, or within any two values; while the thickness of the second plate (the plate having a heating layer or a cooling layer) is 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, or within any two values.

[0246] F. Cooling layer with high K and / or high thermal conductivity ratio (KC ratio).

[0247] Because any heat conduction through non-sample materials wastes energy, and because lateral heat conduction has a much longer thermal path than vertical heat conduction, the energy wasted in lateral heat conduction within non-sample materials should be minimized. One way to minimize this wasted energy is to use a material with high thermal conductivity (high K) or, more precisely, a high thermal conductivity-to-capacity ratio (K / C ratio) as the cooling layer. For a given thermal conductivity, a given temperature change, and a given geometry, high K and / or high K / C ratio materials will require significantly less energy to heat than low K and / or low K / C ratio materials.

[0248] In some embodiments, the KC ratio of the material used for the cooling layer is equal to or greater than 0.1 cm² / sec, 0.2 cm² / sec, 0.3 cm² / sec, 0.4 cm² / sec, 0.5 cm² / sec, 0.6 cm² / sec, 0.7 cm² / sec, 0.8 cm² / sec, 0.9 cm² / sec, 1 cm² / sec, 1.1 cm² / sec, 1.2 cm² / sec, 1.3 cm² / sec, 1.4 cm² / sec, 1.5 cm² / sec, 1.6 cm² / sec, 2 cm² / sec, 3 cm² / sec, or within any two values.

[0249] In some preferred embodiments, the KC ratio of the cooling layer is 0.5 cm² / sec and 0.7 cm² / sec, 0.7 cm² / sec and 0.9 cm² / sec, 0.9 cm² / sec and 1 cm² / sec, 1 cm² / sec and 1.1 cm² / sec, 1.1 cm² / sec and 1.3 cm² / sec.

[0250] Within the range of 1.3 cm² / sec and 1.6 cm² / sec, 1.6 cm.

[0251] In some embodiments, a high thermal conductivity (i.e., high K) material is used for the cooling layer. The high K material has a thermal conductivity equal to or greater than 50 W / (m·K), 80 W / (m·K), 100 W / (m·K), 150 W / (m·K), 200 W / (m·K), 250 W / (m·K), 300 W / (m·K), 350 W / (m·K), 400 W / (m·K), 450 W / (m·K), 500 W / (m·K), 600 W / (m·K), 1000 W / (m·K), 5000 W / (m·K), or within any two values.

[0252] In some preferred embodiments, a high thermal conductivity (i.e., high K) material is used for the cooling layer, and the thermal conductivity of the high K material is in the range of 50 W / (m·K) to 100 W / (m·K), 110 W / (m·K) to 200 W / (m·K), 200 W / (m·K) to 400 W / (m·K), 400 W / (m·K) to 600 W / (m·K), or 400 W / (m·K) to 5000 W / (m·K).

[0253] In some embodiments, the high-K material is selected from metals, semiconductors, and allows for a thermal conductivity higher than 50 W / (m·K), and any combination thereof (including any mixture). In some embodiments, the high-K material is selected from gold, copper, silver, and aluminum, and any combination thereof (including any mixture). In some embodiments, the high-K material is selected from carbon particles, carbon nanotubes, graphite, silicon, and any combination thereof (including any mixture).

[0254] G-1. The area of ​​the cooling zone is larger than the area of ​​the laterally correlated sample and the area of ​​the heating zone.

[0255] To effectively cool the sample while minimizing wasted energy in non-sample materials, in some embodiments, high-K and / or high-K-C ratio materials (referred to as "high-K materials") are used as the primary channel for removing heat from the sample. The area of ​​the high-K cooling zone (layer) should be larger than the relevant lateral dimension of the sample.

[0256] In some embodiments, the area of ​​the cooling zone (layer) is 1.5, 2, 3, 4, 5, 10, 20, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, or within any two values ​​larger than the lateral area of ​​the relevant sample.

[0257] In a preferred embodiment, the area of ​​the cooling zone (layer) is 1.5 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000 to 10,000, or 10,000 to 100,000 times larger than the lateral area of ​​the relevant sample.

[0258] To improve cooling speed and thermal circulation efficiency, in some embodiments, the area of ​​the high-K cooling layer (zone) should be larger than the area of ​​the heating zone.

[0259] In some embodiments, the area of ​​the cooling zone (layer) is 1.1, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 5,000, 10,000, 100,000, or within any two values ​​larger than the area of ​​the heating zone (layer).

[0260] In a preferred embodiment, the area of ​​the cooling zone (layer) is 1.1 to 1.5, 1.5 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000 to 10,000, or 10,000 to 100,000 larger than the lateral area of ​​the heating zone (layer).

[0261] G-2. The areas of the cooling zone and heating zone are the same as the areas of the transversely correlated samples.

[0262] In some embodiments, the area of ​​the cooling zone and the area of ​​the heating zone are the same as the area of ​​the laterally correlated sample, which is much smaller than the total sample area on the plate and smaller than the area of ​​the plate. The area of ​​the cooling zone is 1 mm², ...

[0263] The cooling zones can have different shapes. In some embodiments, there are more than one cooling zone on a plate, and the cooling zones are separated from each other by a material with low thermal conductivity (such as air or plastic).

[0264] H. Heating zone with high K and / or high thermal conductivity to volume ratio (KC ratio)

[0265] Because any heat conduction in the non-sample material through lateral heat conduction has a much longer thermal path than through vertical heat conduction, the energy wasted in lateral heat conduction in the non-sample material should be minimized. One way to minimize this wasted energy is to use a material with a thermal conductivity to capacity (KC) ratio in the heating zone, which would require much less thermal energy for a given thermal conductivity, a given temperature change, and a given geometry.

[0266] In some embodiments, the KC ratio material used for the heating layer is equal to or greater than 0.1 cm² / sec, 0.2 cm² / sec, 0.3 cm² / sec, 0.4 cm² / sec, 0.5 cm² / sec, 0.6 cm² / sec, 0.7 cm² / sec, 0.8 cm² / sec, 0.9 cm² / sec, 1 cm² / sec, 1.1 cm² / sec, 1.2 cm² / sec, 1.3 cm² / sec, 1.4 cm² / sec, 1.5 cm² / sec, 1.6 cm² / sec, 2 cm² / sec, 3 cm² / sec, or within any two values.

[0267] In some preferred embodiments, the KC ratio of the heating layer is 0.5 cm² / sec and 0.7 cm² / sec, 0.7 cm² / sec and 0.9 cm² / sec, 0.9 cm² / sec and 1 cm² / sec, 1 cm² / sec and 1.1 cm² / sec, 1.1 cm² / sec and 1.3 cm² / sec.

[0268] Within the range of 1.3 cm² / sec and 1.6 cm² / sec, 1.6 cm² / sec and 2 cm² / sec, or 2 cm² / sec and 3 cm² / sec.

[0269] In some embodiments, a high thermal conductivity (i.e., high K) material is used for the heating layer, and the thermal conductivity of the high K material is equal to or greater than 50 W / (m·K), 80 W / (m·K), 100 W / (m·K), 150 W / (m·K), 200 W / (m·K), 250 W / (m·K), 300 W / (m·K), 350 W / (m·K), 400 W / (m·K), 450 W / (m·K), 500 W / (m·K), 600 W / (m·K), 1000 W / (m·K), 5000 W / (m·K), or within any two values.

[0270] In some preferred embodiments, a high thermal conductivity (i.e., high K) material is used for the heating layer, and the thermal conductivity of the high K material is in the range of 50 W / (m·K) to 100 W / (m·K), 110 W / (m·K) to 200 W / (m·K), 200 W / (m·K) to 400 W / (m·K), 400 W / (m·K) to 600 W / (m·K), or 400 W / (m·K) to 5000 W / (m·K).

[0271] In some embodiments, the high-K material is selected from metals, semiconductors, and allows for a thermal conductivity higher than 50 W / (m·K), and any combination thereof (including any mixture). In some embodiments, the high-K material is selected from gold, copper, silver, and aluminum, and any combination thereof (including any mixture). In some embodiments, the high-K material is selected from carbon particles, carbon nanotubes, graphite, silicon, and any combination thereof (including any mixture).

[0272] To receive light energy through the heated zone (layer), a thermal radiation-enhanced surface (on one or both sides of the heated zone) will be used. A thermal radiation absorption-enhanced surface can be achieved by directly altering the surface structure (e.g., patterning nanostructures), coating with a high thermal radiation material (e.g., coating with a black paint), or both.

[0273] The thermally enhanced surface has a high average light absorption rate (e.g., the black paint used in our experiments). In some embodiments, the average light absorption rate of the heated area surface is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or within any two values.

[0274] In some preferred embodiments, the average absorbance of the surface of the heated zone is in the range of 30% to 40%, 40% to 60%, 60% to 80% to 90%, or 90% to 100%.

[0275] In some preferred embodiments, the average absorbance of the surface of the heated zone is in the range of 30% to 100%, 50% to 100%, 70% to 100%, or 80% to 100%.

[0276] In some embodiments, the average light absorptivity of the surface of the heated zone is obtained by averaging the wavelength ranges of 400 nm to 800 nm, 700 nm to 1500 nm, 900 nm to 2000 nm, or 2000 nm to 20000 nm.

[0277] Increased thermal radiation cooling

[0278] In some embodiments, rapid temperature cycling is achieved by increasing the percentage of radiative cooling (i.e., removing heat to the environment) in the total cooling of the sample and sample holder during thermal cycling, preferably by using a material with high thermal conductivity as the radiative cooling material. One reason is that cooling via lateral thermal conduction requires heating multiple non-sample materials, wasting energy. Another reason is that radiative cooling is proportional to the fourth power of temperature and can be more efficient than thermal conduction in thin films.

[0279] To enhance thermal radiation cooling, in some embodiments, thermal radiation cooling utilizes a cooling layer (cooling zone) that is enhanced for thermal radiation cooling. This enhancement includes (i) increasing the thermal conductivity of the cooling zone (layer), (ii) increasing the area of ​​the cooling zone (layer), (iii) enhancing the surface thermal radiation of the cooling zone, and (iv) combinations thereof.

[0280] Examples of materials with high thermal conductivity are metals (such as gold, silver, CoBr, aluminum), half-metals, semiconductors (such as silicon), or combinations thereof.

[0281] To further enhance the thermal radiation of the cooling zone (layer), a thermal radiation-enhanced surface (on one or both sides of the cooling zone) will be used. A thermal radiation-enhanced surface can be achieved by directly altering the surface structure (e.g., patterning nanostructures), coating with a high thermal radiation material (e.g., applying a black coating), or both.

[0282] The thermally enhanced surface has a high average light absorptivity (e.g., the black paint used in our experiments). In some embodiments, the average light absorptivity of the surface in the cooling zone is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or within any two values.

[0283] In some preferred embodiments, the average light absorption rate of the surface of the cooling zone is in the range of 30% to 40%, 40% to 60%, 60% to 80% to 90%, or 90% to 100%.

[0284] In some preferred embodiments, the average light absorption rate of the surface of the cooling zone is in the range of 30% to 100%, 50% to 100%, 70% to 100%, or 80% to 100%.

[0285] In some embodiments, the average light absorption rate of the surface of the cooling zone is obtained by averaging the wavelength ranges of 400 nm to 800 nm, 700 nm to 1500 nm, 900 nm to 2000 nm, or 2000 nm to 20000 nm.

[0286] In some embodiments, the surface thermal radiation enhancement layer is a black coating, a plasma structure, a nanostructure, or any combination thereof.

[0287] High heat radiation materials are polymer mixtures that appear black to the human eye (often referred to as "black paint"). High heat radiation materials include, but are not limited to, mixtures of polymers and nanoparticles. Examples of nanoparticles include black carbon nanoparticles, carbon, nanotubes, graphite particles, graphene, metal nanoparticles, semiconductor nanoparticles, or combinations thereof.

[0288] High thermal radiation materials also include materials deposited or formed on the surface of a layer and appearing black to the human eye. These materials include, but are not limited to, carbon black nanoparticles, carbon, nanotubes, graphite particles, graphene, metal nanoparticles, semiconductor nanoparticles, or combinations thereof.

[0289] Plasma structures include nanostructured plasma structures.

[0290] In some embodiments, the cooling layer comprises a high thermal conductivity metal layer (50 W / (m·K) or higher) having a surface thermal radiation enhancement layer. In some embodiments, the surface thermal radiation enhancement layer has a low lateral thermal conductivity, which is attributed to the ultrathin layer, low thermal conductivity, or both.

[0291] Percentage of thermal radiation cooling.

[0292] In some embodiments, thermal radiative cooling is achieved by increasing the area of ​​the radiative cooling layer (i.e., a high-K material, unless otherwise stated), and the area of ​​the radiative cooling layer is a multiple of the lateral area of ​​the relevant sample by 1.2, 1.5, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 200, 300, 400, 500, 600, 700, 800, 800, 1,000, 2,000, 5,000, 10,000, 100,000, or within any two values.

[0293] In a preferred embodiment, the area of ​​the radiative cooling zone (layer) is 1.2 to 3, 3 to 5, 5 to 10, 10 to 50, 50 to 100, 100 to 500, 500 to 1,000, 1,000 to 10,000, or 10,000 to 100,000 times larger than the lateral area of ​​the relevant sample.

[0294] In some embodiments, during thermal cycling, the ratio of thermal radiation cooling by the cooling zone (layer) to the total cooling of the sample and sample holder is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or within any two values.

[0295] In some preferred embodiments, during thermal cycling, the ratio of thermal radiation cooling of the cooling zone (layer) to the total cooling of the sample and sample holder is in the range of 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 99%.

[0296] J. Cooling layer thickness control

[0297] In some embodiments, the thickness of the cooling layer is configured to facilitate localized optimization of heating and / or energy efficiency. If the cooling zone (layer) is too thick, a larger percentage of heating energy will be wasted by the cooling layer, prolonging the heating time (for a given heating power). On the other hand, if the cooling zone is too thin, the cooling time will be significantly longer. Therefore, the cooling layer thickness should be optimized for rapid heating and cooling.

[0298] Experiments have shown that the thickness of the high-K cooling layer can adjust the cooling rate. By selecting an appropriate high-K cooling layer thickness and a suitable LED power density, rapid heating and cooling can be achieved.

[0299] Since the thermal conductivity of a layer is proportional to the thermal conductivity of the material multiplied by the layer thickness, the product should be optimized.

[0300] In some embodiments, the thermal conductivity of the cooling zone (layer) multiplied by its thickness is 6×10⁻⁵ W / K, 9×10⁻⁵ W / K, 1.2×10⁻⁴ W / K, 1.5×10⁻⁴ W / K, 1.8×10⁻⁴ W / K, 2.1×10⁻⁴ W / K, 2.7×10⁻⁴ W / K, 3×10⁻⁴ W / K, 1.5×10⁻⁴ W / K, or within any two values.

[0301] In some preferred embodiments, the thermal conductivity of the cooling zone (layer) multiplied by its thickness is in the range of 6×10⁻⁵ W / K to 9×10⁻⁵ W / K, 9×10⁻⁵ W / K to 1.5×10⁻⁴ W / K, 1.5×10⁻⁴ W / K to 2.1×10⁻⁴ W / K, 2.1×10⁻⁴ W / K to 2.7×10⁻⁴ W / K, 2.7×10⁻⁴ W / K to 3×10⁻⁴ W / K, or 3×10⁻⁴ W / K to 1.5×10⁻⁴ W / K.

[0302] In some preferred embodiments, the thermal conductivity of the cooling zone (layer) multiplied by its thickness is in the range of 9×10⁻⁵ W / K to 2.7×10⁻⁴ W / K, 9×10⁻⁵ W / K to 2.4×10⁻⁴ W / K, 9×10⁻⁵ W / K to 2.1×10⁻⁴ W / K, or 9×10⁻⁵ W / K to 1.8×10⁻⁴ W / K.

[0303] In one embodiment, the cooling region comprises a gold layer with a thickness ranging from 200 nm to 800 nm. In another embodiment, the cooling region comprises a gold layer with a thickness ranging from 300 nm to 700 nm.

[0304] K. High electrical conductivity between the sample and the heating or cooling zone.

[0305] For rapid heating and cooling of the sample, the thermal conductivity per unit area between the sample and the heating and / or cooling layers should be high. Thermal conductivity per unit area equals the conductivity (per unit volume) divided by the material thickness between the HC layer and the sample. For example, for a 100 nm thick PS as a second plate with an HC layer on one surface and the sample on the other, the electrical conductivity between the HC layer and the sample is -1000 W / (m²·K).

[0306] Based on experiments, in some embodiments of the RHC card, the material between the heating zone and the associated sample has a thermal conductivity and thickness configured to be about 1000 W / (m2·K) or higher.

[0307] In some embodiments of the RHC card, the material between the heating zone and the associated sample has thermal conductivity and thickness configured to have a conductivity per unit area equal to or greater than 1000 W / (m²·K), 2000 W / (m²·K), 3000 W / (m²·K), 4000 W / (m²·K), 5000 W / (m²·K), 7000 W / (m²·K), 10000 W / (m²·K), 20000 W / (m²·K), 50000 W / (m²·K), 50000 W / (m²·K), 100000 W / (m²·K), or within any of these values.

[0308] The preferred electrical conductivity per unit area of ​​the material between the heating zone and the relevant sample is in the range of 1000 W / (m2·K) to 2000 W / (m2·K), 2000 W / (m2·K) to 4000 W / (m2·K), 4000 W / (m2·K) to 10000 W / (m2·K), or 10000 W / (m2·K) to 100000 W / (m2·K).

[0309] In another preferred embodiment, the distance between the heating zone and the associated sample is zero, so the electrical conductivity of the material per unit area between the heating zone and the associated sample is infinite.

[0310] In some embodiments, the heating or cooling layer is separated from the relevant sample by a thin plastic sheet (or film) with a thermal conductivity ranging from 0.1 W / (m·K) to 0.3 W / (m·K), and the thickness of the thin plastic layer is 0 nm, 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2.5 μm, 5 μm, 10 μm, 25 μm, 50 μm, 75 nm, 100 μm, 150 μm, or within any two values.

[0311] In some preferred embodiments, the thickness of the thin plastic sheet (or film) separating the relevant sample from the heating or cooling layer is 0 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 μm, 1 μm to 5 μm, 5 μm to 10 μm, 10 μm to 25 μm, 25 μm to 50 μm, 50 μm to 75 μm, 75 μm to 100 μm, or 100 μm to 150 μm.

[0312] In a preferred embodiment of the RHC card, the thickness of the thin plastic sheet (or film) separating the relevant sample from the heating or cooling layer is 1 nm, 10 nm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 20 μm, 25 μm, or within any two values.

[0313] L. Relatively small lateral diffusion relative to the reagent

[0314] To ensure that biochemical reactions are substantially homogeneous within the relevant sample volume during temperature changes or thermal cycling, the average lateral area of ​​the relevant sample should be significantly larger than the lateral diffusion of nucleic acids and / or other reagents used for molecular amplification and / or reactions. This way, during temperature changes or thermal cycling, most molecules within the relevant sample volume do not have sufficient time to diffuse out of the relevant sample volume, while most molecules outside the relevant sample volume do not have sufficient time to diffuse into the relevant sample volume.

[0315] Considering a molecule with a molecular weight of approximately 600 Da, a thermal cycling duration of 3 minutes, and a diffusion constant of -1×

[0316] 10⁻⁶ cm² / s, diffusion length -130 μm.

[0317] In some embodiments, during thermal cycling or reaction, the ratio of the average lateral dimension of the relevant sample volume to the diffusion length of the reagent is equal to or greater than 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 5000, 10000, 100000, or within any two values.

[0318] In some preferred embodiments, during thermal cycling or reaction, the ratio of the average lateral dimension of the relevant sample volume to the diffusion length of the reagent is in the range of 5 to 10, 10 to 30, 30 to 60, 6 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 5000, 5000 to 10000, or 10000 to 100000.

[0319] In some preferred embodiments, during thermal cycling or reaction, the ratio of the average lateral dimension of the relevant sample volume to the diffusion length of the reagent is in the range of 5 to 10, 10 to 30, 30 to 60, 6 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 5000, 5000 to 10000, or 10000 to 100000.

[0320] In some preferred embodiments, the average lateral dimension of the relevant volume is 1 mm, 2 mm, 3 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 12 mm, 15 mm, 20 mm, 30 mm, 40 mm, 50 mm, 70 mm, 100 mm, 200 mm, or within any two values.

[0321] In some preferred embodiments, the average lateral dimension of the relevant volume is in the range of 1 mm to 5 mm, 5 mm to 10 mm, 10 mm to 20 mm, 20 mm to 40 mm, 40 mm to 70 mm, 70 mm to 100 mm, or 100 mm to 200 mm.

[0322] In another preferred embodiment, the average lateral dimension of the relevant volume is in the range of 1 mm to 5 mm, 1 mm to 10 mm, or 5 mm to 20 mm.

[0323] M. No edge banding or simple edge banding

[0324] To simplify the operation and reduce the cost of the sample holder, in some embodiments, there is no seal between the two plates defining the sample; that is, the sample sandwiched between the plates can evaporate from the sample edge into the environment. However, in our experiments, we found that in our sample card configuration, such evaporation is negligible relative to the total sample volume due to the large ratio of the lateral sample area to the sample edge area; these plates prevent most of the evaporation.

[0325] In some embodiments, closed-loop spacers or discontinuous spacer walls may be placed on one or two plates to reduce or eliminate sample evaporation.

[0326] P-2 Forced Air Cooling

[0327] In some embodiments, a forced air cooling / circulation system is present near the RHC card to accelerate the cooling process. Examples of forced air cooling systems include, but are not limited to, fans that circulate cool air near the card, a plurality of fans that circulate cool air near the card, a cooling source that cools the air near the card, a cooling pad that directly contacts the card, or a combination thereof.

[0328] In some embodiments, a forced air cooling / recirculation system is present to cool the air on the top surface of the card.

[0329] In some embodiments, a forced air cooling / circulation system is present to cool the air on the bottom surface of the card.

[0330] In some embodiments, there is a forced air cooling / recirculation system that cools the air surrounding all surfaces of the card.

[0331] 2. Mechanical Structure Design

[0332] N. Movable plates and extrusion open flows, hinges, open notches, groove edges, and sliders.

[0333] To simplify sample loading, in some embodiments of the invention, the two plates of the RHC card can be moved relative to each other in different configurations. The sample is deposited in the open configuration of the plates, and then the plates are pressed into a closed configuration. During pressing, the sample flows into a thin layer between the plates, and this flow is referred to as “compressed open flow” because there is sufficient space between the plates to allow sample flow.

[0334] In some embodiments, space for adjusting sample thickness is added to one or two plates, so the device for rapidly changing the temperature of the fluid sample includes:

[0335] The structure comprises a first plate (10), a second plate (20), a heating layer (112-1), and a cooling layer (112-2), wherein:

[0336] The first and second plates can be moved relative to each other to form different configurations;

[0337] Each of the first and second plates has a sample contact area on its respective inner surface for contacting the fluid sample; wherein the sample contact areas face each other, are separated by an average spacing of 200 μm or less, and are capable of clamping the sample between them;

[0338] Heating layer:

[0339] It is positioned on the inner surface, outer surface, or inner side of one of the plates.

[0340] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0341] Cooling layer:

[0342] Located on the inner surface, outer surface, or inner side of one of the plates;

[0343] Configured for cooling-related sample volumes; and

[0344] It includes a material layer with a thermal conductivity and heat capacity ratio of 0.6 cm² / sec or greater;

[0345] One of the constructions is an open construction, wherein the two plates are partially or completely separated and the average spacing between the plates is at least 300 μm;

[0346] Another type of configuration among these is a closed configuration, which is configured after a fluid sample has been deposited onto one or two sample contact areas of an open configuration; and in the closed configuration: at least a portion of the sample is defined as a layer by two plates, wherein the average sample thickness is 200 μm or less; and

[0347] In some embodiments, the heating layer and the cooling layer are layers of the same material having heating and cooling zones, and the heating and cooling zones may have the same area or different areas.

[0348] In some embodiments, the sample holder with a movable plate (also referred to as an "RHC card" or "Q card") further includes hinges, notches, and grooves that facilitate manipulation of the sample holder and measurement of the sample. Additionally, the sample holder can slide into a slider. The structure, material, function, variation, and dimensions of hinges, notches, grooves, sliders, and extrusion open fluids are disclosed, described, and summarized herein in PCT Application No. PCT / US2016 / 045437 filed August 10, 2016, PCT Application No. PCT / US0216 / 051775 filed September 14, 2016, U.S. Provisional Application No. 62 / 456065 filed February 7, 2017, U.S. Provisional Application No. 62 / 456287 filed February 8, 2017, and U.S. Provisional Application No. 62 / 456504 filed February 8, 2017, respectively, and the entire contents of all such applications are incorporated herein for all purposes.

[0349] Spacer (13)

[0350] In some embodiments, the spacer, as described in Example SH-5, will be used to adjust the sample thickness and make the thickness uniform. Even when both plates are very thin (e.g., 25 μm thick or less), the spacer allows for a uniform sample thickness.

[0351] In some embodiments, the spacer is fixed to one or two plates. In some embodiments, the spacer is mixed with the sample. In some embodiments, the spacer has a uniform height, and the spacer, together with the first and second plates, adjusts the sample layer. In some embodiments, the thickness of the sample layer is substantially equal to the height of the spacer.

[0352] In some embodiments, these plates are flat (e.g., as shown in the figure). Figure 12A As shown). In some embodiments, one or both plates include holes (e.g., as shown). Figure 12B(As shown). For example, in some embodiments, the width of the aperture may be less than 500 μm, 200 μm, 100 μm, 50 μm, 25 μm, 10 μm, 5 μm, 2.5 μm, 1 μm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or within any two values. In some embodiments, the depth of the aperture may be less than 500 μm, 200 μm, 100 μm, 50 μm, 25 μm, 10 μm, 5 μm, 2.5 μm, 1 μm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 20 nm, 10 nm, 5 nm, 2 nm, or 1 nm, or within any two values.

[0353] In some embodiments, one or two plates have holes, and most or all of the sample is contained within the hole of one plate and covered by the other plates (not shown).

[0354] P. Sample box and thermal insulation

[0355] In some embodiments, an RHC card (sample holder) may be further mounted on a sample cartridge. The cartridge may be configured to slide in or out of a base (also referred to as an "adapter"). The base houses a power supply, temperature sensor and controller, signal measurement devices, and slots for the sample holder, with or without the cartridge, to slide in or out of the base.

[0356] In some embodiments, the sample holder, housing (i.e., sample holder support) or both are “thermally conductive isolated,” meaning they have little or no thermal conduction to the environment during thermal cycling. In this case, cooling during thermal cycling is primarily achieved through thermal radiation (this is referred to as “non-conductive heat transfer”). In some embodiments, this “thermally conductive isolation” is achieved in the sample holder, housing, or both by configuring their materials, geometries (including reduced thickness), or both.

[0357] Q. The above combination

[0358] Implementations of the RHC card can be any combination of the specifications described in SH-1, SH-2, SH-3 and subsections A to P.

[0359] R. Heating source

[0360] The heating layer or heating / cooling layer in the RHC card is configured to be heated by a heating source, wherein the heating source transfers thermal energy to the heating / cooling layer through light, electricity, radio frequency (RF) radiation, or a combination thereof.

[0361] S. Base (i.e., adapter)

[0362] In some embodiments, the device further includes a base (adapter) configured to receive a sample card, a heating source, a temperature sensor, a portion of a temperature control system (in some embodiments including a smartphone), an additional heat sink (optionally), a fan (optionally), or a combination thereof. In some embodiments, the adapter includes a card slot into which a sample card or sample box can be inserted. In some embodiments, the sample card or sample box is stabilized and held in place without any movement after being fully inserted into the slot or after reaching a predetermined position in the slot.

[0363] T. Smartphone

[0364] In some embodiments, a smartphone is used to image the sample card, control heating and / or cooling, sense signals, monitor operation using a camera, provide light / energy with a flash, communicate with a local or remote device integrated through a base (adapter) in the system, or a combination thereof.

[0365] Applications of U. Isothermal Nucleic Acid Amplification

[0366] With slight modifications, the present invention also provides useful apparatus and methods for isothermal nucleic acid amplification, wherein the sample temperature needs to be raised from ambient temperature to an elevated temperature (i.e., 65°C) and maintained at that temperature for a period of time (i.e., 5-10 minutes). In some embodiments, one of the modifications required for isothermal nucleic acid amplification detection is to reduce or eliminate the cooling zone / layer, thereby reducing heat loss from the sample and / or sample holder to the environment.

[0367] With slight modifications, this invention provides useful apparatus and methods for reverse transcription polymerase chain reaction, which include an isothermal process prior to conventional PCR, wherein the sample temperature is required to rise from ambient temperature to an elevated temperature (i.e., 50°C) and maintained at that temperature for a period of time (i.e., 5-10 minutes). With slight modifications, this invention also provides useful apparatus and methods for minimizing PCR cross-contamination, such as methods using dUTP and uracil-DNA N-glycosylation enzymes, wherein the sample temperature is required to rise from ambient temperature to an elevated temperature (i.e., 50°C) and maintained at that temperature for a period of time (i.e., 1-20 minutes).

[0368] Experiments in some embodiments

[0369] Some embodiments of the present invention have been tested experimentally. Some experimental results are presented herein.

[0370] In some of our experiments, Figure 18A The device shown includes a sample holder (e.g., an RHC card), an LED light source (i.e., an energy source) focused by a lens onto a (~5mm × 5mm) area of ​​the sample holder, and a sample holder support made of a thermally conductive and insulating material. Figure 18A(Not shown in the image). The sample holder support supports the edges of the second plate at two opposite edges (e.g., around the perimeter of the second plate) with a thickness of -2 mm. There are no additional heat sinks, and heat is primarily radiated into the open environment (e.g., a room).

[0371] In the experiments described in this section, the sample holder comprises a first plate, a second plate, and a heating / cooling layer. One of the plates has a spacer. The first and second plates can be moved relative to each other into different configurations; one of these configurations is an open configuration, in which the two plates are separated by an average distance of at least 300 μm. In the open configuration, the sample is deposited on one of the plates. Another card is then placed on top of the sample, and the two plates are pressed together by hand to form a closed configuration. In the closed configuration, the spacer adjusts the distance between the two plates, thus adjusting the sample thickness through the two plates and the spacer. By using appropriate spacers and plates (see other parts of the instruction manual), the sample thickness in the closed configuration can be uniform over a large area and close to the height of the spacer. Experiments have shown that even with variations in hand pressure, force, and sequence (pressing one area of ​​the RHC card first, then rubbing another area of ​​the RHC card), the sample thickness remains uniform.

[0372] The first plate is made of a poly(methyl methacrylate) (PMMA) film with a thickness of -50μm, a width of 20mm, and a length of 20mm.

[0373] The second plate is a 20mm wide square polyethylene terephthalate (PET) film with a thickness of 25μm. The inner surface of the second plate has a periodic array of columnar spacers, each 30μm high, 30μm × 40μm in size, and with a spacer spacing of 80μm. The spacers have a uniform height and a flat top surface (note that other types of spacers can be used, and will be described later). The spacers fixed to the plate are manufactured by direct imprinting onto a flat PMMA sheet (other manufacturing methods are also possible).

[0374] Experiments tested various heating / cooling layers of different materials and geometries on the outer or inner surface of the second plate. An example (e.g.) Figure 18A (As shown) The heating / cooling layer is located on the outer surface of the second plate and covers the entire outer surface of the second plate. The heating / cooling layer consists of an Au (gold) film and a black coating layer. One surface of the gold film is in contact with the outer surface of the second plate, while the other surface is coated with the black coating. The black coating is a commercially available product consisting of a film composed of a mixture of black carbon nanoparticles and polymers. The average thickness of the black coating is -9 μm (with a thickness variation of -2 μm). The black coating layer can be directly faced with the incident LED light, such as... Figure 19As shown, a 5 nm Ti adhesion layer exists between the Au film and the outer surface of the second plate, which improves the adhesion between the Au and the second plate. However, the adhesion layer is optional, and due to its thin thickness, it has little or no effect on the thermal performance of the sample holder.

[0375] The heating source can be a blue light-emitting diode (LED) with a center wavelength of 450nm. For example... Figure 19 As shown, according to some embodiments, a lens is used to project light from the LED onto the heating / cooling layer, but only onto the central region of the heating / cooling layer, and the size (i.e., area) of the LED spot on the heating / cooling layer is typically approximately 5 mm × 5 mm. As shown later, for a given sample card and sample thickness, only the sample at the LED heating point can be changed and / or reach the designed temperature. Therefore, the heating area is approximately 5 mm × 5 mm. In some embodiments, the cooling area can be approximately 16 times larger than the heating area (i.e., a high KC / H ratio = 16) compared to the entire area of ​​the first plate.

[0376] The LED heating source is powered by a power supply capable of changing the LED current in less than 100 ms. In some embodiments, an aspherical condenser lens is used to focus the LED light, and the lens has a diameter of 12 mm, a focal length of 10.5 mm, and a numerical aperture (NA) of 0.54.

[0377] A temperature-sensitive dye (LDS698) monitors the temperature of the sample in the heating zone (i.e., the area directly irradiated by the LED). A photodetector is used to monitor the temperature-sensitive dye and provide feedback to control the LED current and thus the LED heating source and the temperature of the heating zone.

[0378] In the experiments described below (Experiments 1 through 12), unless otherwise stated, the sample holder is supported by a -2mm edge at two opposite edges of the second plate. Therefore, the sample holder is thermally isolated from the outside, and cooling of the sample holder is primarily achieved through thermal radiation. Thermal radiation cooling is mainly provided by the H / C layer, as the sample and plate are poor radiators of heat and have much lower thermal conductivity than the H / C layer. Thermal radiation cooling radiates heat energy into the open environment (i.e., the room).

[0379] In our experiments, a liquid sample of -5 μL with thermal properties close to that of water was deposited between two plates, approximately in the central region of the plate surface. The sample was first dropped onto one plate of the RHC card, then the other card was placed on top of the sample, and the two plates were manually pressed into a closed configuration. Due to the spacers on the plates, the spacing between the two plates was adjusted to 30 μm when in the closed configuration by an array of spacers with a height of 30 μm, and uniform sample thickness was found even under different hand-pressing pressures, forces, and sequences (first pressing one area, then rubbing against another area of ​​the RHC card). Several advantages are provided in the practical use of this invention for achieving good sample thickness through manual pressing.

[0380] For a -5 μL sample between two plates, the sample has a thickness of 30 μm and an area of ​​-166 mm² (approximately -13 mm by -13 mm²). The lateral shape of the sample is affected by the spacers on the plates, such as... Figure 18B (As shown in the top view). In some embodiments, the total sample area is more than -6.6 times larger than the heating zone area (-5mm × 5mm). Experiments have shown that with this setup, only the beverage sample above the heating zone is heated to the desired temperature. That is, the area (volume) of the heated sample portion is approximately 1 / 6 of the total sample area (volume).

[0381] Furthermore, in this setup, there are no physical walls at the edge of the sample tray, only air. However, as described later, we found that the diameter of the sample tray did not change much before and after 30 cycles of PCR (i.e., the difference was almost imperceptible to the naked eye), which means that even without physical walls (except for the air-liquid interface) to enclose the liquid sample, sample evaporation was negligible.

[0382] All the spacers used in this experimental section are fixed to a plate and have a post with a flat top that can contact another plate.

[0383] In our experiments, a liquid sample is deposited on one plate, and then a second plate is placed on top of the sample. The plates are pressed together by hand. During this hand pressure, the sample spreads out to form a film between the plates. Due to the spacers (with uniform height) on the plates, the final sample thickness is uniform even under hand pressure and is regulated by the height of the two plates and the spacers. Furthermore, after the sample reaches its final thickness and the hand pressure is removed, the two plates of the sample holder "self-"hold" each other by the capillary force of the liquid sample to maintain a constant sample thickness. Moreover, even during thermal cycling at 65–95°C, the capillary force maintains a constant sample thickness. This self-sampling holding mechanism, which does not use any clamps, greatly simplifies device operation and reduces costs.

[0384] Experiment 1

[0385] Light absorption of different H / C layer materials

[0386] In one experiment, the effect of the material used for the H / C layer on the light absorption of LEDs was investigated.

[0387] The optical absorption spectra of different materials used in the heating / cooling (H / C) layer were experimentally tested for four different H / C layer materials used for 450 nm LED illumination: Au (gold) only 500 nm thick (i.e., without black coating), Al (aluminum) only 400 nm thick, Au (500 nm thick) with black coating (9 μm thick), and Al (400 nm thick) with black coating (9 μm thick). We found that, as Figure 20 As shown, the black coatings of Au and Al exhibit light absorption of -99% across the entire wavelength range of 400 to 800 nm, with a maximum of 73% (at a wavelength of -490 nm), and only Au shows significantly lower absorption beyond 490 nm; and for Al alone, the absorption is 0.1% across the 400 nm to 800 nm bandwidth. This indicates that the 9 μm thick black coating used in our experiments greatly enhanced the light absorption and radiation of the H / C layer.

[0388] Experiment 2

[0389] Heating zone area measurement

[0390] In another experiment, the area of ​​the heated region on the HC layer was measured. The experiment revealed that the vertical heat transfer from the HC layer to the plate and sample is several orders of magnitude better than the lateral heat conduction in the plate, or even in samples with an HC layer. The area of ​​the heated region in the sample is approximately the same as the LED illumination area on the HC layer.

[0391] Through experiments, the sample holder card (such as...) Figure 18A(As shown) A first plate consists of a 50 μm thick PMMA substrate, a 25 μm thick PET substrate, a 30 μm thick sample notch controlled by spacers, and a gold H / C layer on the outer surface of the second plate. The first plate, the second plate, and the gold / black HC layer have the same area of ​​20 mm × 20 mm. The HC layer contains a 500 nm thick Au (gold) film and a black coating layer. One surface of the gold film is in contact with the outer surface of the second plate, while the other surface is coated with the black coating. The black coating is a commercially available product consisting of a film composed of black carbon nanoparticles and a polymer mixture. The average thickness of the black coating is -9 μm (with a thickness variation of -2 μm). The LED heating power projected onto the -5 mm × 5 mm heating zone of the H / C layer is 300 mW. The sample is a 5 μL liquid temperature-sensitive dye, LDS698, with a concentration of 2 mg / mL in 60% water and 40% DMSO. The temperature-sensitive dye allows us to optically measure the sample temperature. The 5 μL sample on the RHC card is much larger than the heating zone area, with a thickness of 30 μm and an area of ​​-167 mm². Thermal cycling was performed between 65 °C and 95 °C.

[0392] We have experimentally observed that, under given conditions, during thermal cycling (65-95°C), based on measurements using a temperature-sensitive dye, for a sample area of ​​167 mm², only the sample area directly irradiated by the LED at the top (-5 mm × 5 mm) experiences thermal cycling (65-95°C), while the rest of the sample area remains at a nearly constant temperature close to room temperature (i.e., ambient temperature, e.g., -20°C). The thermally cycled zone in the sample is approximately 1 / 6 of the total sample area. The transition distance from the thermally cycled zone to the sample area at ambient temperature, measured by the temperature-sensitive dye, is approximately 2-3 mm. The experiment also shows that, for a 20 mm × 20 mm area gold / black HC layer, only the sample area directly irradiated by the LED (-5 mm × 5 mm) is heated. That is, the heated zone is only 1 / 16 of the total HC layer (i.e., high KC / H ratio = 16). This is because, in a given RHC card, the vertical heat transfer from the HC layer to the plate and sample is several orders of magnitude better than the lateral heat conduction in the plate and sample with the HC layer.

[0393] Experiment 3

[0394] Effect of HC layer area on heating and cooling time

[0395] In another experiment, the effect of the H / C region area on heating and cooling times was investigated. Two types of RHC cards were studied.

[0396] Type 1 RHC cards use a disk-shaped HC layer. A Type 1 RHC card may comprise a first plate of 100 μm thick PMMA (polymethyl methacrylate), a second plate of 50 μm thick PET (polyethylene terephthalate), a 30 μm thick sample notch controlled by spacers, and an H / C layer of 700 nm thick gold film on the outer surface of the second plate. The first and second plates have a square shape and identical area of ​​20 mm × 20 mm. The second plate has a periodic array of flat-topped columnar spacers with a uniform height of 30 μm, dimensions of 30 μm x 40 μm, and spacer spacing of 80 μm on its inner surface. The HC layer located at the center of the outer surface of the second plate is a 700 nm thick Au layer and a disk shape with different disk diameters for different RHC cards.

[0397] Type 2 RHC cards use square HC layers. A Type 2 RHC card may include a first plate of 50 μm thick PMMA, a second plate of 50 μm thick PET, 30 μm high spacers to control sample thickness to 30 μm, and an H / C layer of 500 nm thick gold film on the outer surface of the second plate. The first plate has a square shape, an area of ​​20 mm × 20 mm, and a periodic array of flat-topped columnar spacers with a uniform height of 30 μm, dimensions of 30 μm × 40 μm, and spacer spacing of 80 μm on its inner surface. The second plate has a square shape and four different areas for the four different HC layers. The two second plates have 20 mm × 20 mm areas for HC layer areas of 10 mm × 10 mm and 20 mm × 20 mm, respectively; however, the other two plates have areas of 30 mm × 30 mm and 40 mm × 40 mm, respectively, the same as the HC layer area in the HC layer region.

[0398] When testing two types of RHC cards, LED heating power was projected onto a -5mm × 5mm area H / C layer to form a heating zone with a power of 300mW. The sample was a 5μL liquid temperature-sensitive dye, LDS698, at a concentration of 2mg / mL in 60% water and 40% DMSO. The temperature-sensitive dye allows us to optically measure the local temperature of the sample. The 5μL sample on the RHC card, much larger than the heating zone area, had a thickness of 30μm (adjusted by spacers) and an area of ​​-167mm². Thermal cycling was conducted between 65°C and 95°C.

[0399] Experimental data (such as) Figure 22 and Figure 23 As shown in the figure, as the H / C layer area increases, the heating time increases, but the cooling time decreases. For Type 1 RHC cards, the HC layer does not have direct physical contact with the mechanical support of the card (e.g., sample holder), so the reduction in cooling cycle time is mainly due to the increased thermal radiation cooling of the HC layer caused by the increased radiative cooling area of ​​the HC layer.

[0400] Experiment 4

[0401] Achieving 0.6s heating, 0.75s cooling, and 500mW heating using a 500nm AuH / C layer.

[0402] In another experiment, the RHC card of a 5 μL water-like sample with a thickness of 30 μm and an LED power of 500 mW was studied (compared to...). Figure 18A The heating and cooling cycle is the same as that shown in the RHC card. Figure 21 The experimental data shown represents 10 cycles between 65°C and 93°C, with a heating time of 0.65 seconds (average temperature rise rate of 43°C / second) and a cooling time of 0.75 seconds (average temperature fall rate of 37°C / second).

[0403] Experiment 5

[0404] (The effect of H / C layer thickness on heating and cooling time)

[0405] In one experiment, the effect of the layer thickness of H / C gold on heating and cooling time was investigated.

[0406] An exemplary RHC card has a first plate made of 100 μm thick PMMA, a second plate made of 50 μm thick PET, an array of 30 μm thick spacers to control sample thickness, and a gold-coated H / C layer on the outer surface of the second plate. The first plate, the second plate, and the gold-coated HC layer all have the same area of ​​20 mm × 20 mm. An LED heating power of 300 mW is projected onto a -5 mm × 5 mm heating zone of the H / C layer. A 5 μL water-like sample on the RHC card is much larger than the heating zone area, having a thickness of 30 μm and an area of ​​-167 mm². Thermal cycling is performed between 65°C and 95°C.

[0407] Figure 24A and 24B The experimental data shown indicate that as the gold thickness of the HC layer changes from 300 nm to 700 nm, the heating time in the thermal cycle increases slightly (from 1.75 seconds to 1.90 seconds), but the cooling time in the thermal cycle decreases with increasing gold thickness (from 1.5 seconds to 1.3 seconds).

[0408] Cooling cycle time decreases with gold thickness. This indicates that (a) thermal radiation cooling of the HC layer of gold material is important for sample cooling, and (b) thermal radiation cooling involves heat conduction from the sample to the gold surface for radiation. The thicker the gold, the better the heat conduction from the sample to the edge of the HC layer of gold material.

[0409] The heating cycle time increases with increasing gold thickness. Clearly, thicker gold increases the total heating energy. However, in this experiment, the LED only heated the relevant sample area (-5mm × 5mm) and the HC layer area of ​​the gold material to the maximum cycle temperature, and the thermal mass of gold is relatively small (due to its thinness). Therefore, the increase in total heating energy is small, resulting in a slight increase in heating cycle time with increasing gold thickness.

[0410] Experiment 6

[0411] (The effect of the distance between the heating / cooling layer and the sample on heating and cooling time)

[0412] In another experiment, the effect of the distance between the HC layer and the sample on heating and cooling time was investigated.

[0413] An exemplary RHC card has a first plate of 100 μm thick PMMA film, a second plate of PET film with varying thicknesses depending on the RHC card, a sample thickness of 30 μm controlled by spacers, and an HC layer made of bare 0.5 μm thick gold on the outer surface of the second plate. The first plate, the second plate, and the gold HC layer all have the same area of ​​20 mm × 20 mm. LED heating power of 300 mW is projected onto a -5 mm × 5 mm heating zone of the H / C layer. A 5 μL water sample on the RHC card is much larger than the heating zone area, having a thickness of 30 μm and an area of ​​-167 mm². Thermal cycling is performed between 65°C and 95°C.

[0414] The distance between the HC layer and the sample is the distance between the gold surface in contact with the surface of the second plate and the sample surface in contact with another surface of the second plate (i.e., the distance between the gold and the sample).

[0415] Figure 25B and 25A The sample data shown indicates that when the thickness of the second plate changes from 25 μm (which is the distance from gold to the sample) to 1000 μm, both the heating cycle time and the cooling cycle time increase. However, the heating cycle time increases much more significantly with the thickness of the second plate than the cooling cycle time.

[0416] Data shows that as the thickness of the second plate increases, the energy required to heat and cool the second plate increases significantly, and the thermal conductivity between the sample and the HC layer decreases significantly.

[0417] To facilitate rapid heating and cooling, the thickness of the second plate (which is physically sandwiched between the sample and the HC layer) should be minimized; the second plate should be as thin as possible. A preferred thickness for the second plate is 25 nm or less. Another preferred thickness is 10 nm or less.

[0418] Experiment 7

[0419] (The effect of sample thickness on heating and cooling time)

[0420] In another experiment, the effect of the sample thickness sandwiched between two plates on heating and cooling time was investigated.

[0421] An exemplary RHC card has a first plate of 100 μm thick PMMA, a second plate of 25 μm thick PET film, a periodic array of spacers for controlling sample thickness, and an HC layer made of bare 0.5 μm thick gold on the outer surface of the second plate. For each different RHC card, the water-like sample has a different notch (i.e., thickness). The first plate, the second plate, and the gold HC layer all have the same area of ​​20 mm × 20 mm. LED heating power of 300 mW is projected onto a -5 mm × 5 mm heating zone of the H / C layer. The water-like sample on the RHC card is much larger than the heating zone area, having an area of ​​-167 mm². Thermal cycling is performed between 65°C and 95°C.

[0422] Figure 27A and 27B The results showed that when the sample thickness changed from 10 μm to 100 μm, both the heating cycle time and the cooling cycle time increased. However, the heating cycle time increased much more significantly with the increase of the second plate thickness than the cooling cycle time.

[0423] Data shows that increasing sample thickness leads to a significant increase in the energy required to heat and cool the sample.

[0424] To facilitate rapid heating and cooling, the sample thickness should be minimized. A preferred sample thickness is 30 μm or less. Another preferred sample thickness is 10 μm or less. Yet another preferred sample thickness is 5 μm or less.

[0425] Experiment 8

[0426] The effect of LED power on heating and cooling time

[0427] In another experiment, the effect of LED power on heating and cooling time was investigated. An exemplary RHC card has a first plate of 50 μm thick PMMA, a second plate of 25 μm thick PET, and an HC layer on the outer surface of the second plate. The first plate, the second plate, and the gold / black HC layer have the same area of ​​20 mm × 20 mm. The first plate has a periodic array of spacers with a height of 30 μm, a cross-sectional size of 30 μm × 40 μm, and a spacer spacing of 80 μm on its inner surface. The HC layer comprises a 500 nm thick Au (gold) film and a black coating layer. One surface of the gold film contacts the outer surface of the second plate, while the other surface is coated with the black coating. The black coating is a commercially available product consisting of a film composed of black carbon nanoparticles and a polymer mixture. The average thickness of the black coating is -9 μm (with a thickness variation of -2 μm).

[0428] Heating power provided by a blue (450nm peak wavelength) LED was projected onto a -5mm × 5mm heating zone in the H / C layer, with the power varying from 100mW to 500mW. The sample was a 5μL liquid temperature-sensitive dye, LDS698, at a concentration of 2mg / mL in 60% water and 40% DMSO. The temperature-sensitive dye allowed us to optically measure the sample temperature. The 5μL sample on the RHC card was much larger than the heating zone area, with a thickness of 30μm and an area of ​​-167mm².

[0429] Figure 27A The experimental data shown illustrates the relationship between heating time and heating source power, demonstrating the experimental data on the time required to heat an LED from 65°C to 93°C using heating LED power intensities ranging from 100mW to 500mW on an RHC card.

[0430] Figure 27B The experimental data shown illustrates the relationship between cooling time and heating source power, illustrating the time required to cool from 93°C to 65°C. Heating / cooling time results are also shown in Table 1.

[0431] Experimental data show that, for a given sample holder (i.e., RHC card), as the LED power increases from 100mW to 500mW, the thermal cycle time decreases from 14 seconds to 0.4 seconds, while the cooling cycle time remains almost constant.

[0432] Experimental data show that for low heating power (i.e., low heating power density), delivering a fixed amount of energy will take longer, and this longer time will increase energy losses in heat conduction and radiation, thus increasing wasted energy. For cooling, since the amount of thermal energy stored in a fixed volume of sample at a given temperature is constant, the cooling time is almost independent of the heating cycle time, regardless of how long it takes to reach that temperature.

[0433] Experiments show that heating power should be increased in order to reduce the total thermal cycle time.

[0434] Table 1. Effect of LED power on RHC heating and cooling time

[0435]

[0436] Experiment 9

[0437] The Influence of H / C Layer Material on Heating and Cooling Time

[0438] In another experiment, the effect of the H / C layer material on heating and cooling times was investigated. An exemplary RHC card had a first plate of 50 μm thick PMMA, a second plate of 25 μm thick PET film, a periodic array of spacers that conditioned the water-like sample to a 30 μm thick spacer, and an HC layer on the outer surface of the second plate. The HC layer had a different material for each different RHC card. The first plate, second plate, and HC layer all had the same area of ​​20 mm × 20 mm. LED heating power of 300 mW was projected onto a -5 mm × 5 mm heating zone of the H / C layer. The 5 μL water-like sample on the RHC card was much larger than the heating zone area, having a thickness of 30 μm and an area of ​​-167 mm². Thermal cycling was conducted between 65 °C and 93 °C.

[0439] Figure 28A and 28B Experimental data show that for the three different HC layer materials tested, the heating cycle and cooling cycle time were 0.75 seconds and 0.75 seconds for the sample holder with an HC layer of Au (500 nm thick) plus a 9 μm black coating, respectively; 1 second and 1.1 seconds for the sample holder with an HC layer of Al (500 nm thick) plus a 9 μm black coating, respectively; and 1.75 seconds and 1 second for the sample holder.

[0440] This experiment demonstrates the importance of good lateral thermal conductivity in radiative cooling. Compared to gold with a black coating, aluminum with a black coating almost completely absorbs the light (and therefore emits radiation), but its lateral thermal conductivity is much worse. This results in a much smaller effective radiative surface area because heat cannot diffuse laterally as much as possible.

[0441] Experiments show that the preferred embodiment of the material used for the HC layer is a thin gold film plus a black coating.

[0442] Experiment 10

[0443] Demonstration of a 0.73-second thermal cycle time (0.23 seconds heating time and 0.5 seconds cooling time)

[0444] In another experiment, for thermal cycling between 65°C and 93°C, the RHC card (card B) showed a thermal cycling time of 0.73 seconds (0.23 seconds of heating time and 0.5 seconds of cooling time), while another RHC card (card A) showed a thermal cycling time of 0.9 seconds (0.3 seconds of heating time and 0.6 seconds of cooling time).

[0445] Figure 29AA sample holder with two plates, each plate being a high-density polyethylene (HDPE) film having a thickness of approximately 10 μm, a width of approximately 20 mm, and a length of approximately 20 mm, is shown according to some embodiments. The spacers controlling the sample thickness are soda-lime balls with a diameter of approximately 24 μm and a concentration of approximately 60 mg / mL. The spherical spacers are mixed with the sample.

[0446] Figure 29B A sample holder is shown consisting of a first plate with a 25 μm thick polymethyl methacrylate (PMMA) film and a second plate with a 10 μm thick high-density polyethylene (HDPE) film. Both plates have the same 20 mm × 20 mm area. The first plate has a periodic array of spacers on its inner surface, each with a height of 10 μm, dimensions of 30 μm × 40 μm, and an internal spacer spacing of 80 μm.

[0447] Figure 29A and 29B The two sample holder embodiments shown have an H / C layer covering the entire outer surface of the second plate. The H / C layer comprises an Au film with a thickness of 500 nm, one surface of which contacts the outer surface of the second plate, and the other surface is coated with a black paint. The black paint is a commercially available product consisting of a film composed of a mixture of black carbon nanoparticles and polymers, and the coated film has an average thickness of 9 μm with a thickness variation of 2 μm.

[0448] The sample was a 2 mg / mL liquid temperature-sensitive dye, LDS698, in 60% water and 40% DMSO. The sample volume was... Figure 29A The sample holder in the sample is 5 μL, for Figure 29B The sample holder in the sample is 3 μL.

[0449] The heating source is a blue light-emitting diode (LED) with a center wavelength of 450nm, which projects 500mW of energy onto the black coating layer of the HC layer, forming a heating area of ​​-5mm×5mm in the center of the second plate.

[0450] Experimental data (Table 2) show that for thermal cycling from 65℃ to 93℃, Figure 29A The sample holder has a heating cycle time of 0.3s and a cooling time of 0.60s, so the total thermal cycle time is 0.90s. Figure 29B The sample holder has a heating cycle time of 0.23 s and a cooling time of 0.50 s, so the total thermal cycle time is 0.73 s.

[0451] Table 2.1 RHC Parameters

[0452]

[0453] Table 2.2 Heating / Cooling Performance

[0454]

[0455] Experiment 11

[0456] The effect of using sample holders and sample adapters on heating and cooling times

[0457] In another experiment, the effects of using a sample holder support and a sample adapter on heating and cooling times were investigated.

[0458] In the experiment, the RHC card was placed on a mechanical sample card support (referred to as the "card support"), and then the card support was slid into the adapter. The thermal cycling time was measured for the following cases: (a) RHC card only, (b) RHC card on the card support, and (c) both the card support and the RHC card on the card support were slid into the adapter.

[0459] In some embodiments, a sample holder (such as...) Figure 30A and 30B The first plate (shown) comprises a poly(methyl methacrylate) (PMMA) film having a thickness of 10 μm to 50 μm, a width of 22 mm, and a length of 27 mm. The first plate has a periodic array of spacers on its inner surface, the spacers having a height of 30 μm, a cross-sectional dimension of 30 μm × 40 μm, and a spacer spacing of 80 μm.

[0460] In some embodiments, the second plate is a polyethylene terephthalate (PET) film or a high-density polyethylene film having a thickness of 10 μm to 50 μm, a width of 20 mm, and a length of 27 mm. A heating / cooling layer covers the entire outer surface of the second plate. The heating / cooling layer comprises an Au film with a thickness of 100 nm to 500 nm, one surface of which contacts the outer surface of the second plate, and the other surface is coated with a black paint. The average thickness of the black paint is 9 μm.

[0461] According to some embodiments, card support (such as...) Figure 30A and 30B (As shown) comprises a 1mm thick PMMA board, 24mm wide and 32mm long, with a 15mm x 15mm square hole in the center. The RHC card and card support are bonded together using an adhesive 10 to 15μm thick, with the adhesive positioned between the black coating of the RHC card and the surface of the card support, as shown. Figure 30B As shown.

[0462] According to some embodiments, the card adapter comprises an assembly of two U-shaped frames into which a sample card can slide in or out. One of the U-shaped frames is made of plastic and the other of aluminum, with the two U-shaped frames assembled parallel to a notch between them, and the notch being a slot for sliding the sample card. An example of a card adapter is a conventional SD card connector cut into a U-shape (cut from the rear).

[0463] When testing the thermal cycling time of RHC cards with a 50 nm thick first card and a 25 nm thick PET second card (both with an area of ​​20 mm × 20 mm), the sample was a 5 μL liquid temperature-sensitive dye LDS698 at a concentration of 2 mg / mL in 60% water and 40% DMSO. A blue 450 nm LED was projected onto a black coating with an area of ​​5 mm × 5 mm (forming a heating zone) and a power of 300 mW.

[0464] Figure 31 The experimental data shown indicate that for thermal cycling between 65°C and 93°C, (a) for RHC cards only, the heating cycle time is 0.67 seconds, the cooling cycle time is 0.9 seconds, and the total thermal cycle time is 1.57 seconds; (b) for RHC cards on card supports, the heating cycle time is 0.77 seconds, the cooling cycle time is 0.87 seconds, and the total thermal cycle time is 1.64 seconds; (c) for RHC cards on card supports with the card support sliding into the adapter, the heating cycle time is 0.93 seconds, the cooling cycle time is 0.7 seconds, and the total thermal cycle time is 1.63 seconds.

[0465] Experimental data show that by cooling the RHC card primarily based on thermal radiation, the RHC card can be supported by a card support, which can be inserted into the adapter, while increasing the thermal cycling time to less than 4%.

[0466] Experiment 12

[0467] Additional heatsink usage effect

[0468] In another experiment, the effect of adding an external heat sink on heating and cooling time was investigated. In this experiment, instead of allowing heat from the RHC card to radiate into the environment, the Peltier cooling device was brought into contact with the edge of the HC layer of the RHC card.

[0469] The RHC card has a first plate made of 50 μm thick PMMA film, a second plate made of 50 μm thick PET film, a periodic array of spacers controlling the sample thickness to 30 μm, and an HC layer made of exposed 0.3 μm thick gold on the outer surface of the second plate. The first plate has an area of ​​20 mm × 20 mm. The second plate and the gold HC layer have the same area of ​​30 mm × 30 mm.

[0470] The LED heating power projected onto the -5mm × 5mm heating zone of the H / C layer was 500mW. The 5μL water sample on the RHC card was much larger than the heating zone area, with a thickness of 30μm and an area of ​​-167mm². Thermal cycling was performed between 65℃ and 93℃.

[0471] In some setups, a Peltier cooler, providing a 0°C radiator, contacts or is close to the HC layer by overlapping its 3mm edge with the second plate. In the reference setup, there is no Peltier cooler. The sample is a 5 μL liquid temperature-sensitive dye, LDS698, at a concentration of 2 mg / mL in 60% water and 40% DMSO.

[0472] Experimental data (Table 3) show that without a Peltier cooler, the time for the liquid inside the RHC card to heat from 65°C to 93°C is 0.63 s, while the time to cool from 93°C to 65°C is 1.2 s, with the Peltier cooler in contact with the Au membrane. With the Peltier cooler, the time for the liquid inside the RHC card to heat from 65°C to 93°C increases to 0.73 s, while the time to cool from 93°C to 65°C decreases to 0.93 s. Using the Peltier cooler, the total thermal cycle time decreases from 1.83 to 1.66. This is achieved by slightly increasing the heating cycle time while significantly reducing the cooling cycle time.

[0473] Table 3. Effects of using an RHC card with an additional heatsink

[0474]

[0475] Sample card (i.e., RHC card)

[0476] The following are some exemplary embodiments of key components of the sample card (i.e., the RHC card).

[0477] Sample thickness

[0478] To reduce the thermal mass of the sample and to reduce heat convection losses in the sample, in some embodiments, the average sample thickness in the region heated by the heating / cooling layer is 500 μm or less, 200 μm or less, 100 μm or less, 50 μm or less, 20 μm or less, 10 μm or less, 5 μm or less, 2 μm or less, 1 μm or less, 500 nm or less, 300 nm or less, 100 nm or less, 50 nm or less, or any two of these values.

[0479] A preferred average sample thickness in the area heated by the heating / cooling layer is 0.1 μm to 0.5 μm, 0.5 μm to 10 μm, 10 μm to 20 μm, 20 μm to 30 μm, 30 μm to 50 μm, 50 μm to 80 μm, 80 μm to 100 μm, or 100 μm to 150 μm.

[0480] Experiment 13

[0481] Example and system of real-time PCR amplification using RHC cards

[0482] The RHC card used in this experiment has a first plate of 50 μm thick PMMA, a second plate of 25 μm thick PET, and an H / C layer on the outer surface of the second plate. The gold / black coating HC layer has an area of ​​10 mm in diameter. The first plate has a periodic array of spacers on its inner surface. The HC layer comprises a thin Au (gold) film and a black coating layer. The gold film has one surface in contact with the outer surface of the second plate and another surface in contact with the black coating. The black coating is a commercially available product consisting of a film composed of black carbon nanoparticles and a polymer mixture. The average thickness of the black coating is -9 μm (with a thickness variation of -2 μm).

[0483] The PCR (real-time PCR) reagent for amplifying 20 μL of Staphylococcus aureus genomic DNA contains MSSA forward primers, MSSA reverse primers, and Cy5-labeled DNA probes, as well as AptaTaq DNA buffer, AptaTaq polymerase, MgCl2, dNTPs, bovine serum albumin (BSA), template DNA, and ddH2O.

[0484] In real-time PCR experiments, two positive RHC cards showed a significant increase in fluorescence signal relative to the cycle number, especially after 20 cycles of amplification. However, a single negative RHC card did not show a significant increase in fluorescence signal relative to the cycle number. After 40 cycles of amplification in the RHC system, the PCR products from the RHC cards were extracted and subjected to nucleic acid gel electrophoresis to confirm that only the two positive RHC cards had successful amplification bands.

[0485] Sample well

[0486] In some embodiments, one or two plates have sample holes, wherein the holes regulate the maximum volume of the sample in the holes and prevent the sample from flowing into other parts of the plates.

[0487] Plate thickness

[0488] To reduce the thermal mass of the first and second plates and to reduce lateral heat conduction losses in the plates, the thickness of the first and second plates is preferably thin.

[0489] In some embodiments, the thickness of the first or second plate is 2 nm or less, 10 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 1000 nm or less, 2 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 10 mm or less, or within any two of these values.

[0490] In some embodiments, the thickness of the first or second plate is 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2.5 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 200 μm or 500 μm, 1000 μm, or within any two values.

[0491] The first and second plates can have the same thickness or different thicknesses, and can be made of the same material or different materials.

[0492] In some preferred embodiments, the thickness of the first or second plate is 10 nm and 500 nm, 500 nm and 1 μm, 1 μm and 2.5 μm, 2.5 μm and 5 μm, 5 μm and 10 μm, 10 μm and 25 μm, 25 μm and 50 μm, 50 μm and 100 μm, 100 μm and 200 μm, or 200 μm and 500 μm, or 500 μm and 1000 μm.

[0493] The preferred thickness of the first or second plate is 10 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 1000 nm or less, 2 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, or within any two of these values.

[0494] In some preferred embodiments, the plate with the heating / cooling layer is thinner than the other plate without a heater.

[0495] In some preferred embodiments, the thickness of the first plate is 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 250 μm, or within any two of these values; while the thickness of the second plate is 25 μm, 50 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 250 μm, 500 μm, 1 mm, 1.5 mm, 2 mm, or within any two of these values.

[0496] In some embodiments, the average thickness of at least one plate is 1 to 1000 μm, 10 to 900 μm, 20 to 800 μm, 25 to 700 μm, 25 to 800 μm, 25 to 600 μm, 25 to 500 μm, 25 to 400 μm, 25 to 300 μm, 25 to 200 μm, 30 to 200 μm, 35 to 200 μm, 40 to 200 μm, 45 to 200 μm, or 50 to 200 μm.

[0497] In some embodiments, the average thickness of at least one plate is in the range of 50 to 75 μm, 75 to 100 μm, 100 to 125 μm, 125 to 150 μm, 150 to 175 μm, or 175 to 200 μm.

[0498] In some embodiments, the average thickness of at least one plate is about 50 μm, about 75 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, or about 200 μm.

[0499] Board area. In some embodiments, the lateral area of ​​the first board and / or the second board is 1 mm² or less, 10 mm² or less, 25 mm² or less, 50 mm² or less, 75 mm² or less, 1 cm² or less, 2 cm² or less, 3 cm² or less, 3 cm² or less, 5 cm² or less, 10 cm² or less, 20 cm² or less, 30 cm² or less, 50 cm² or less, 100 cm² or less, 500 cm² or less, 1000 cm² or less, 5000 cm² or less, 10,000 cm² or less, or any value within any two of these values.

[0500] In a preferred embodiment, the lateral area of ​​the first plate and / or the second plate is in the range of 1 mm² to 10 mm², 10 mm² to 50 mm², 50 mm² to 100 mm², 1 cm² to 5 cm², 5 cm² to 20 cm², 20 cm² to 50 cm², 50 cm² to 100 cm², 100 cm² to 500 cm², 500 cm² to 1000 cm², or 1000 cm² to 10,000 cm².

[0501] In some embodiments, the first plate and the second plate have the same lateral dimensions. In some embodiments, one plate has an area that differs from the other plates by 10% or less, 30% or less, 50% or less, 80% or less, 90% or less, 95% or less, 99% or less, or within any two values ​​(the plate with the largest area is the basis for calculating the different percentages).

[0502] In some embodiments, the first plate and / or the second plate have a width or length of 5 mm, 10 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 75 mm, 100 mm, or within any two of these values.

[0503] In a preferred embodiment, the first plate and / or the second plate have a width or length in the range of 5 mm to 10 mm, 20 mm to 30 mm, 30 mm to 50 mm, 50 mm to 75 mm, or 75 mm to 100 mm.

[0504] In one preferred embodiment, the width or length of the plate is in the range of 5 mm to 50 mm. In another preferred embodiment, the plate has a width in the range of 5 mm to 50 mm and a length in the range of 6 mm to 70 mm.

[0505] Board materials

[0506] In some embodiments, the materials used for the first and second plates include, but are not limited to, polymers (e.g., plastics) or amorphous organic materials. Polymer materials include, but are not limited to, acrylate polymers, vinyl polymers, olefin polymers, cellulose polymers, non-cellulose polymers, polyester polymers, nylon, cyclic olefin copolymers (COC), poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefin polymers (COP), liquid crystal polymers (LCP), polyamide (PA), polyethylene (PE), polyimide (PI), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyetheretherketone (PEEK), polyethersulfone (PES), polyethylene phthalate (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkylene (PFA), polydimethylsiloxane (PDMS), rubber, or any combination thereof.

[0507] In some embodiments, the materials used for the first plate and the second plate include, but are not limited to, inorganic materials, including dielectric materials such as silicon dioxide, porcelain, ceramic enamel, mica, glass, and oxides of various metals.

[0508] In some embodiments, the materials used for the first plate and the second plate include, but are not limited to, inorganic materials, including alumina, aluminum chloride, cadmium sulfide, gallium nitride, gold chloride, indium arsenide, lithium borohydride, silver bromide, sodium chloride, graphite, carbon nanotubes, carbon fibers, etc.

[0509] In some embodiments, the materials used for the first plate and the second plate include, but are not limited to, metals (e.g., gold, copper, aluminum, etc.) and alloys.

[0510] In some embodiments, the materials used for the first plate and the second plate are made of the multilayer materials and / or mixtures of materials listed above.

[0511] Heating layer and cooling layer

[0512] In some embodiments, the heating layer (112-1) and the cooling layer (112-2) comprise high-k materials and / or high-k-C ratio materials. High-k and / or high-k-C ratio materials include materials / structures such as, but not limited to, metal films, semiconductors, half-metals, plasma surfaces, metamaterials (e.g., nanostructures), black silicon, graphite, carbon nanotubes, silicon interlayers, graphene, superlattices, plasma materials, any material / structure capable of effectively absorbing electromagnetic waves and converting the absorbed energy into heat, and any combination thereof.

[0513] For a heating layer heated by an optical heating source, the heating layer comprises a material layer that significantly absorbs radiant energy from the optical heating source. Significant absorption means that the heating / cooling layer absorbs radiant energy from the optical heating source more significantly than the sample and plate.

[0514] In some embodiments, the thickness of the heating / cooling layer is in the range of 50 nm to 15 μm. In some embodiments, the heating / cooling layer comprises a high-k layer with a thickness in the range of 100 nm to 1 μm.

[0515] In some embodiments, the size of the photoheating region is approximately 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1 mm, 2 mm, 5 mm, 10 mm, 20 mm, 50 mm, or 100 mm, or within any two values. In various embodiments, the size and shape of the photoheating region can vary.

[0516] In some embodiments, the heating / cooling layer includes a coupled point-pole antenna (D2PA) array, such as, but not limited to, the D2PA arrays described in U.S. Provisional Patent Application No. 61 / 347,178, filed May 21, 2010; U.S. Provisional Patent Application No. 61 / 622,226, filed April 10, 2012; U.S. PCT Application No. PCT / US2011 / 037455, filed May 20, 2011; PCT Application No. PCT / US2013 / 032347, filed March 15, 2013; and U.S. Patent Application No. 13 / 699,270, filed June 13, 2013, the entire disclosure of which is incorporated herein by reference for all purposes.

[0517] In some embodiments, more than one heating / cooling layer may be present. For example, at least two surfaces of any first or second plate may have a heating / cooling layer.

[0518] In some embodiments, the heating / cooling layer may be two layers of material: one for heating and one for cooling, and both layers may be on the same surface of either the first or second plate. For the sample, the heating layer may be on the outer surface of the second plate, while the cooling layer may be on the outer or inner surface of the first plate. Even the cooling layer may be on the outer surface of the first plate, provided that the first plate has a thin thickness (e.g., 25 μm or less), which should be effective for cooling the sample.

[0519] spacers

[0520] In some embodiments of the invention, a spacer exists between the two plates. In some embodiments, at least one spacer is located in the sample contact area. In some embodiments, the spacer has a uniform height. In some embodiments, the thickness of the sample is the sample thickness as the height of the spacer. In some embodiments, the spacer is fixed to one of the plates.

[0521] Spacer Function. In this invention, the spacer is configured to have one or any combination of the following functions and properties: the spacer is configured to (1) control the thickness of the sample or the relevant volume of the sample together with the plate (preferably, the thickness control is precise or uniform or both in the relevant area); (2) give the sample a squeeze-regulated open flow (CROF) on the plate surface; (3) not occupy a large surface area (volume) in a given sample area (volume); (4) reduce or improve the sedimentation effect of particles or analytes in the sample; (5) change and / or control the wettability of the inner surface of the plate; (6) identify the position, size ratio and / or plate-related information of the plate, or (7) perform any combination of the above.

[0522] Spacer Structure and Shape. In some embodiments, spacers are fixed to their respective plates to achieve the desired sample thickness reduction and control. Typically, spacers can have any shape, as long as they can adjust the sample thickness during the CROF process, but certain shapes are preferred to achieve certain functions, such as better uniformity, less overshoot, etc.

[0523] A spacer can be a single spacer or multiple spacers (e.g., an array). Some embodiments of multiple spacers are arrays of spacers (e.g., columns) where the spacer spacing is periodic or aperiodic, or periodic or aperiodic in certain areas of the plate, or with different distances in different areas of the plate.

[0524] There are two types of spacers: open spacers and closed spacers. An open spacer allows sample flow through it (i.e., the sample flows around and through the spacer; for example, a column acts as a spacer). A closed spacer, on the other hand, prevents sample flow (i.e., the sample cannot flow beyond the spacer; for example, a ring-shaped spacer, and the sample is within the ring). Both types of spacers use their height to adjust the final sample thickness in a closed configuration.

[0525] In some embodiments, the spacer is simply an open spacer. In some embodiments, the spacer is simply a closed spacer. In some embodiments, the spacer is a combination of an open spacer and a closed spacer.

[0526] The term "columnar spacer" refers to a spacer having a columnar shape, and a columnar shape refers to an object having a height and lateral shape that allows a sample to flow around it during squeeze open flow. In some embodiments, the spacer has a flat top (e.g., a column having a flat top to contact a plate).

[0527] In some embodiments, the lateral shape of the columnar spacer is a shape selected from the group consisting of: (i) circles, ovals, rectangles, triangles, polygons, rings, stars, letter shapes (e.g., L-shapes, C-shapes, letters from A to Z), and number shapes (e.g., shapes resembling 0, 1, 2, 3, 4, ... to 9); (ii) shapes in group (i) having at least one rounded corner; (iii) shapes in group (i) having a zigzag shape or rough edges; and (iv) any superposition of (i), (ii), and (iii). For multiple spacers, different spacers may have different lateral shapes and dimensions as well as different distances from adjacent spacers.

[0528] In some embodiments, spacers may be and / or may include pillars, columns, beads, spheres, and / or other suitable geometries. The lateral shape and size of the spacers (i.e., transverse to the respective plate surface) may be any shape and size except for the following limitations in some embodiments: (i) the spacer geometry does not cause significant errors when measuring sample thickness and volume; or (ii) the spacer geometry does not prevent sample flow between the plates (i.e., it is not a closed form). However, in some embodiments, some spacers are required to be closed spacers to restrict sample flow.

[0529] In some embodiments, the spacer is shaped with rounded corners. For example, a rectangular spacer has one, several, or all of its rounded corners (e.g., circles instead of 90-degree angles). Rounded corners generally make the spacer easier to manufacture and, in some cases, cause less damage to the biomaterial.

[0530] The sidewalls of a column can be straight, curved, inclined, or of different shapes at different portions. In some embodiments, the spacer is a column having various lateral shapes, sidewalls, and column height to column lateral area ratios.

[0531] In a preferred embodiment, the spacer has a column shape for allowing open flow.

[0532] Spacer material. In this invention, the spacer is generally made of any material with a thickness capable of being used together with the two plates to adjust the relevant volume of the sample. In some embodiments, the material of the spacer is different from the material of the plates. In some embodiments, the material used for the spacer is at least a portion of the same as the material used for at least one plate.

[0533] The spacers are made of a single material, a composite material, multiple materials, multilayer materials, alloys, or combinations thereof. Each material used for the spacers is an inorganic material, an organic material, or a mixture thereof, with examples of materials given in paragraphs Mat-1 and Mat-2. In a preferred embodiment, the spacers are made of the same material as the plates used in CROF.

[0534] Mechanical strength and flexibility of the spacers. In some embodiments, the mechanical strength of these spacers is sufficient such that during the extrusion of the plates and in a closed configuration, the height of the spacers is the same as or substantially the same as the height of the plates in an open configuration. In some embodiments, the difference between the open and closed configurations of these spacers can be characterized and predetermined.

[0535] The material used for spacers can be rigid, flexible, or any adaptation in between. Rigidity is relative to the given pressure used to place the plate in the closed configuration: if the spacer does not deform more than 1% over its height under pressure, the spacer material is considered rigid; otherwise, it is flexible. When spacers are made of flexible materials, the final sample thickness under the closed configuration can still be predetermined based on the pressure and mechanical properties of the spacer.

[0536] Spacers within the sample. To achieve desired sample thickness reduction and control, particularly for good sample thickness uniformity, spacers are placed within the sample or its relevant volume in some embodiments. In some embodiments, one or more spacers with suitable spacer spacing exist within the sample or its relevant volume. In some embodiments, there is at least one spacer within the sample, at least two spacers within the sample or its relevant volume, or at least "n" spacers within the sample or its relevant volume, where "n" may be determined by the sample thickness uniformity or the sample flow characteristics required during CROF.

[0537] Spacer height. In some embodiments, all spacers have the same predetermined height. In some embodiments, spacers have the same predetermined height. In some embodiments, spacers may be divided into groups or regions, wherein each group or region has its own spacer height. And in some embodiments, the predetermined height of the spacers is the average height of the spacers. In some embodiments, the heights of the spacers are approximately the same. In some embodiments, a percentage of the number of spacers has the same height. In some embodiments, on the same board, the spacer height in one region differs from the spacer height in another region. In some cases, having boards with different spacer heights in different regions has the advantage of measurement.

[0538] The height of the spacer is selected by adjusting the final sample thickness and the residual sample thickness as desired. The spacer height (predetermined spacer height) and / or sample thickness is 3 nm or less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 μm or less, 2 μm or less, 3 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less, 4 mm or less, or within any two of these values.

[0539] The spacer height and / or sample thickness are 1 nm to 100 nm in one preferred embodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to 1000 nm in a separate preferred embodiment, 1 μm (i.e., 1000 nm) to 2 μm in another preferred embodiment, 2 μm to 3 μm in a separate preferred embodiment, 3 μm to 5 μm in another preferred embodiment, 5 μm to 10 μm in a separate preferred embodiment, and 10 μm to 50 μm in another preferred embodiment, and 50 μm to 100 μm in a separate preferred embodiment.

[0540] In some embodiments, the spacer height and / or sample thickness (i) are equal to or slightly larger than the minimum size of the analyte, or (ii) are equal to or slightly larger than the maximum size of the analyte. “Slightly larger” means approximately 1% to 5% larger, and is any value between the two.

[0541] In some embodiments, the height of the spacer, the spacing between the plates, and / or the sample thickness are greater than the minimum size of the analyte (e.g., the analyte has an anisotropic shape), but less than the maximum size of the analyte.

[0542] For example, a red blood cell has a disk shape with a minimum size of 2 μm (disc thickness) and a maximum size of 11 μm (disc diameter). In embodiments of the invention, spacers are selected such that the inner surface spacing of the plates in the relevant area is 2 μm in one embodiment (equal to the minimum size), 2.2 μm in another embodiment, or 3 μm in yet another embodiment (50% larger than the minimum size), but smaller than the maximum size of the red blood cell. This embodiment has certain advantages in blood cell counting. In one embodiment, for red blood cell counting, by making the inner surface spacing 2 μm or 3 μm, and any value between the two, the undiluted whole blood sample is confined within this spacing, and on average, each red blood cell (RBC) does not overlap with other red blood cells, thus allowing for visually accurate counting of red blood cells. (Too much overlap between RBCs can lead to serious errors in counting).

[0543] In some embodiments of this invention, when the plate is in a closed configuration, the plate and spacers are used to adjust not only the thickness of the sample, but also the orientation and / or surface density of the analyte / entity in the sample. When the plate is in a closed configuration, the thinner the sample, the less analyte / entity per surface area (i.e., the lower the surface concentration).

[0544] Lateral dimensions of spacers. For open spacers, the lateral dimensions can be characterized by their lateral dimensions (sometimes called width) in the two orthogonal directions, x and y. The lateral dimensions of a spacer may be the same or different in each direction.

[0545] In some embodiments, the ratio of the lateral dimensions in the x and y directions is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000, or within any two values. In some embodiments, different ratios are used to adjust the sample flow direction; the larger the ratio, the flow is along one direction (the larger dimension direction).

[0546] In some embodiments, different lateral dimensions of the spacer in the x and y directions are used as (a) to use the spacer as a scale marker to indicate the orientation of the plate, (b) to use the spacer to generate more sample flow in a preferred direction, or both.

[0547] In a preferred embodiment, the period, width, and height are specified.

[0548] In some embodiments, all spacers have the same shape and size. In some embodiments, each spacer has a different lateral dimension.

[0549] For the sealing spacer, in some embodiments, the internal transverse shape and size are selected based on the total volume of the sample to be sealed by the sealing spacer, wherein the volume dimensions have been described in this disclosure; and in some embodiments, the external shape and size are selected based on the required strength to support the pressure of liquid against the spacer and the squeezing pressure of the press plate.

[0550] The aspect ratio of the height of the columnar spacer to the average lateral dimension. In some embodiments, the aspect ratio of the height of the columnar spacer to the average lateral dimension is 100,000, 10,000, 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, or within any two of these values.

[0551] Spacer height accuracy. The height of the spacers should be precisely controlled. The relative accuracy of the spacers (i.e., the ratio of deviation to the desired spacer height) is 0.001% or less, 0.01% or less, 0.1% or less; 0.5% or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, 99.9% or less, or within any range of values.

[0552] Spacer spacing. A spacer can be a single spacer or multiple spacers in a relevant area on a plate or sample. In some embodiments, spacers on the plate are configured and / or arranged in an array, and the array is periodic, non-periodic, or periodic in some locations on the plate, while being non-periodic in others.

[0553] In some embodiments, the periodic array of spacers is arranged as a lattice of square, rectangle, triangle, hexagon, polygon or any combination thereof, wherein a combination means that different positions of the plate have different spacer lattices.

[0554] In some embodiments, the spacer spacing of the spacer array is periodic (i.e., uniform spacer spacing) in at least one direction of the array. In some embodiments, the spacer spacing is configured to improve the uniformity between the plate spacings in a closed configuration.

[0555] The distance between adjacent spacers (i.e., spacer spacing) is 1 μm or less, 5 μm or less, 7 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 40 μm or less, 50 μm or less, 60 μm or less, 70 μm or less, 80 μm or less, 90 μm or less, 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, or within any two values.

[0556] In some embodiments, the spacer spacing is within a range of 400 μm or less, 500 μm or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm or less, 10 mm or less, or any value between these ranges. In some embodiments, the spacer spacing is within a range of 10 mm or less, 20 mm or less, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, or any value between these ranges.

[0557] The distance between adjacent spacers (i.e., the spacer-to-spacer distance) is selected such that, for a given characteristic of the plate and the sample, in the closed configuration of the plate, in some embodiments, the sample thickness between two adjacent spacers varies by at most 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or within any range of values; or in some embodiments, at most 80%, 100%, 200%, 400%, or within any two of these values.

[0558] Clearly, in order to maintain a given sample thickness variation between two adjacent spacers, closer spacer spacing is required when using more flexible plates.

[0559] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are columns with a height of 2 to 4 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 1 μm to 100 μm.

[0560] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are columns with a height of 2 to 4 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 100 μm to 250 μm.

[0561] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are columns with a height of 4 to 50 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 1 μm to 100 μm.

[0562] In a preferred embodiment, the spacers are a periodic square array, wherein the spacers are columns with a height of 4 to 50 μm, an average lateral dimension of 5 to 20 μm, and a spacer spacing of 100 μm to 250 μm.

[0563] The period of the spacer array is 1 nm to 100 nm in one preferred embodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to 1000 nm in a separate preferred embodiment, 1 μm (i.e., 1000 nm) to 2 μm in another preferred embodiment, 2 μm to 3 μm in one preferred embodiment, 3 μm to 5 μm in another preferred embodiment, 5 μm to 10 μm in one preferred embodiment, and 10 μm to 50 μm in another preferred embodiment, 50 μm to 100 μm in a separate preferred embodiment, 100 μm to 175 μm in a separate preferred embodiment, and 175 μm to 300 μm in a separate preferred embodiment.

[0564] Spacer density. The spacers are arranged on the plates with a surface density greater than 1 / μm², greater than 1 / 10μm², greater than 1 / 100μm², greater than 1 / 500μm², greater than 1 / 1000μm², greater than 1 / 5000μm², greater than 1 / 0.1mm², greater than 1 / 1mm², greater than 1 / 5mm², greater than 1 / 10mm², greater than 1 / 100mm², greater than 1 / 1000mm², greater than 1 / 10000mm², or greater than 1 / 10000mm², or within any two of these values.

[0565] These spacers are configured not to occupy a large surface area (volume) in a given sample region (volume);

[0566] The ratio of spacer volume to sample volume. In many embodiments, the ratio of spacer volume (i.e., the volume of the spacer) to sample volume (i.e., the volume of the sample), and / or the ratio of spacer volume to sample volume within a relevant volume of the sample, is controlled to achieve certain advantages. These advantages include, but are not limited to, uniformity of sample thickness control, uniformity of analytes, and sample flow characteristics (i.e., flow rate, flow direction, etc.).

[0567] In some embodiments, the ratio of the spacer volume (r) to the sample volume, and / or the ratio of the spacer volume to the sample volume within the relevant volume of the sample, is less than 100%, at most 99%, at most 70%, at most 50%, at most 30%, at most 10%, at most 5%, at most 3%, at most 1%, at most 0.1%, at most 0.01%, at most 0.001%, or within any range of values.

[0568] Spacers fixed to the plate. The spacing and orientation of the spacers, which play a key role in this invention, are preferably maintained during the process of changing the plate from an open structure to a closed structure, and / or preferably predetermined before the process of changing from an open structure to a closed structure.

[0569] In some embodiments of the invention, the spacers are fixed to one of the plates before the plates are converted into a closed configuration. The term "spacer fixed to its corresponding plate" means that the spacer is attached to the plate and retains that attachment at least during the use of the plate. An example of "spacer fixed to its corresponding plate" is that the spacer is made integrally from a single piece of material of the plate, and the position of the spacer relative to the plate surface does not change. An example of "spacer not fixed to its corresponding plate" is that the spacer is bonded to the plate by adhesive, but during the use of the plate, the adhesive cannot hold the spacer in its original position on the plate surface (i.e., the spacer moves away from its original position on the plate surface).

[0570] In some embodiments, at least one spacer is fixed to its respective plate. In some embodiments, two spacers are fixed to their respective plates. In some embodiments, most spacers are fixed with their respective plates. In some embodiments, all spacers are fixed to their respective plates.

[0571] In some embodiments, the spacer is integrally fixed to the plate.

[0572] In some embodiments, the spacer is fixed to its respective plate by one or any combination of the following methods and / or constructions: attaching, joining, fusing, embossing, and etching.

[0573] The term "embossing" refers to the process of fixing spacers and a board integrally by embossing (i.e., printing) a piece of material to form spacers on the surface of the board. The material can be a single layer or multiple layers.

[0574] The term "etching" refers to the process of fixing spacers and a plate together by etching a piece of material to form spacers on the surface of the plate. The material can be a single layer or multiple layers.

[0575] The term "fusion" refers to the process of fixing a spacer and a plate together as a whole by attaching them together, so that the original materials of the spacer and the plate are fused together and a clear material boundary exists between the two materials after fusion.

[0576] The term "bonded to" refers to the integral fixation of spacers and plates by adhesive bonding.

[0577] The term "attached to" refers to the connection of spacers and plates together.

[0578] In some embodiments, the spacer and the plate are made of the same material. In other embodiments, the spacer and the plate are made of different materials. In another embodiment, the spacer and the plate are formed integrally. In yet another embodiment, one end of the spacer is fixed to its respective plate, while the other end is open to accommodate different configurations of the two plates.

[0579] In another embodiment, each spacer is independently attached to, bonded to, fused to, imprinted to, and etched into the corresponding plate at least once. The term "independently" means securing a spacer to its corresponding plate by the same or different methods, selected from those methods of attaching, bonding to, fusing to, imprinting to, and etching into the corresponding plate.

[0580] In some embodiments, at least a distance between two spacers is predetermined (“predetermined spacer spacing” means that the distance is known when the user uses the board).

[0581] In some embodiments of all the methods and apparatuses described herein, additional spacers exist in addition to the fixed spacers.

[0582] In a preferred embodiment, spacers are integrally manufactured on a plate by using a die-imprinting (e.g., nanoimprinting) of a thin plastic film, and the spacers are made of the same material, and the plate thickness is 50 μm to 500 μm.

[0583] In a preferred embodiment, spacers are integrally manufactured on a plate by using a die-imprinting (e.g., nanoimprinting) of a thin plastic film, and the spacers are made of the same material, and the plate thickness is 50 μm to 250 μm.

[0584] In a preferred embodiment, the spacer is integrally manufactured on the plate and made of the same material, and the plate has a thickness of 50 μm to 500 μm.

[0585] In a preferred embodiment, the spacer is integrally formed into a thin plastic film on a plate using a mold, and is made of the same material, and the plate has a thickness of 50 μm to 250 μm.

[0586] In a preferred embodiment, spacers are integrally manufactured on a plate by using a die-stamping (e.g., nano-imprinting) of a thin plastic film, and the spacers are made of the same material, wherein the plastic film is PMMA (polymethyl methacrylate) of PS (polystyrene).

[0587] In a preferred embodiment, spacers are integrally manufactured on a plate by using a die-stamping (e.g., nano-imprinting) of a thin plastic film, and the spacers are made of the same material, wherein the plastic film is PS (polystyrene) in the form of PMMA (polymethyl methacrylate), and the plate thickness is from 50 μm to 500 μm.

[0588] In a preferred embodiment, spacers are integrally manufactured on a plate by using a die-stamping (e.g., nano-imprinting) of a thin plastic film, and the spacers are made of the same material, wherein the plastic film is PS (polystyrene) in the form of PMMA (polymethyl methacrylate), and the plate thickness is from 50 μm to 250 μm.

[0589] In a preferred embodiment, the spacers are integrally manufactured on a plate by using a die-stamping (e.g., nano-imprinting) of a thin plastic film and are made of the same material, wherein the plastic film is PS (polystyrene) of PMMA (polymethyl methacrylate), and the spacers have a square or rectangular shape and have the same spacer height.

[0590] In a preferred embodiment, the spacer has a square or rectangular shape (with or without rounded corners).

[0591] In a preferred embodiment, the spacer has a column width (spacer width in each lateral direction) between 1 μm and 200 μm; a column period (i.e., spacer period) between 2 μm and 2000 μm; and a column height (i.e., spacer height) between 1 μm and 100 μm, which are square or rectangular columns.

[0592] In a preferred embodiment, the spacer made of PMMA or PS has a column width (spacer width in each lateral direction) between 1 μm and 200 μm; a column period (i.e., spacer period) of 2 μm to 2000 μm; and a column height (i.e., spacer height) of 1 μm to 100 μm, which are square or rectangular columns.

[0593] In a preferred embodiment, the spacer is integrally manufactured on a plate and made of plastic material, and the spacer has a column width (spacer width in each transverse direction) between 1 μm and 200 μm; a column period (i.e., spacer period) of 2 μm to 2000 μm; and a column height (i.e., spacer height) of 1 μm to 100 μm, which are square or rectangular columns.

[0594] In a preferred embodiment, the spacers are integrally manufactured on a plate and made of the same material, and the spacers have a column width (spacer width in each transverse direction) between 1 μm and 200 μm; a column period (i.e., spacer period) of 2 μm to 2000 μm; and a column height (i.e., spacer height) of 1 μm to 10 μm, which are square or rectangular columns.

[0595] In a preferred embodiment, the spacer is integrally manufactured on a plate and made of the same material selected from PS or PMMA or other plastics, and the spacer has a column width (spacer width in each transverse direction) between 1 μm and 200 μm; a column period (i.e., spacer period) of 2 μm to 2000 μm; and a column height (i.e., spacer height) of 10 μm to 50 μm, which are square or rectangular columns.

[0596] Specific sample thickness. In this invention, it has been observed that a larger plate holding force (i.e., the force that holds two plates together) can be achieved by using a smaller plate spacing (for a given sample area) or a larger sample area (for a given plate spacing) or both.

[0597] In some embodiments, at least one plate is transparent in the region surrounding the relevant area, each plate has an inner surface configured to contact the sample in a closed configuration; in the closed configuration, the inner surfaces of the plates are substantially parallel to each other; the inner surfaces of the plates are substantially planar except at locations with spacers; or any combination thereof.

[0598] Final sample thickness and uniformity. In some embodiments, a distinctly flat ratio relative to the final sample thickness is determined, and depending on the embodiment and application, the ratio to the sample thickness is less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, or less than 10%, or within any two of these values.

[0599] In some embodiments, the flatness relative to the sample thickness is less than 0.01%, 0.1%, less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 50%, less than 70%, less than 80%, less than 100%, or within any two of these values.

[0600] In some embodiments, apparent flatness may refer to a surface flatness variation (measured from the average thickness) of less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, or less than 10%, or within any two of these values. Typically, flatness relative to plate thickness may be less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 50%, or less than 100%, or within any two of these values.

[0601] The height of the spacer is selected by adjusting the required spacing between the plates and / or adjusting the final sample thickness relative to the remaining sample thickness. The spacer height (predetermined spacer height), the spacing between the plates, and / or the sample thickness are 3 nm or less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 μm or less, 2 μm or less, 3 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less, 4 mm or less, or within any two of these values.

[0602] The spacer height, the spacing between plates, and / or the sample thickness are 1 nm to 100 nm in one preferred embodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to 1000 nm in a separate preferred embodiment, 1 μm (i.e., 1000 nm) to 2 μm in another preferred embodiment, 2 μm to 3 μm in a separate preferred embodiment, 3 μm to 5 μm in another preferred embodiment, 5 μm to 10 μm in a separate preferred embodiment, and 10 μm to 50 μm in another preferred embodiment, and 50 μm to 100 μm in a separate preferred embodiment.

[0603] In some embodiments, the spacers may be spherical beads and randomly distributed in the sample.

[0604] In some embodiments, the QMAX device is fully transparent or partially transparent to reduce heat absorption by the card itself, wherein transparency is defined as greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or within any two of these values.

[0605] In some embodiments, the QMAX device is partially reflective to reduce heat absorption by the card itself. The reflectivity of the surface is greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or within any two values.

[0606] In some embodiments, the QMAX device and fixture are coated with a thermal insulation layer to reduce heat absorption by the card itself. This thermal insulation layer comprises a material including the aforementioned low thermal conductivity material.

[0607] In some embodiments, the clamp covers and seals all QMAX cards in a closed configuration.

[0608] In some embodiments, the clamp in the closed configuration only covers and seals the periphery of the QMAX card.

[0609] In some embodiments, the clamp in the closed configuration only covers and seals the periphery of the QMAX card, without covering and sealing the heating and cooling area.

[0610] In some embodiments, the clamp covers some surfaces of the QMAX card in a closed configuration.

[0611] In some embodiments, the clamp has a transparent window to allow light to enter and exit the QMAX card.

[0612] In some embodiments, the clamp is completely transparent to allow light to enter and exit the QMAX card.

[0613] The transparency of the fixture refers to a value higher than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or within any two values.

[0614] In some embodiments, air or liquid exists between the clamp in the closed configuration and the QMAX device. In some embodiments, the liquid includes, but is not limited to, water, ethane, methane, oil, benzene, hexane, heptane, silicone oil, polychlorinated biphenyls, liquid air, liquid oxygen, liquid nitrogen, etc.

[0615] In some embodiments, after the clamp is closed, the pressure applied by the clamp to the surface of the QMAX card is 0.01 kg / cm², 0.1 kg / cm², 0.5 kg / cm², 1 kg / cm², 2 kg / cm², 5 kg / cm², 10 kg / cm², 20 kg / cm², 30 kg / cm², 40 kg / cm², 50 kg / cm², 60 kg / cm², 100 kg / cm², 150 kg / cm², 200 kg / cm², 500 kg / cm², or within any two values; and a preferred range of 0.1 kg / cm² to 0.5 kg / cm², 0.5 kg / cm² to 1 kg / cm², 1 kg / cm² to 5 kg / cm², 5 kg / cm² to 10 kg / cm² (pressure).

[0616] In some embodiments, after the clamp is closed, the pressure applied by the clamp to the surface of the QMAX card is at least 0.01 kg / cm², 0.1 kg / cm², 0.5 kg / cm², 1 kg / cm², 2 kg / cm², 5 kg / cm², 10 kg / cm², 20 kg / cm², 30 kg / cm², 40 kg / cm², 50 kg / cm², 60 kg / cm², 100 kg / cm², 150 kg / cm², 200 kg / cm², or 500 kg / cm².

[0617] like Figure 2A and Figure 2B As shown in the cross-sectional view of the device, the heating / cooling layer 112 spans the sample contact area. However, it should be noted that the lateral area of ​​the heating / cooling layer may also occupy only a portion of the sample contact area, in percentages of approximately 1% or more, 5% or more, 10% or more, 20% or more, 50% or more, 80% or more, 90% or more, 95% or more, 99% or more, 85% or less, 75% or less, 55% or less, 40% or less, 25% or less, 8% or less, or 2.5% or less. In some embodiments, to facilitate temperature change of the sample, in some embodiments, the lateral area of ​​the heating / cooling layer is configured such that the sample 90 receives thermal radiation from the heating / cooling layer 112, which substantially uniformly spans the lateral dimension of the sample 90 over the sample contact area.

[0618] In some embodiments, the radiation absorption area is 10%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the total plate area, or within any two values.

[0619] In some embodiments, the thickness of the heating / cooling layer 112 is 10 nm or greater, 20 nm or greater, 50 nm or greater, 100 nm or greater, 200 nm or greater, 500 nm or greater, 1 μm or greater, 2 μm or greater, 5 μm or greater, 10 μm or greater, 20 μm or greater, 50 μm or greater, 100 μm or greater, 75 μm or less, 40 μm or less, 15 μm or less, 7.5 μm or less, 4 μm or less, 1.5 μm or less, 750 μm or less, 400 nm or less, 150 nm or less, 75 nm or less, 40 nm or less, or 15 nm or less, or within any two values. In some embodiments, the thickness of the heating / cooling layer 112 is 100 nm or less.

[0620] In some embodiments, the area of ​​the sample layer and the heating / cooling layer 112 is substantially greater than the uniform thickness. Here, the term "substantially greater than" means that the uniform diameter or diagonal distance of the sample layer and / or the heating / cooling layer is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 times the uniform thickness. 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1500 times, 2000 times, 2500 times, 3000 times, 3500 times, 4000 times, 4500 times, or 5000 times, or within any two values.

[0621] Figure 11A and 11B An exemplary embodiment of the first plate and the heating / cooling layer of the present invention is shown. Figure 11A It is a top view, and Figure 11B It is a cross-sectional view. Figure 12A and 12B Cross-sectional views of two exemplary embodiments of the invention are shown, illustrating a first plate, a second plate, and a heating / cooling layer. As a whole, the first and second plates, and optionally the heating / cooling layer, can be considered as sample holders, relating not only to the embodiments shown and / or described herein, but also to other embodiments capable of compressing at least a portion of a liquid sample into a layer of uniform thickness.

[0622] like Figure 11A and 11B As shown, in some embodiments, the heating / cooling layer is in contact with the first plate. However, it should be noted that in some embodiments, the heating / cooling layer may be in contact with the second plate 20. Additionally, in some embodiments, the heating / cooling layer is not in contact with any plate. In some embodiments, there is no separate structure for the heating / cooling layer; the first plate and / or the second plate 20 and / or the sample itself may absorb electromagnetic radiation, allowing the sample temperature to rise.

[0623] In some embodiments, the area of ​​the heating / cooling layer is less than 1000 mm², 900 mm², 800 mm², 700 mm², 600 mm², 500 mm², 400 mm², 300 mm², 200 mm², 100 mm², 90 mm², 80 mm², 75 mm², 70 mm², 60 mm², 50 mm², 40 mm², 30 mm², 25 mm², 20 mm², 10 mm², 5 mm², 2 mm², 1 mm², 0.5 mm², 0.2 mm², 0.1 mm², or 0.01 mm², or within any two values. In some embodiments, the heating / cooling layer has an area substantially smaller than that of the first plate (and / or the second plate). For example, in some embodiments, the percentage of the area of ​​the heating / cooling layer that occupies only a portion of the area of ​​the first plate (or the second plate; or the sample contact area of ​​the first or second plate) is about 1% or more, 5% or more, 10% or more, 20% or more, 50% or more, 80% or more, 90% or more, 95% or more, 99% or more, 85% or less, 75% or less, 55% or less, 40% or less, 25% or less, 8% or less, or 2.5% or less.

[0624] In some embodiments, the heating / cooling layer has a substantially uniform thickness. In some embodiments, the thickness of the heating / cooling layer is less than 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, or 10 mm, or within the range of any two values.

[0625] The heating / cooling layer can take any shape. For example, from a top view, the heating / cooling layer can be square, circular, elliptical, triangular, rectangular, parallelogram, trapezoidal, pentagonal, hexagonal, octagonal, polygonal, or various other shapes.

[0626] In some embodiments, the thickness of the first or second plate is 2 nm or less, 10 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 1000 nm or less, 2 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 10 mm or less, 20 mm or less, 50 mm or less, 100 mm or less, 500 mm or less, or less, or within any two of these values.

[0627] In some embodiments, the lateral area of ​​the first plate and the second plate is 1 mm² or less, 10 mm² or less, 25 mm² or less, 50 mm² or less, 75 mm² or less, 1 cm² or less, 2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm² or less, 10 cm² or less, 100 cm² or less, 500 cm² or less, 1000 cm² or less, 5000 cm² or less, 10,000 cm² or less, 10,000 cm² or less, or within any two of these values.

[0628] In some embodiments, the spacer spacing (ISD) divided by the plate thickness (h) and the fourth power of Young's modulus (E) (ISD4 / (hE)) is 5 × 106 μm3 / GPa or less;

[0629] In some embodiments, the product of the column contact fill factor and the Young's modulus of the spacer is 2 MPa or greater, wherein the column contact fill factor is the ratio of the area of ​​the plate being contacted by the column to the total area of ​​the plate (in the column region).

[0630] In some embodiments, the spacers have a predetermined substantially uniform height and a predetermined constant spacer spacing that is at least about twice the size of the analyte and at most 200 μm, and at least one of the spacers is located within the sample contact area.

[0631] In some embodiments, the plates (first plate, second plate, or both plates) having the heating / cooling layer are thin, allowing for rapid temperature changes in the sample. For example, in some embodiments, the thickness of the plate in contact with the heating / cooling layer is equal to or less than 500 μm, 200 μm, 100 μm, 50 μm, 25 μm, 10 μm, 5 μm, 2.5 μm, 1 μm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or a range between any two values. In some embodiments, if only one plate is in contact with the heating / cooling layer, the plate in contact with the heating / cooling layer is substantially thinner than the plate not in contact with the heating / cooling layer. For example, in some embodiments, the thickness of the plate in contact with the heating / cooling layer is less than 1 / 1,000,000, 1 / 500,000, 1 / 100,000, 1 / 50,000, 1 / 10,000, 1 / 5,000, 1 / 1,000, 1 / 500, 1 / 100, 1 / 50, 1 / 10, 1 / 5, or 1 / 2 of the thickness of the plate in contact with the heating / cooling layer, or within a range between any two values.

[0632] In some embodiments, the sample layer is thin, allowing for rapid temperature changes. In some embodiments, the thickness of the sample layer is equal to or less than 100 μm, 50 μm, 25 μm, 10 μm, 5 μm, 2.5 μm, 1 μm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm, or within any two values.

[0633] In various embodiments, the positioning of the heating / cooling layer can also vary. In some embodiments, such as Figure 12A As shown in 12B, the heating / cooling layer is located on the inner surface of the first plate. Here, the inner surface is defined as the surface in contact with the sample when the sample is compressed into a layer. The other surface is the outer surface. In some embodiments, the heating / cooling layer is located on the inner surface of the first plate. In some embodiments, the heating / cooling layer is located on the inner surface of the second plate. In some embodiments, the heating / cooling layer is located on the outer surface of the first plate. In some embodiments, the heating / cooling layer is located inside one or both plates. In some embodiments, the heating / cooling layer is located on the outer surface of the second plate. In some embodiments, at least two heating / cooling layers are present on the inner and / or outer surfaces of the first and / or second plates.

[0634] As shown and described herein, in some embodiments, the sample holder is configured to compress the fluid sample into a thin layer, thereby reducing the thermal mass of the sample. However, if the thermal mass is reduced, a small amount of energy can rapidly change the temperature of the sample. Furthermore, by limiting the sample thickness, thermal conduction is also restricted.

[0635] In some embodiments, sample contact areas exist on the respective surfaces of the first plate 10 and the second plate 20. The sample contact areas can be any portion of the surfaces of the first plate 10 and / or the second plate 20. In some embodiments, the heating / cooling layer at least partially overlaps with the sample contact areas. In the overlapping portion, the sample is rapidly heated due to their proximity and small thermal mass.

[0636] In some embodiments, the sample holder 100 is a squeeze-regulated open flow (CROF, also known as QMAX) device, such as, but not limited to, the CROF devices described in the following patent applications: U.S. Provisional Patent Application No. 62 / 202,989, filed August 10, 2015; U.S. Provisional Patent Application No. 62 / 218,455, filed September 14, 2015; U.S. Provisional Patent Application No. 62 / 293,188, filed February 9, 2016; U.S. Provisional Patent Application No. 62 / 305,123, filed March 8, 2016; and U.S. Provisional Patent Application No. 62 / 369, filed July 31, 2016. The entire disclosures of U.S. Provisional Patent Application No. 62 / 394,753, filed September 15, 2016; PCT Application No. PCT / US2016 / 045437, filed August 10, 2016; PCT Application No. PCT / US2016 / 051775, filed September 14, 2016; PCT Application No. PCT / US2016 / 051794, filed September 15, 2016; and PCT / US2016 / 054025, filed September 27, 2016, are incorporated herein by reference for all purposes.

[0637] Sealing to reduce sample evaporation

[0638] When two plates clamp the sample in a shape with a large lateral / vertical ratio (e.g., 15 mm to 30 μm = 500), the evaporation of the sample during thermal cycling is greatly reduced because the sample surface covered by the two plates is 500 times larger. Experiments showed no visible change in sample volume over 30 temperature cycles (approximately 60 seconds).

[0639] In another aspect, in some embodiments, a sealing element is present that contacts the two plates to form a closed chamber preventing sample vapor from escaping. Besides reducing or eliminating sample evaporation, such a sealing element can also reduce sample contamination. The sealing element can be a strip, a plastic seal, an oil seal, or a combination thereof.

[0640] In some embodiments, the sealing element does not reach the sample, but it contacts both plates to form a closed chamber that prevents sample vapor from escaping. In some embodiments, the sealing element may be used as a spacer to adjust the thickness of the relevant sample.

[0641] In some embodiments, such as Figure 7 As shown, the sample holder 100 includes a sealing element 30 configured to seal the gap 102 between the first plate 10 and the second plate 20 outside the media contact area in a closed configuration. In some embodiments, the sealing element 30 encloses the sample 90 within a region (e.g., a sample receiving region) such that the entire lateral area of ​​the sample 90 is clearly defined and measurable. In some embodiments, the sealing element 30 improves the uniformity of the sample 90, particularly the thickness of the sample layer.

[0642] In some embodiments, such as Figure 7 As shown, the sealing element 30 includes an adhesive applied between the first plate 10 and the second plate 20 in a closed configuration. The adhesive is selected from materials such as, but not limited to: starch, dextrin, gelatin, tar, bitumen, polyisoprene natural rubber, resin, shellac, cellulose and its derivatives, vinyl derivatives, acrylic derivatives, reactive acrylics, polychloroprene, styrene-butadiene, styrene-diene-styrene, polyisobutylene, acrylonitrile-butadiene, polyurethane, polysulfide, silicone, aldehyde condensation resin, epoxy resin, amino resin, polyester resin, polyolefin polymer, soluble silicate, phosphate cement, or any other adhesive material, or any combination thereof. In some embodiments, the adhesive is a dry adhesive, pressure-sensitive adhesive, contact adhesive, heat-sensitive adhesive, or a one-component or multi-component reactive adhesive, or any combination thereof. In some embodiments, the adhesive is a natural adhesive or a synthetic adhesive, or from any other source, or any combination thereof. In some embodiments, the adhesive is spontaneously curing, heat-curing, UV-curing, or cured by any other treatment, or any combination thereof.

[0643] In some embodiments, such as Figure 7 As shown, the sealing element 30 includes a closed spacer (hole). For example, in a top view, the closed spacer has a circular shape (or any other closed shape) and surrounds the sample 90, essentially confining the sample 90 together with the first plate 10 and the second plate 20. In some embodiments, the closed spacer (hole) also serves as a spacer mechanism 40. In such embodiments, the closed spacer seals the lateral boundaries of the sample 90 and adjusts the thickness of the sample layer.

[0644] In some embodiments, during heating, an "anti-evaporation ring" is present outside the liquid area (e.g., the sample area) to prevent or reduce the escape of liquid vapor from the plate.

[0645] In some embodiments, a clamp is present on the outside of the QMAX card to secure the QMAX card to its closed configuration during heating.

[0646] In some embodiments, the two plates are pressed with imprecise pressure, neither set to a precise level nor set to be substantially uniform. In some embodiments, the two plates are pressed directly by hand.

[0647] In some embodiments, the QMAX card / RHC, which includes plates and spacers, is made of a material with low thermal conductivity to reduce heat absorption by the card itself.

[0648] In some embodiments, a clamp is present on the outside of the QMAX card to hold the QMAX card in its closed configuration during heating (i.e., the clamp surrounds only the edge of the plate, not the center of the plate pair). This clamp is also made of a material with low thermal conductivity to reduce heat absorption by the card itself.

[0649] Heating source, additional radiator, temperature sensor and temperature control

[0650] The heating layer or heating / cooling layer in the RHC card is configured to be heated by a heating source, wherein the heating source transfers thermal energy to the heating / cooling layer through light, electricity, radio frequency (RF) radiation, or a combination thereof.

[0651] Optical heating source. In some embodiments, when the heating layer is optically heated by a heating source, the heating source includes a light source, including but not limited to an LED (light-emitting diode), a laser, a lamp, or a combination thereof.

[0652] In order to allow more light to reach the heating layer from the light source in the optical heating source, some embodiments of the heating source use optical lenses, light guides, or combinations thereof.

[0653] In some embodiments, the wavelength of the electromagnetic wave is 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1μm, 10μm, 25μm, 50μm, 75μm, or 100μm, or within any two values. In some embodiments, the wavelength of the electromagnetic wave is 100nm to 300nm, 400nm to 700nm (visible range), 700nm to 1000nm (IR range), 1μm to 10μm, 10μm to 100μm, or within any two values.

[0654] The lens's NA (numerical aperture) is 0.001, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.5, or within any two values.

[0655] In a preferred embodiment, the NA of the lens is 0.01 to 0.1, 0.1 to 0.4, 0.4 to 0.7, 0.7 to 1.0, or 1.0 to 1.5.

[0657] In some embodiments, the light guide (also known as an optical collimator) that collimates the light source into the heating zone / plate comprises a hollow tube with reflective walls.

[0658] One embodiment of a light guide tube comprises a hollow dielectric tube having reflective walls (i.e., its inner wall, outer wall, or both are reflective). The hollow dielectric tube may be made of glass, plastic, or a combination thereof. The reflective walls may be a thin light-reflective coating on the wall of the hollow tube. The reflective coating may be a thin metal film, such as gold, aluminum, silver, copper, or any mixture or combination thereof. Figure 17 A perspective view of an embodiment of a light guide tube is shown, comprising a hollow tube and a reflective material coated on the outer wall of the tube. The reflective coating may also be in the inner wall. The reflective wall may also be made of a multilayer interference material that reflects associated light. The light guide tube may be a block of material with a hollow tube and reflective walls.

[0659] In some embodiments, the hollow tube has a length in the range of 1 mm to 70 mm, an internal dimension (diameter or width) in the range of 1 mm to 40 mm, and a wall thickness in the range of 0.01 mm to 10 mm.

[0660] In some preferred embodiments, the inner diameter (or average width) of the hollow tube used for the light guide is in the range of 1 mm to 5 mm, 5 mm to 10 mm, 10 mm to 15 mm, 15 mm to 20 mm, 20 mm to 30 mm, or 30 mm to 50 mm.

[0661] In some preferred embodiments, the wall thickness (or average width) of the hollow tube used for the light guide is in the range of 0.001 mm to 0.01 mm, 0.01 mm to 0.1 mm, 0.1 mm to 0.5 mm, 0.5 mm to 1 mm, 1 mm to 2 mm, or 2 mm to 50 mm.

[0662] Electric heating source. In some embodiments, when the heating layer or heating / cooling layer is electrically heated by the heating source, the electric heating source includes a power source that transmits electrical energy to the heating / cooling layer via wires.

[0663] Additional heat sink. In some embodiments, heat is removed from the sample and sample holder into the environment, but in some embodiments, an additional heat sink will be used to accelerate heat removal. The additional heat sink may be a Peltier cooler, a passive heat sink, or both. In some embodiments, a fan will be used to generate air convection to accelerate sample cooling (directly to the sample and sample holder, directly to the additional heat sink, or both).

[0664] Figure 6A and 6B Perspective views and cross-sectional views of several embodiments of a thermal cycling system are also shown, which includes a sample holder 100 in a closed position and a thermal control unit 200. The sample holder 100 may include a first plate 10, a second plate 20, and a spacer mechanism (not shown). The thermal control unit 200 may include a heating source 202 and a controller 204.

[0665] like Figure 6B As shown, the thermal control unit 200 may include a heating source 202 and a controller 204. In some embodiments, the thermal control unit 200 provides energy in the form of electromagnetic waves for changing the temperature of the sample.

[0666] Refer to two Figure 6A and 6B The heating source 202 is configured to project electromagnetic waves 210 onto the heating / cooling layer 112 of the sample holder 100. The heating / cooling layer 112 is configured to absorb the electromagnetic waves 210 and convert most of them into heat to generate thermal radiation, which raises the temperature of the portion of the sample 90 closest to the heating / cooling layer 112. In other words, the coupling between the heating source 202 and the heating / cooling layer 112 is configured to provide the thermal energy required to promote a temperature change in the sample 90.

[0667] In some embodiments, radiation from the heating source 202 includes radio waves, microwaves, infrared waves, visible light, ultraviolet light, X-rays, gamma rays, or thermal radiation, or any combination thereof. In some embodiments, the heating / cooling layer 112 has a preferred wavelength range in which its absorption efficiency is high. In some embodiments, the heating source 202 is configured to project electromagnetic waves within, overlap with, or cover the preferred wavelength range of the heating / cooling layer 112. In other embodiments, to facilitate temperature change, the wavelength is rationally designed to be far from the preferred wavelength of the heating / cooling layer.

[0668] In some embodiments, the heating source 202 includes a laser source that provides laser light within a narrow wavelength range. In other embodiments, the heating source 202 includes one of an LEDs (light-emitting diodes).

[0669] Temperature sensor. The temperature of the sample can be controlled by delivering pre-calibrated energy to the heating zone / layer using a real-time temperature sensor, or by using a real-time temperature sensor, or both.

[0670] Real-time temperature sensors can be thermometers, thermocouples, radiation temperature sensors, temperature-sensitive dyes (which change light intensity or color or both with temperature), or combinations thereof.

[0671] like Figure 7 As shown, in some embodiments, the thermal control unit 200 includes a thermometer 206. In some embodiments, the thermometer 206 provides a monitoring and / or feedback mechanism to control / monitor / regulate the temperature of the sample 90. For example, in some embodiments, the thermometer 206 is configured to measure the temperature at or near the sample contact area. In some embodiments, the thermometer 206 is configured to directly measure the temperature of the sample 90. In some embodiments, the thermometer 206 is selected from fiber optic thermometers, infrared thermometers, liquid crystal thermometers, pyrometers, quartz thermometers, silicon bandgap temperature sensors, temperature bars, thermistors, and thermocouples. In some embodiments, the thermometer 206 is an infrared thermometer.

[0672] In some embodiments, thermometer 206 is configured to send a signal to controller 204. This signal contains information related to the temperature of sample 90, causing controller 204 to make corresponding changes. For example, in a PCR process, for the denaturation step, a target temperature is set to 95°C; after measurement, the thermometer sends a signal to controller 204 indicating that the measured temperature of sample 90 is actually 94.8°C; controller 204 then changes the output of heating source 202, which projects electromagnetic waves or adjusts specific parameters (e.g., intensity or frequency) of existing electromagnetic waves, causing the temperature of sample 90 to increase to 95°C. This measurement signal modulation cycle is applied to any step in any reaction / assay.

[0673] Controller. Referring to Figures (A) and (B) of Figure 4, controller 204 is configured to control electromagnetic waves 210 projected by heating source 202 for temperature changes of the sample. Parameters of the electromagnetic waves 210 controlled by controller 204 include, but are not limited to, presence, intensity, wavelength, incident angle, and any combination thereof. In some embodiments, the controller is manually operated; for example, it simply controls the on and off of the heating source, as easily as a manual switch, thereby controlling the presence or absence of electromagnetic waves projected by the heating source. In other embodiments, the controller includes hardware and software configured to automatically control the electromagnetic waves according to one or more predetermined programs.

[0674] In some embodiments, the predetermined program refers to a time schedule, wherein parameters of the electromagnetic wave 210 (e.g., presence, intensity, and / or wavelength) are set to predetermined levels for corresponding predetermined time periods. In other embodiments, the predetermined program refers to a time schedule, wherein the temperature of the sample 90 is set to predetermined levels for corresponding predetermined time periods, and time periods for the sample temperature to change from one predetermined level to another are also set separately. In some embodiments, the controller 204 is configured to be programmable, meaning that the controller 204 includes hardware and software configured to receive and execute predetermined programs of the system provided by the system operator.

[0675] Figure 7 A cross-sectional view of an embodiment of the invention is shown, illustrating a thermal cycler system and illustrating additional components that facilitate temperature change and control. Figure 7 As shown, the thermal cycler system includes a sample holder 100 and a thermal control unit 200. The sample holder 100 includes a first plate 10, a second plate 20, a spacer mechanism 40, and a sealing element 30; the thermal control unit 200 includes a heating source 202, a controller 204, a thermometer 206, and an expander 208.

[0676] Figure 7 A sample holder 100 in a closed configuration is shown, wherein the inner surfaces 11 and 21 of a first plate 10 and a second plate 20 face each other, and the spacing 102 between the two plates is adjusted by a spacer mechanism 40. If a sample 90 has already been deposited on one or both plates in the open configuration, when switching to the closed configuration, pressing the first plate 10 and the second plate 20 with a hand or other mechanism compresses the sample 90 into a thin layer by the two plates. In some embodiments, the thickness of the layer is uniform and the same as the spacing 102 between the two plates. In some embodiments, the spacing 102 (and therefore also the thickness of the sample layer) is adjusted by the spacer mechanism 40. In some embodiments, the spacer mechanism includes closed spacers fixed to one of the plates. In some embodiments, the spacer mechanism 40 includes a plurality of columnar spacers fixed to one or both plates. Here, the term "fixed" means that the spacers are attached to the plates and remain attached at least during the use of the plates.

[0677] In some embodiments, controller 204 is configured to adjust the temperature of the sample according to a predetermined procedure to facilitate a determination and / or reaction involving sample 90. In some embodiments, the determination and / or reaction is PCR. In some embodiments, controller 204 is configured to control the presence, intensity, and / or frequency of electromagnetic waves from heating source 206.

[0678] Sample signal monitoring

[0679] As shown in Figures 11 and 12, the signal sensor can be used to detect signals from the sample in the sample holder (as well as reaction products during temperature changes).

[0680] In some embodiments, the signal sensor is an optical sensor configured to image a fluid sample. For example, the optical sensor is a photodetector, a camera, or a device capable of capturing images of the fluid sample. In some embodiments, the optical sensor may be a camera. In some embodiments, the camera is a camera integrated into a mobile device (e.g., a smartphone or tablet). In some embodiments, the camera is separate from other parts of the system. In some embodiments, one or more light sources are used to excite the sample (and products from the reaction during temperature changes) to generate a signal.

[0681] In some embodiments, the signal sensor is an electrical sensor configured to detect electrical signals from the device. In some embodiments, the signal sensor is a mechanical sensor configured to detect mechanical signals from the device.

[0682] In some embodiments, the signal sensor is configured to monitor the amount of analyte in the sample. In some embodiments, the signal sensor is located outside the chamber and receives optical signals from the sample through an optical aperture on the chamber.

[0683] Base and System

[0684] In some embodiments, the device further includes a base (adapter) configured to receive a sample card, a heating source, a temperature sensor, a portion of a comprehensive temperature control system (in some embodiments including a smartphone), an additional heat sink (optionally), a fan (optionally), or a combination thereof. In some embodiments, the adapter includes a card slot into which a sample card can be inserted. In some embodiments, the sample card is stabilized and held in place without any movement after being fully inserted into the slot, or after reaching a predetermined position within the slot.

[0685] In some embodiments, the base (adapter) is configured to position the sample card and the sample within it within the field of view of an optical sensor (e.g., a camera) such that the sample can be imaged. In some embodiments, the camera is part of a mobile device (e.g., a smartphone). In some embodiments, the adapter includes a slider in a slot. In some embodiments, the sample card can be placed on the slider, which can slide into or out of the slot in the adapter. In some embodiments, the adapter includes a card support. In some embodiments, the sample card can be placed on the card support and does not need to be moved before imaging.

[0686] In some embodiments, the adapter is configured to connect to an optical sensor such that the optical sensor (e.g., a mobile device; for example, a smartphone) and the sample card are fixed. In some embodiments, the adapter may include a replaceable connecting member (as an example) that is directly attached to the mobile device. The connecting member can slide onto the mobile device and securely attach the adapter to the mobile device, optimally positioning the sample card to be imaged or for the detection and / or measurement of analytes. In some embodiments, the connecting member is replaceable, such that different connecting members can be used for different mobile devices.

[0687] In some embodiments, the adapter includes a radiation aperture that allows electromagnetic waves to pass through, either for heating or cooling the sample. In some embodiments, the adapter includes an optical aperture that allows imaging of the sample. In some embodiments, the adapter serves as a heating slot for a sample card. Figure 13 and 14 Other embodiments of the system are provided. Figure 13 A cross-sectional view of an exemplary embodiment of the present invention, demonstrating a system for rapidly changing sample temperature, is shown. Figure 13 The detailed components of a heating source according to one embodiment are shown.

[0688] like Figure 13 and Figure 14 As shown, in some embodiments, the system includes a sample holder and a heating source. In some embodiments, as described herein, the sample holder includes a first plate, a second plate, and / or a heating / cooling layer. The heating source emits electromagnetic waves that reach the sample and can be converted into heat to raise the sample temperature. In some embodiments, the conversion occurs through the heating / cooling layer. When there is no specific heating / cooling layer, the conversion occurs through other parts of the sample holder.

[0689] like Figure 13 and Figure 14 As shown, in some embodiments, the system includes a chamber that encloses a sample holder. In some embodiments, the chamber is... Figure 1 Examples of additional heat sinks are provided. In some embodiments, the chamber includes an optical aperture configured to allow imaging of the sample. In some embodiments, the chamber includes a radiation aperture configured to allow electromagnetic waves to pass from a heating source to the heating / cooling layer. In some embodiments, a window is located at the radiation aperture to allow electromagnetic waves to pass through. In some embodiments, a filter (e.g., a bandpass filter) is positioned at the optical aperture to allow the sample to be imaged in the sample holder.

[0690] In some embodiments, the chamber is used to absorb heat from a sample and / or a heating source. In some embodiments, the chamber comprises a metal housing. In some embodiments, the chamber comprises an outer layer. In some embodiments, the outer layer is black. In some embodiments, the outer layer is made of a ferrous metal. In some embodiments, the chamber comprises an inner layer. In some embodiments, the inner layer is made of a non-reflective material. In some embodiments, the inner layer is black. In some embodiments, the inner layer is made of a ferrous metal.

[0691] like Figure 13 and Figure 14 As shown, in some embodiments, the system includes an optical sensor configured to capture images of a fluid sample in a sample holder. In some embodiments, the system also includes a light source, which may be integrated with the optical sensor in some cases and may be spaced apart in others. In some embodiments, the light source is configured to provide excitation light that can reach the sample. In some embodiments, the sample may provide signal light that can be captured by the optical sensor for image capture.

[0692] like Figure 13 As shown, in some embodiments, the heating source comprises an LED or a laser diode. In some embodiments, the heating source also comprises a fiber optic coupler and an optical fiber that guides light from the LED / laser diode to the sample holder.

[0693] Figure 14 A cross-sectional view of an exemplary embodiment of the present invention, demonstrating a system for rapidly changing sample temperature, is shown. Figure 14 Detailed components of a heating source according to one embodiment are shown. Figure 14 As shown, in some embodiments, the heating source includes an LED or a laser diode. In some embodiments, the heating source also includes one or more focusing lenses that focus electromagnetic waves from the heating source onto the sample in the sample holder.

[0694] like Figure 7 As shown, the thermal control unit 200 includes a beam expander 208 configured to expand electromagnetic waves from the heating source 202 from a smaller diameter to a larger diameter. In some embodiments, the electromagnetic waves projected from the heating source 202 are sufficient to cover the entire sample contact area; however, in some embodiments, it is necessary to expand the coverage area of ​​the electromagnetic waves projected by the heating source 202 to generate expanded electromagnetic waves 210, providing a heat source for all sample contact areas. The beam expander 208 employs any known technology, including but not limited to the beam expanders described in U.S. Patent Nos. 4,545,677, 4,214,813, 4,127,828, and 4,016,504, and U.S. Patent Publications 2008 / 0297912 and 2010 / 0214659, the entire contents of which are incorporated herein by reference for all purposes.

[0695] smartphones

[0696] In some embodiments, the sample card is imaged by a mobile device. In some embodiments, the mobile device is a smartphone, which can be used as an example.

[0697] In some embodiments, the smartphone includes a camera for imaging samples in a sample card. In some embodiments, an adapter is used to hold the sample card, and the adapter is configured to attach to the smartphone such that the sample card (and the sample therein) can be placed within the camera's field of view.

[0698] In some embodiments, the smartphone can also be used as a control unit configured to control the device. For example, the smartphone can be used to control the heating and / or cooling of a sample card. In some embodiments, the smartphone is connected to a heating source and controls electromagnetic waves from the heating source. In some embodiments, the smartphone controls the presence, intensity, wavelength, frequency, and / or angle of the electromagnetic waves. In some embodiments, the smartphone receives temperature data from a thermometer that measures the sample temperature. In some embodiments, the smartphone controls the electromagnetic waves based on the temperature data.

[0699] In some embodiments, the smartphone can also be used as a data processing and communication device. For example, after imaging a sample, the image can be saved on the smartphone. In some embodiments, saving the image can be processed by software or an application on the smartphone. For example, the presence and / or quantity of an analyte can be inferred from the image by software or an application on the smartphone. In some embodiments, the processing results can be displayed on the smartphone screen. In some embodiments, the processing results can be sent to the user, for example, using email or other messaging software. In some embodiments, the processing results can be sent to a third party, such as a healthcare professional, who can further diagnose and / or process the data in additional steps. In some embodiments, an unprocessed image can be displayed and / or sent. In some embodiments, the image is displayed on the smartphone screen. In some embodiments, the image is sent to the user, for example, via email or other messaging software. In some embodiments, the image can be sent to a third party, such as a remote server, which can further process the image. In some embodiments, the results and / or images are compressed and / or encrypted before being sent.

[0700] RHC Card Usage

[0701] The RHC card in the instruction manual can be used as one step in a series of steps for testing the sample, or as one step in performing the entire test.

[0702] In some embodiments, the RHC card is used for a so-called "one-step assay," in which all reagents and samples for analysis are loaded onto the RHC card, subjected to thermal cycling or temperature changes, and the signal is observed during the thermal cycling or temperature changes.

[0703] Other embodiments

[0704] Example 1:

[0705] One embodiment includes the apparatus of embodiments SH-1 to SH-6, wherein the first plate and the second plate are flexible plastic films and / or thin glass films, each plate having a substantially uniform thickness of a value selected from the range of 1 μm to 25 μm.

[0706] The area of ​​each board ranges from 1 cm² to 16 cm².

[0707] The sample sandwiched between the two plates has a thickness of 40 μm or less.

[0708] The ratio of relevant samples to the whole sample (RE ratio) is 12% or less.

[0709] The cooling zone is at least nine times larger than the heating zone.

[0710] The thermal mass ratio of the sample to the non-sample is 2.2 or greater.

[0711] In some embodiments, the RHC does not have spacers, but in others it does.

[0712] The STC ratio is and the cooling zone contains a material layer with a thermal conductivity of 70 W / mK or higher and a thermal conductivity multiplied by its thickness.

[0713] Example 2:

[0714] For the embodiments of SH-1 to SH-x, they have the following parameters arranged for rapid thermal cycling.

[0715] The first and second plates are made of plastic or thin glass. The thickness of the first and second plates is 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, or within any two values.

[0716] The thickness of the sample between the two plates is 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, or within any two values.

[0717] The distance from the H / C layer to the sample is 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, or within any two values.

[0718] The ratio of the cooling zone area to the relevant sample area is 16, 9, 4, 2, or within any two values.

[0719] The ratio of the cooling zone area to the heating zone area is 16, 9, 4, 2, or within any two values.

[0720] The distance between the H / C layer and the heating source (e.g., LED) is 5 mm, 10 mm, 20 mm, 30 mm, or within any two values.

[0721] Example 3:

[0722] For the embodiments of SH-1 to SH-x, they have the following parameters arranged for rapid thermal cycling.

[0723] The first and second plates are made of plastic or thin glass. The thickness of the first plate is 10 μm, 25 μm, 50 μm, or within any two values; while the thickness of the second plate (the plate with a heating or cooling layer) is 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, or within any two values.

[0724] The thickness of the sample between the two plates is 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, or within any two values.

[0725] The distance between the H / C layer and the sample is 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, or any two values ​​in between.

[0726] The ratio of the cooling zone area to the relevant sample area is 16, 9, 4, 2, or within any two values.

[0727] The ratio of the cooling zone area to the heating zone area is 16, 9, 4, 2, or within any two values.

[0728] The distance between the H / C layer and the heating source (e.g., LED) is 5 mm, 10 mm, 20 mm, 30 mm, or within any two values.

[0729] Example 4:

[0730] For the embodiments of SH-1 to SH-x, they have the following parameters arranged for rapid thermal cycling.

[0731] The first and second plates are made of plastic or thin glass. The thickness of the first and second plates is 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 175 μm, 250 μm, or within any two values.

[0732] The thickness of the sample between the two plates is 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 175 μm, 250 μm, or within any two values.

[0733] The distance between the H / C layer and the sample is 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 25 μm, 50 μm, 100 μm, 175 μm, 250 μm, or any two values ​​in between.

[0734] The ratio of the cooling zone area to the relevant sample area is 100, 64, 16, 9, 4, 2, 1, 0.5, 0.1, or within any two values.

[0735] The ratio of the cooling zone area to the heating zone area is 100, 64, 16, 9, 4, 2, 1, 0.5, 0.1, or within any two values.

[0736] The distance between the H / C layer and the heating source (e.g., LED) is 500 μm, 1 mm, 3 mm, 5 mm, 10 mm, 20 mm, 30 mm, or within any two values.

[0737] Example 5:

[0738] For the embodiments of SH-1 to SH-5, they have the following parameters arranged for rapid thermal cycling.

[0739] The light guide calibrates the light from a light source (e.g., an LED) into the heating zone. The light stack comprises a structure with a hollow hole (e.g., a tube or milled hole structure) with reflective walls. The light stack has a lateral dimension of 1 mm to 8 mm and a length of 2 mm to 50 mm.

[0740] Example 6:

[0741] For the embodiments of SH-1 to SH-5, they have the following parameters arranged for rapid thermal cycling.

[0742] The first and second plates are made of plastic or thin glass. The thickness of the first and second plates is 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, or within any two values.

[0743] The thickness of the sample between the two plates is 1 to 5 μm, 5 μm to 10 μm, 10 to 30 μm, or 30 μm to 50 μm.

[0744] The distance from the H / C layer to the sample is in the range of 10nm to 100nm, 100nm to 500nm, 500nm to 1μm, 1μm to 5μm, 5μm to 10μm, or 10μm to 25μm.

[0745] The ratio of the cooling zone area to the relevant sample area is 16, 9, 4, 2, or within any two values.

[0746] The ratio of the cooling zone area to the heating zone area is 16, 9, 4, 2, or within any two values.

[0747] The distance between the H / C layer and the heating source (e.g., LED) is 5 mm, 10 mm, 20 mm, 30 mm, or within any two values.

[0748] The KC ratio of the cooling layer is in the range of 0.5 cm² / sec and 0.7 cm² / sec, 0.7 cm² / sec and 0.9 cm² / sec, 0.9 cm² / sec and 1 cm² / sec, 1 cm² / sec and 1.1 cm² / sec, 1.1 cm² / sec and 1.3 cm² / sec, 1.3 cm² / sec and 1.6 cm² / sec, 1.6 cm² / sec and 2 cm² / sec, or 2 cm² / sec and 3 cm² / sec.

[0749] The thermal mass ratio of the sample to the non-sample is in the range of 0.2 to 0.5, 0.5 to 0.7, 0.7 to 1, 1 to 1.5, 1.5 to 5, 5 to 10, 10 to 30, 30 to 50, or 50 to 100.

[0750] Example 7:

[0751] For the embodiments of SH-1 to SH-5 and Examples 1 to 6, they have the following parameters arranged for rapid thermal cycling:

[0752] The transverse area of ​​the first and / or second plates is 1 mm² to 10 mm², 10 mm² to 50 mm², 50 mm² to 100 mm², 1 cm² to 5 cm², 5 cm² to 20 cm², or 20 cm² to 50 cm².

[0753] The scaled thermal conductivity (STM ratio) is in the range of 10 to 20, 30 to 50, 50 to 70, 70 to 100, 100 to 1000, 1000 to 10000, or 10000 to 1000000; and the thermal conductivity of the cooling zone (layer) multiplied by its thickness is 6×10⁻⁵ W / K, 9×10⁻⁵ W / K, 1.2×10⁻⁴ W / K, 1.5×10⁻⁴ W / K, 1.8×10⁻⁴ W / K, 2.1×10⁻⁴ W / K, 2.7×10⁻⁴ W / K, 3×10⁻⁴ W / K, 1.5×10⁻⁴ W / K, or in any range between two values.

[0754] The sample holder (RHC card) has no significant thermal conduction to the environment during thermal cycling.

[0755] Sample type

[0756] The apparatus, systems, and methods disclosed herein can be used for samples, such as, but not limited to, diagnostic samples, clinical samples, environmental samples, and food samples. Sample types include, but are not limited to, those listed, described, and summarized in PCT application (designated U.S.) No. PCT / US2016 / 045437, filed August 10, 2016, and PCT application (designated U.S.) No. PCT / US0216 / 051775, filed September 14, 2016, the entire contents of which are incorporated herein by reference.

[0757] For example, in some embodiments, the apparatus, systems, and methods disclosed herein are used for samples comprising cells, tissues, body fluids, and / or mixtures thereof. In some embodiments, the sample comprises human body fluids. In some embodiments, the sample comprises at least one of cells, tissues, body fluids, feces, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, earwax, chyle, chyme, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled breath condensate.

[0758] In some embodiments, the apparatus, systems, and methods disclosed herein are used for environmental samples obtained from any suitable source, such as, but not limited to, rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, etc.; the solid samples are from soil, compost, sand, rock, concrete, wood, brick, sewage, etc.; and the gaseous samples are from air, underwater vents, industrial exhaust gases, vehicle exhaust gases, etc. In some embodiments, the environmental samples are fresh; in some embodiments, the environmental samples are processed. For example, samples in non-liquid form are converted into liquid form before applying the apparatus, systems, and methods.

[0759] In some embodiments, the apparatus, systems, and methods disclosed herein are used for food samples that are suitable for or may become suitable for animal consumption, such as human consumption. In some embodiments, the food sample includes raw materials, cooked or processed foods, plant and animal-derived foods, pre-processed foods, and partially or fully processed foods. In some embodiments, the sample, which is not in liquid form, is converted into a liquid form before the apparatus, systems, and methods are applied.

[0760] The apparatus, system, and method described herein can be used to analyze samples of any volume. Examples of volumes include, but are not limited to, about 10 mL or less, 5 mL or less, 3 mL or less, 1 microliter (μL, also referred to herein as “μL”) or less, 500 μL or less, 300 μL or less, 250 μL or less, 200 μL or less, 170 μL or less, 150 μL or less, 125 μL or less, 100 μL or less, 75 μL or less, 50 μL or less, 25 μL or less, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less, 0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1 pL or less, or any range between any two of these values.

[0761] In some embodiments, the sample volume includes, but is not limited to, about 100 μL or less, 75 μL or less, 50 μL or less, 25 μL or less, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less, 0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1 pL or less, or any two of these values. In some embodiments, the sample volume includes, but is not limited to, about 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less, 0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1 pL or less, or any two of these values.

[0762] In some embodiments, the sample volume is approximately one drop of liquid. In some embodiments, the sample volume refers to the amount collected from a punctured finger or fingertip. In some embodiments, the sample volume refers to the amount collected from a microneedle, micropipette, or intravenous aspiration.

[0763] In some embodiments, the sample holder is configured to hold a fluid sample. In some embodiments, the sample holder is configured to compress at least a portion of the fluid sample into a thin layer. In some embodiments, the sample holder includes structures configured to heat and / or cool the sample. In some embodiments, a heating source provides electromagnetic waves that can be absorbed by certain structures in the sample holder to change the temperature of the sample. In some embodiments, a signal sensor is configured to detect and / or measure a signal from the sample. In some embodiments, a signal sensor is configured to detect and / or measure an analyte from the sample. In some embodiments, a heat sink is configured to absorb heat from the sample holder and / or the heating source. In some embodiments, the heat sink includes a chamber that at least partially surrounds the sample holder.

[0764] application

[0765] The apparatus, systems, and methods disclosed herein can be used for a wide range of biological / chemical sampling, sensing, measurement, and applications, including those listed, described, and summarized in PCT application (designated United States) No. PCT / US2016 / 045437, filed August 10, 2016, the entirety of which is incorporated herein by reference.

[0766] In some embodiments, the apparatuses, systems, and methods disclosed herein are used in a variety of applications across various fields where it is necessary to detect the presence, quantify, and / or amplify one or more analytes in a sample. For example, in some embodiments, the apparatuses, systems, and methods are used to detect proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, organic compounds, bacteria, viruses, cells, tissues, nanoparticles, and other molecules, compounds, mixtures, and substances. Various fields in which the apparatuses, systems, and methods can be used include, but are not limited to: diagnosis, management, and / or prevention of human diseases and conditions; diagnosis, management, and / or prevention of animal diseases and conditions; diagnosis, management, and / or prevention of plant diseases and conditions; agricultural uses; veterinary uses; food testing; environmental testing and remediation; drug testing and prevention, etc.

[0767] Applications of this invention include, but are not limited to: (a) detecting, purifying, quantifying, and / or amplifying compounds or biomolecules associated with certain diseases (e.g., infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental disorders, neuropsychiatric disorders, and organic diseases (e.g., lung diseases, kidney diseases)) or certain stages of these diseases; (b) detecting, purifying, quantifying, and / or amplifying cells and / or microorganisms (e.g., viruses, fungi, and bacteria) from the environment (e.g., water, soil, or biological samples (e.g., tissues, body fluids)); (c) detecting and quantifying compounds or biological samples (e.g., toxic waste, anthrax) that pose a threat to food safety, human health, or national security; (d) detecting and quantifying vital parameters (e.g., glucose, blood oxygen levels, total blood cell count) in medical or physiological monitors; (e) detecting and quantifying specific DNA or RNA from biological samples (e.g., cells, viruses, body fluids); (f) performing sequencing and comparison of genetic sequences of DNA in chromosomes and mitochondria for genomic analysis; or (g) detecting and quantifying reaction products, for example, during the synthesis or purification of pharmaceuticals.

[0768] In some embodiments, the apparatus, systems, and methods are used to detect nucleic acids, proteins, or other molecules or compounds in a sample. In some embodiments, the apparatus, systems, and methods are used for rapid clinical detection and / or quantification of one, two, or more, or three or more disease biomarkers in biological samples, for example, for the diagnosis, prevention, and / or management of disease conditions in subjects. In some embodiments, the apparatus, systems, and methods are used to detect and / or quantify one, two, or more, or three or more environmental biomarkers in environmental samples, such as samples obtained from rivers, oceans, lakes, rain, snow, sewage, wastewater treatment runoff, agricultural runoff, industrial runoff, tap water, or drinking water. In some embodiments, the apparatus, systems, and methods are used to detect and / or quantify one, two, or more, or three or more food biomarkers from food samples obtained from tap water, drinking water, prepared food, processed food, or unprocessed food.

[0769] In some embodiments, the apparatus, system, and method of the present invention can be used to detect analytes. In some embodiments, the analyte is a pathogen. Exemplary detectable pathogens include, but are not limited to: varicella-zoster virus; Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus cephalosporin, Staphylococcus warwickii, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus mimicus, Streptococcus pneumoniae, and Candida albicans; gonorrhea (Neisseria gonorrhoeae), syphilis (Treponema pallidum), Chlamydia trachomatis, and non-gonococcal urethritis (Ureaplasma). Urzyplasmum), chancroid (Haemophilus ducreyi), trichomonas (trichomonal vaginitis); Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MSRA), Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Stenotrophomonas maltophilia, Haemophilus influenzae parainfluenzae, Escherichia coli, Enterococcus faecalis, Serratia marcescens, Haemophilus parahaemolyticus, Enterococcus cloacae, Candida albicans, Moraxella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas fluorescens, Neisseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii, Klebsiella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, and Mycobacterium tuberculosis, etc.

[0770] In some embodiments, the apparatus, system, and method of the present invention can be used to detect analytes as diagnostic markers. In some embodiments, the diagnostic markers are selected from any of the following tables.

[0771] Table 4.1: Diagnostic Markers

[0772]

[0773]

[0774]

[0775] Table 4.2: Diagnostic Markers

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795]

[0796] In some embodiments, the apparatus, system, and method of the present invention can be used to inform a subject of the health status of a sample obtained therefrom. Health conditions that can be diagnosed or measured by the present method, apparatus, and system include, but are not limited to: chemical balance; nutritional health; exercise; fatigue; sleep; stress; prediabetes; allergies; aging; exposure to environmental toxins, pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause; and male menopause. Table 4.3 provides exemplary diagnostic markers that can be detected using the present invention and their associated health conditions.

[0797] Table 4.3: Diagnostic Markers

[0798]

[0799]

[0800]

[0801]

[0802]

[0803]

[0804]

[0805]

[0806]

[0807]

[0808] In other embodiments, diagnostic markers detectable by this method may be antibodies in a sample (e.g., a diagnostic sample) that can be used to diagnose a disease or health condition in a subject from whom the sample was obtained. Table 4.4 provides a list of five autoantibody targets that can be used, in whole or as epitope fragments, as capture agents in this method to measure the amount of epitope-binding antibody analytes in a sample, thereby diagnosing related diseases or health conditions, such as autoimmune diseases. In some cases, the disease or health condition is related to an immune response to an allergen. Table 4.5 provides a list of allergens that can be used, in whole or as epitope fragments, as capture agents in this method to measure the amount of epitope-binding antibody analytes in a sample, thereby diagnosing related diseases or health conditions, such as allergies. In some cases, the disease or health condition is related to an infectious disease, in which the source of infection can be diagnosed based on information including the measured amount of antibodies against one or more epitopes derived from the source of infection (e.g., lipopolysaccharides, toxins, proteins, etc.). Table 4.6 provides a list of infectious source-derived epitopes that can be used in whole or as epitope fragments as capture agents in the methods of the present invention to measure the amount of epitope-bound antibody analytes in a sample and thereby diagnose related diseases or health conditions (e.g., infections). Other epitopes or antigens suitable for this diagnostic method are described, for example, in PCT application publication number WO2013164476, which is incorporated herein by reference.

[0809] Table 4.4: Diagnostic autoantibody epitopes

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817] Table 4.5: Epitopes of Allergens

[0818]

[0819]

[0820]

[0821]

[0822] Table 4.6: Epitopes derived from infectious agents

[0823]

[0824]

[0825] In some embodiments, the apparatus, system, and method of the present invention can be used to detect diagnostic markers, which are microRNA (miRNA) biomarkers associated with diseases or health conditions. Table 4.7 provides an exemplary list of miRNA biomarkers that can be used and their associated diseases / health conditions.

[0826] Table 4.7: Diagnostic miRNA Markers

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835] * miRNA marker downregulated in parentheses

[0836] In some embodiments, the apparatus, system, and method of the present invention can be used to detect or analyze environmental samples. Environmental samples can be obtained from any suitable source, such as rivers, oceans, lakes, rain, snow, sewage, wastewater treatment runoff, agricultural runoff, industrial runoff, water, tap water, or drinking water, etc.

[0837] In some embodiments, the analytes that can be detected or analyzed using the apparatus, systems, and methods of the present invention are environmental markers. Environmental markers can be any suitable marker that can be captured by a trapping agent that specifically binds the environmental marker to an apparatus configured with the trapping agent. In some embodiments, the apparatus, systems, and methods of the present invention detect the concentration of lead or toxins in water. In some embodiments, the presence, absence, or quantitative level of an environmental marker in a sample can indicate the state of the environment from which the sample was obtained. In some embodiments, environmental markers can be substances that are toxic or harmful to organisms exposed to the environment, such as humans, companion animals, plants, etc. In some embodiments, environmental markers can be allergens that can cause allergic reactions in some individuals exposed to the environment. In some embodiments, the presence, absence, or quantitative level of an environmental marker in a sample can be related to the general health of the environment. In this case, the overall health of the environment can be measured over a period of time (e.g., a week, months, years, or decades).

[0838] In some embodiments, the apparatus, system, and method of the present invention further include receiving or providing a report indicating the safety or hazard to a subject exposed to the environment from which the sample was obtained, based on information including the amount of environmental markers measured. Information used to assess the safety risk or health of the environment may include data in addition to the type and amount of environmental markers measured. This other data may include, for example, location, altitude, temperature, time of day / month / year, pressure, humidity, wind direction and speed, weather, etc. Data may be represented, for example, as an average or trend over a specific time period (minutes, hours, days, weeks, months, years, etc.) or as instantaneous values ​​over shorter time periods (milliseconds, seconds, minutes, etc.).

[0839] In some embodiments, the report may be generated by a device configured to read data, or it may be generated at a remote location while transmitting data including the amount of environmental markers measured. In some embodiments, an expert may be at a remote location or may have access to data transmitted to a remote location, and may analyze or review that data to generate the report. In some embodiments, the expert may be a scientist or manager from a government agency (such as the U.S. Centers for Disease Control and Prevention (CDC) or the U.S. Environmental Protection Agency (EPA)), a research institution (such as a university), or a private company. In some embodiments, the expert may issue instructions or recommendations to a user based on data transmitted by the device and / or analyzed at a remote location.

[0840] A list of exemplary environmental markers is set forth in Table 8 of U.S. Provisional Application Serial No. 62 / 234,538, filed September 29, 2015, which is incorporated herein by reference.

[0841] Other exemplary environmental markers are listed in Table 4.8.

[0842] Table 4.8: Environmental Markers

[0843]

[0844]

[0845]

[0846]

[0847]

[0848]

[0849] In some embodiments, the apparatus, systems, and methods of the present invention can be used to detect or analyze food samples. Food samples can be obtained from any suitable source, such as from raw food, processed food, cooked food, drinking water, etc. In some embodiments, the analytes that can be detected or analyzed using the apparatus, systems, and methods of the present invention are food markers. Food markers can be, for example, any suitable markers that can be captured by a capture agent, as shown in Table 4.9, which specifically binds to the food marker in an apparatus equipped with the capture agent. In some embodiments, the presence or 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, a food marker is a substance derived from a pathogen or microorganism that indicates the presence or absence of organisms in the food from which the sample was obtained. In some embodiments, a food marker is a toxic or harmful substance if consumed by a subject. In some embodiments, a food marker is a bioactive compound that may unintentionally or accidentally alter physiology if consumed by a subject.

[0850] In some embodiments, food markers are indicators of how the food was obtained (e.g., growth, acquisition, capture, harvesting, processing, cooking, etc.). In some embodiments, food markers indicate the nutritional content of the food. In some embodiments, if the food sample is consumed by a subject, the food marker is an allergen that can induce an allergic reaction.

[0851] In some embodiments, the apparatus, systems, and methods of the present invention further include receiving or providing a report that indicates the safety or hazard of a subject consuming a food sample obtained from, based on information including measurement levels of food markers. Information used to assess the safety of food intended for consumption may include data different from the type and amount of food markers measured. This other data may include any health conditions relevant to the consumer (allergies, pregnancy, chronic or acute illnesses, currently prescribed medications, etc.).

[0852] The report can be generated by a device configured to read data, or it can be generated at a remote location while transmitting data including measurements of food markers. In some cases, food safety experts may be at a remote location or have access to data transmitted to a remote location, and may analyze or view that data to generate a report. In some embodiments, food safety experts may be scientists or managers at government agencies (e.g., the U.S. Food and Drug Administration (FDA) or the CDC), research institutions (e.g., universities), or private companies. In some embodiments, food safety experts may issue instructions or recommendations to users based on data transmitted by the device and / or analyzed at a remote location.

[0853] Exemplary food markers are listed in Table 9 of U.S. Provisional Application Serial No. 62 / 234,538, filed September 29, 2015, which is incorporated herein by reference.

[0854] Other exemplary food labels are listed in Table 4.9.

[0855] Table 4.9: Food Labels

[0856]

[0857]

[0858]

[0859] In some embodiments, the present invention relates to a kit comprising the apparatus of the present invention. In some embodiments, the kit includes means configured to specifically bind the analytes described herein. In some embodiments, the kit includes instructions for implementing the methods of the present invention using a handheld device such as a mobile phone. In some embodiments, the instructions may be present in the kit in a variety of forms, one or more of which may be present in the kit. One form of these instructions may be printed information on a suitable medium or substrate (e.g., a sheet or multiple sheets of paper on which information is printed, in the packaging of the kit, in a packaging insert, etc.). Another means may be a computer-readable medium on which information is recorded or stored, such as a disk, CD, DVD, Blu-ray, computer-readable storage device, etc. Another means may also be present is a website address, which can be used via the Internet to access information at a removed site. The kit may further include software for implementing the methods provided on a computer-readable medium for measuring the analytes on the apparatus as described herein. Any convenient means may be present in the kit.

[0860] In some embodiments, the kit includes a detection reagent containing a detectable label (e.g., a fluorescently labeled antibody or oligonucleotide that specifically binds to the relevant analyte) for labeling the analyte. The detection reagent may be disposed as a device in a separate container or may be disposed within a device.

[0861] In some embodiments, the kit includes a control sample comprising a known detectable amount of the analyte to be detected in the sample. The control sample may be provided in a container and in a solution of known concentration, or in a dry form (e.g., lyophilized or freeze-dried). If provided in a dry form, the kit may also include a buffer for dissolving the control sample.

[0862] In some embodiments, the apparatus, system, and method of the present invention can be used for simple and rapid blood cell counting using a smartphone. In some embodiments, the first and second plates are selected from thin glass slides (e.g., 0.2 mm thick) or thin plastic films (e.g., 15 mm thick) with relatively flat surfaces, and each has an area of ​​about 0.5 cm to 10 cm in length and width. In some embodiments, the spacer is made of glass, plastic, or other materials that do not deform significantly under pressure. In some embodiments, the spacer is placed on the first plate, the second plate, or both prior to sample deposition; and the first plate, the second plate, or both may optionally be coated with a reagent that promotes blood counting (staining dyes and / or anticoagulants). In some embodiments, the first and second plates may be sealed in a bag for easy transport and to achieve a longer shelf life.

[0863] In some embodiments of blood cell counting tests, only about 1 μL (microliter) (or about 0.1 μL to 3 μL) of blood, which can be taken from, for example, a finger or other body part, is required as a sample. In some embodiments, the blood sample can be deposited directly from the body (e.g., a finger) onto a first and second plate without any dilution. In such embodiments, the first and second plates can face each other, such that the blood sample is located between the inner surfaces of the first and second plates. In some embodiments, reagents (staining dyes or anticoagulants) are pre-deposited on the inner surfaces to mix with the sample. The first and second plates can then be pressed by a finger or a simple mechanical device (e.g., using a spring-loaded clip). Under pressure, the inner gap decreases, eventually stopping at a value set by the height of the spacer, and a final sample thickness is reached, which is typically equal to the final inner gap. Because the final inner gap is known, the final sample thickness becomes known, i.e., quantified (measured) by this method.

[0864] In some embodiments, if the blood sample is undiluted, the final sample thickness can be thin after pressing (sample deformation) the spacer, and therefore the final sample thickness can be, for example, less than 1 μm, less than 2 μm, less than 3 μm, less than 4 μm, less than 5 μm, less than 7 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 30 μm, less than 40 μm, less than 50 μm, less than 60 μm, less than 80 μm, less than 100 μm, less than 150 μm, or any range between any two values. A thin final sample can be useful because if the final sample is thick, many red blood cells may overlap during imaging, which could lead to inaccurate cell counting. For example, an undiluted whole blood sample approximately 4 μm thick will produce approximately one layer of red blood cells.

[0865] After pressing, the sample can be imaged directly via a smartphone or through additional optical elements (such as lenses, filters, or light sources, depending on the need). The image of the sample can be processed to identify the cell type and number. Image processing can be done locally or remotely on the same smartphone that acquired the image, but the final result is transmitted back to the smartphone (where the image is transmitted to a remote location and processed there). The smartphone will display the cell number for a specific cell. In some cases, certain suggestions will be displayed. Suggestions can be stored on the smartphone before testing, or they can come from a remote machine or a professional.

[0866] In some embodiments, the methods and apparatus described herein are used to place the reagent on the inner surface of the first plate and / or the second plate.

[0867] In some embodiments, an apparatus or method for blood testing comprises (a) the apparatus or method described herein and (b) a plate spacing (i.e., the distance between the inner surfaces of two plates) in a closed configuration or using such a spacing in which the average transverse intercellular distance of undiluted whole blood red blood cells (RBCs) in the plate spacing is greater than the average diameter of the RBC disc shape.

[0868] In some embodiments, an apparatus or method for arranging non-spherical cells includes (a) an apparatus or method as described herein, and (b) a plate spacing (i.e., the distance between the inner surfaces of the two plates) in a closed configuration or using such a spacing, wherein the spacing is less than the average size of the cell in its longitudinal direction (which is the direction of the cell's maximum dimension). This arrangement can improve the measurement of sample volume (e.g., red blood cell volume).

[0869] In some embodiments, the analytes in the blood test include a list of protein markers available on the American Association for Clinical Chemistry website.

[0870] Table 4.10 provides other exemplary analytes that can be used in point-of-care (POC) settings and / or when used by non-professional users / subjects.

[0871] Table 4.10: POC Analytes

[0872]

[0873]

[0874]

[0875]

[0876]

[0877]

[0878]

[0879] In some embodiments, the apparatus, system, and method of the present invention can be used to detect or diagnose health conditions. In some embodiments, health conditions include, but are not limited to: chemical balance; nutritional health; exercise; fatigue; sleep; stress; prediabetes; allergies; aging; exposure to environmental toxins, pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause; and male menopause.

[0880] In some embodiments, these methods can be used to obtain and compare the relative levels of nucleic acids in two or more different nucleic acid samples. In these embodiments, the results obtained from the methods described herein are typically normalized to the total amount of nucleic acids (e.g., constitutive RNA) in the samples and compared. This can be done by comparing ratios or by any other means. In a particular embodiment, the nucleic acid profiles of two or more different samples can be compared to identify nucleic acids associated with a specific disease or condition.

[0881] In some embodiments, the apparatus, system, and method of the present invention may include a) obtaining a sample, b) applying the sample to an apparatus containing a trapping agent bound to the associated analyte under conditions suitable for the binding of the analyte in the sample to the trapping agent, c) a washing apparatus, and d) a reading apparatus, thereby obtaining a measurement of the amount of analyte in the sample. In some embodiments, the analyte may be a biomarker, an environmental marker, or a food marker. In some cases, the sample is a liquid sample and may be a diagnostic sample (e.g., saliva, serum, blood, sputum, urine, sweat, tears, semen, or mucus); an environmental sample, taken from a river, ocean, lake, rainwater, snow, sewage, wastewater treatment runoff, agricultural runoff, industrial runoff, tap water, or drinking water; or a food sample obtained from tap water, drinking water, prepared food, processed food, or raw material. In some embodiments, the apparatus may be housed in a microfluidic device, and the application step b) may include applying the sample to the microfluidic device containing the apparatus. In some embodiments, the reading step d) may include detecting a fluorescent or luminescent signal from the device. In some embodiments, reading step d) may include reading the device using a handheld device configured to read the device. The handheld device may be a mobile phone, such as a smartphone. In some embodiments, the device may include a labeling reagent that can bind to the analyte-capture agent complex on the device. In some embodiments, between steps c) and d), the apparatus, system, and method of the present invention may further include applying the labeling reagent that binds to the analyte-capture agent complex on the device to the device and washing the device. In any embodiment, reading step d) may include reading an identifier of the device. The identifier may be an optical barcode, an RFID tag, or a combination thereof. In some embodiments, the apparatus, system, and method of the present invention may further include applying a control sample to a control device containing a capture agent that binds to the analyte, wherein the control sample includes a known detectable amount of the analyte, and reading the control device to obtain a control measurement of a known detectable amount of the analyte in the sample. In some embodiments, the sample may be a diagnostic sample obtained from a subject, the analyte may be a biomarker, and the amount of the analyte measured in the sample may be a diagnosis of a disease or condition.

[0882] In some embodiments, the apparatus, system, and method of the present invention may further include receiving or providing a report to a subject indicating the amount of a biomarker measured and the range of values ​​for the biomarker measured in an individual who does not have or is at low risk of having a disease or condition, wherein the amount of the biomarker measured relative to the range of values ​​is used to diagnose the disease or condition. In some embodiments, the apparatus, system, and method of the present invention may further include diagnosing the subject based on information including the amount of the biomarker measured in a sample. In some embodiments, the diagnostic step includes transmitting data including the amount of the measured biomarker to a remote location and receiving a diagnosis based on information including measurements from the remote location. In some embodiments, the biomarker may be selected from those listed in the table. In some embodiments, the apparatus may include multiple capture agents, each binding to a biomarker described herein, wherein the reading step d) includes obtaining a measurement of the amount of multiple biomarkers in a sample, and wherein the amount of multiple biomarkers in the sample is used to diagnose a disease or condition. In some embodiments, the capture agent may be an antibody epitope, and the biomarker may be an antibody binding to the antibody epitope. In some embodiments, the antibody epitope includes an allergen selected from the table or a fragment thereof. In some embodiments, the antibody epitope includes an allergen selected from the table or a fragment thereof. In some embodiments, antibody epitopes include biomolecules or fragments thereof derived from an infection source selected from the table. In some embodiments, the device may include a plurality of antibody epitopes selected from the table, wherein reading step d) includes obtaining a measurement of the amount of antibody bound to the plurality of epitopes in the sample, and wherein the amount of antibody bound to the plurality of epitopes in the sample can be used to diagnose a disease or condition.

[0883] In some embodiments, the sample may be an environmental sample, and the analyte may be an environmental marker. In some embodiments, the environmental markers described herein. In some embodiments, the method may include receiving or providing a report indicating the safety or harm to a subject exposed to the environment from which the sample was obtained. In some embodiments, the method may include transmitting data containing the measured amount of the environmental marker to a remote location and receiving a report indicating the safety or harm to a subject exposed to the environment from which the sample was obtained. In any embodiment, the apparatus may include multiple traps, each associated with an environmental marker described herein, and the reading step d) may include obtaining measurements of the amounts of multiple environmental markers in the sample.

[0884] In some embodiments, the sample may be a food sample, wherein the analyte may be a food marker, and wherein the amount of the food marker in the sample may be related to the safety of the food for consumption. In some embodiments, the food marker is an example described herein. In any embodiment, the method may include receiving or providing a report instructing a subject on the safety or hazard of consuming the food from which the sample was obtained. In any embodiment, the method may include transmitting data containing the measured amount of the food marker to a remote location and receiving a report instructing a subject on the safety or hazard of consuming the food from which the sample was obtained. In any embodiment, the device array may include a plurality of trapping agents, each incorporating a food marker described herein, wherein the acquisition may include obtaining measurements of the amounts of the plurality of food markers in the sample, and wherein the amounts of the plurality of food markers in the sample may be related to the safety of the food for consumption.

[0885] In some embodiments, the device is part of a microfluidic device. In some embodiments, the device, apparatus, system, and method are used to detect fluorescence or luminescence signals. In some embodiments, the device, system, and method include or are used with a communication device, such as, but not limited to, a mobile phone, tablet computer, and portable computer. In some embodiments, the device, system, and method include or are used with an identifier (such as, but not limited to, an optical barcode, an RFID tag, or a combination thereof).

[0886] In some embodiments, the sample is a diagnostic sample obtained from a subject, the analyte is a biomarker, and the measured amount of the analyte in the sample is a diagnostic conclusion of a disease or condition. In some embodiments, the apparatus, system, and method further include receiving or providing a report to a subject indicating the measured amount of the biomarker and the range of measured values ​​for the biomarker in individuals who do not have or are at low risk of having a disease or condition, wherein the measured amount of the biomarker relative to the range of measured values ​​is a diagnostic conclusion of a disease or condition.

[0887] In some embodiments, the sample is an environmental sample, and the analyte is an environmental marker. In some embodiments, the apparatus, system, and method include receiving or providing a report indicating the safety or hazard of a subject's exposure to the environment from which the sample was obtained. In some embodiments, the apparatus, system, and method include transmitting data containing measured quantities of environmental markers to a remote location and receiving a report indicating the safety or hazard of a subject's exposure to the environment from which the sample was obtained.

[0888] In some embodiments, the sample is a food sample, wherein the analyte is a food marker, and wherein the amount of the food marker in the sample is related to the safety of the food to be consumed. In some embodiments, the apparatus, system, and method include receiving or providing a report indicating the safety or hazard of a subject consuming the food from which the sample was obtained. In some embodiments, the apparatus, system, and method include transmitting data containing measured amounts of food markers to a remote location and receiving a report indicating the safety or hazard of a subject consuming the food from which the sample was obtained.

[0889] Various samples can be used in assays performed using the apparatus, devices, and systems described herein. In some embodiments, the sample contains nucleic acids. In some embodiments, the sample contains proteins. In some embodiments, the sample contains carbohydrates. Current apparatus, devices, and systems can be used to rapidly change and stably maintain the temperature of a sample, thereby providing a rapid and cost-effective method for processing samples. Furthermore, the apparatus, devices, and systems described herein can be used for a variety of applications (e.g., assays). These applications include, but are not limited to, diagnostic testing, health monitoring, environmental testing, and / or forensic identification. Such applications also include, but are not limited to, various biological, chemical, and biochemical assays (e.g., DNA amplification, DNA quantification, selective DNA isolation, genetic analysis, histogentics, oncogene identification, infectious disease testing, genetic fingerprinting, and / or paternity testing).

[0890] In some embodiments, a “sample” can be any nucleic acid, including but not limited to, human fluids such as whole blood, plasma, serum, urine, saliva, and sweat, and cell cultures (mammals, plants, bacteria, fungi). Samples can be freshly obtained or stored or processed in any desired or convenient manner, such as by dilution or addition to buffers or other solutions or solvents. Cellular structures such as human cells, animal cells, plant cells, bacterial cells, fungal cells, and viral particles may be present in the sample.

[0891] As used herein, the term "nucleic acid" refers to any DNA or RNA molecule, or a DNA / RNA hybrid, or a mixture of DNA and / or RNA. Therefore, the term "nucleic acid" is intended to include, but is not limited to, genomic or chromosomal DNA, plasmid DNA, amplified DNA, cDNA, total RNA, mRNA, and small RNA. The term "nucleic acid" is also intended to include native DNA and / or RNA molecules, or synthetic DNA and / or RNA molecules. In some embodiments, cell-free nucleic acids are present in the sample; as used herein, "cell-free" means that the nucleic acids are not contained in any cellular structure. In some other embodiments, the nucleic acids are contained in cellular structures, including, but not limited to, the methods of the present invention applicable to samples of a range of volumes. Samples of different volumes can be introduced onto plates of different sizes.

[0892] As used herein, “nucleic acid amplification” includes any technique used to detect nucleic acids by amplifying (producing large numbers of copies of) a target molecule in a sample, where “target” refers to the sequence or a portion of the relevant nucleic acid. Suitable nucleic acid amplification techniques include, but are not limited to, various polymerase chain reaction (PCR) methods, such as hot-start PCR, nested PCR, falling PCR, reverse transcription PCR, RACEPCR, digital PCR, etc., and isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), strand displacement amplification, helicase-dependent amplification, nickase amplification, rolling circle amplification, recombinase polymerase amplification, etc.

[0893] As used herein, “essential reagents” or “reagents” include, but are not limited to, primers, deoxynucleotides (dNTPs), divalent cations (e.g., Mg2+), monovalent cations (e.g., K+), buffers, enzymes, additives, and reporter molecules. “Essential reagents for nucleic acid amplification” are either in dry form on the inner surface of the first or second plate, or in liquid form encapsulated, embedded, or surrounded in a material (e.g., paraffin) that melts with increasing temperature.

[0894] As used herein, "primer" may, in some embodiments, refer to a pair of forward and reverse primers. In some embodiments, a primer may refer to multiple primers or a set of primers. As used herein, enzymes suitable for nucleic acid amplification include, but are not limited to, DNA-dependent polymerases, or RNA-dependent DNA polymerases, or DNA-dependent RNA polymerases. Examples of suitable DNA-dependent polymerases include, but are not limited to, AptaTaq polymerase, Kapa2G Fast polymerase, Kapa2G Robust, Z-Taq polymerase, Terra PCR direct polymerase, SpeedStar HSDNA polymerase, Phusion DNA polymerase, and high-fidelity DNA polymerase.

[0895] As used herein, “additives” in some embodiments include, but are not limited to, 7-denitro-2'-deoxyguanosine 7-denitro-dGTP, BSA, gelatin, betaine, DMSO, formamide, Tween 20, NP-40, Triton X-100, and tetramethylammonium chloride.

[0896] As used herein, the term "reporter" refers to any tag, label, or dye that can bind to or embed in a nucleic acid molecule or be activated by a byproduct of the amplification process to make the nucleic acid molecule or the amplification process visible. Suitable reporters include, but are not limited to, fluorescent labels or tags or dyes, embedding agents, molecular beacon markers, or bioluminescent molecules, or combinations thereof.

[0897] In some other embodiments, as used herein, “essential reagents” or “reagents” (e.g., for nucleic acid amplification reactions) may also include cell lysis reagents that facilitate the breakdown of cellular structures. Cell lysis reagents include, but are not limited to, salts, detergents, enzymes, and other additives. The term “salt” as used herein includes, but is not limited to, lithium salts (e.g., lithium chloride), sodium salts (e.g., sodium chloride), and potassium salts (e.g., potassium chloride). The term “detergent” as used herein may be ionic, including anionic and cationic, nonionic, or zwitterionic. The term “ionic detergent” as used herein includes any detergent that is partially or wholly ionic when dissolved in water. Suitable anionic detergents include, but are not limited to, sodium dodecyl sulfate (SDS) or other alkali metal alkyl sulfates or similar detergents, Sarkosyl, or combinations thereof. The term “enzyme” as used herein includes, but is not limited to, lysozyme, cellulase, and protease. In addition, cell lysis reagents may also include chelating agents, including but not limited to EDTA, EGTA, and other polyaminocarboxylic acids, and some reducing agents, such as dithiothreitol (dTT). The composition of the essential reagents herein varies depending on the rational design of different amplification reactions. In some embodiments, such as when performing isothermal amplification via LAMP, the sample is heated to 60-65°C for about 1-70 minutes.

[0898] As used herein, "nucleic acid amplification product" refers to various nucleic acids generated through nucleic acid amplification technology. The types of nucleic acid amplification products described herein include, but are not limited to, single-stranded DNA, single-stranded RNA, double-stranded DNA, linear DNA, or circular DNA. In some embodiments, the nucleic acid amplification products may be the same nucleic acid having the same length and structure. In some other embodiments, the nucleic acid amplification products may be multiple nucleic acids with different lengths and structures.

[0899] In some embodiments, a reporter is used to quantify the nucleic acids accumulated after nucleic acid amplification. As defined and used above, the reporter is characterized by quantifiability, which is related to the presence or absence, or the quantity, of nucleic acid amplicon accumulated in the closed chamber.

[0900] As used herein, “cell lysis reagents” include, but are not limited to, salts, detergents, enzymes, and other additives that promote the destruction of cell structure. The term “salt” as used herein includes, but is not limited to, lithium salts (e.g., lithium chloride), sodium salts (e.g., sodium chloride), and potassium salts (e.g., potassium chloride). The term “detergent” as used herein can be ionic, including anionic and cationic, nonionic, or zwitterionic. The term “ionic detergent” as used herein includes any detergent that is partially or wholly ionic when dissolved in water. Suitable anionic detergents include, but are not limited to, sodium dodecyl sulfate (SDS) or other alkali metal alkyl sulfates or similar detergents, Sarkosyl, or combinations thereof. The term “enzyme” as used herein includes, but is not limited to, lysozyme, cellulase, and protease. Furthermore, cell lysis reagents may include chelating agents, including but not limited to EDTA, EGTA, and other polyaminocarboxylic acids, and some reducing agents, such as dithiothreitol (dTT). The composition of the necessary reagents described herein varies depending on the rational design of different amplification reactions.

[0901] As used herein, “essential reagent 2” includes, but is not limited to, primers, deoxynucleotides (dNTPs), divalent cations (e.g., Mg2+), monovalent cations (e.g., K+), buffers, enzymes, and reporters. Essential reagent 2 for nucleic acid amplification may be in a dry form on the inner surface of the first or second plate or both, or in a liquid form encapsulated, embedded, or surrounded by a material (e.g., paraffin) that melts with increasing temperature.

[0902] Rapid heating and cooling system, with a separate heating element located outside the QMAX card.

[0903] In some embodiments, the device further includes a separate heating element external to the RHC card and configured to heat the RHC card when placed near or in contact with it. The separate heating element can attach to or detach the RHC card and draws energy from a heat source in a manner similar to a heating / cooling layer. A separate heating element allows the RHC card to function without a heating / cooling layer. For example, as... Figure 15A and 15B As shown, the heating element is separated from the sample card.

[0904] The terms “CROF card (or card)”, “COF card”, “QMAX card”, “Q card”, “CROF device”, “COF device”, “QMAX device”, “CROF board”, “COF board” and “QMAX board” are interchangeable and can be used to identify embodiments of the devices described herein.

[0905] The term "X-plate" refers to one of the two plates in a CROF card to which a spacer is attached. A more detailed description of the COF card, CROF card, and X-plate is found in Provisional Application Serial No. 62 / 456065, filed February 7, 2017, the entire contents of which are incorporated herein by reference for all purposes.

[0906] RHC cards are QMAX cards, which have or do not have spacers plus a heating / cooling layer on or within one of the boards.

[0907] Figure 5 A device card 100 is shown, comprising a first plate 10 and a second plate 20. In some embodiments, the first plate 10 and the second plate 20 are movable relative to each other into different configurations, including open and closed configurations. In some embodiments, in the open configuration, the two plates are partially or completely separated, and the average spacing between the plates is at least 300 μm. In some embodiments, a sample can be deposited on one or both plates. In some embodiments, in the closed configuration, at least a portion of the sample is compressed into a layer by the two plates, wherein the average sample thickness is 200 μm or less.

[0908] In some embodiments, the QMAX card 100 includes a hinge 103 connecting a first plate 10 and a second plate 20, allowing the two plates to pivot relative to each other. In some embodiments, the QMAX card includes a notch 105 that facilitates switching the card between an open and closed configuration. In some embodiments, one or both plates are transparent. In some embodiments, one or both plates are flexible. In some embodiments, the QMAX card 100 includes a heating / cooling layer 190. In some embodiments, the heating / cooling layer 190 is configured to absorb electromagnetic waves and convert energy to increase the temperature of the sample.

[0909] Figure 4A and 4B Perspective views and cross-sectional views of embodiments of the device of the present invention are shown. Figure 4A A device 100 in an open configuration (also referred to as a "sample holder" of the system) is shown. Figure 4A As shown, the sample holder 100 includes a first plate 10, a second plate 20, and a spacer mechanism (not shown). The first plate 10 and the second plate 20 each include an inner surface (11 and 21, respectively) and an outer surface (12 and 22, respectively). Each inner surface has a sample contact area (not shown) for contacting the fluid sample to be processed and / or analyzed by the device.

[0910] The first plate 10 and the second plate 20 can be moved relative to each other to form different structures. One of these structures is an open structure, in which, for example... Figure 4AAs shown, the first plate 10 and the second plate 20 are partially or completely separated, and the spacing between the first plate 10 and the second plate 20 (i.e., the distance between the inner surfaces 11 of the first plate and 21 of the second plate) is not adjusted by the spacing mechanism. The open structure allows the sample to be deposited on the first plate, the second plate, or both in the sample contact area.

[0911] like Figure 4A As shown, the second plate 20 further includes a heating / cooling layer 112 in the sample contact area. The first plate 10 may also optionally or additionally include a heating / cooling layer 112. In some embodiments, the heating / cooling layer 112 is configured to effectively absorb radiation (e.g., electromagnetic waves) irradiated thereon. The absorption rate is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, 100% or less, 85% or less, 75% or less, 65% or less, or 55% or less, or within any two values. The heating / cooling layer 112 is also configured to convert at least a majority of the absorbed radiant energy into heat (thermal energy). For example, the heating / cooling layer 112 is configured to release the radiation as heat after absorbing energy from the electromagnetic waves. As used herein, the terms “substantial” or “basically” mean 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, 99% or more, or 99.9% or more.

[0912] Figure 3A and 3B The sample card is shown in a closed structure, wherein the heating / cooling layer contains a heating zone that is directly heated / to be heated by a heating source; Figure 3A A perspective view is shown, while Figure 3B A cross-sectional view is shown. In some embodiments, the heating / cooling layer includes a heating zone that is directly heated by a heating source. In some embodiments, the heating source emits electromagnetic radiation (waves) that reach the heating / cooling layer, with or without modulation by a lens or other modulator. The area that directly receives this radiation (wave) is called the heating zone.

[0913] In some embodiments, the heating zone is smaller than the entire area of ​​the heating / cooling layer. In some embodiments, the heating zone occupies approximately 1 / 1000, 1 / 500, 1 / 200, 1 / 100, 1 / 50, 1 / 20, 1 / 10, 1 / 5, 1 / 2, or 2 / 3 of the area of ​​the heating / cooling layer, or within any two values. In some embodiments, when a sample is loaded and pressed into a thin layer by two plates, the volume of the sample directly in contact with the area of ​​the heating zone in the electromagnetic wave path is referred to as the heating volume. In some embodiments, the sample in the heating volume can be rapidly heated to the desired temperature because the sample layer is thin and / or because of the excellent absorption properties of the heating / cooling layer. In some embodiments, the sample in the heating volume can also be rapidly cooled to the desired temperature.

[0914] Biochemistry and assay

[0915] The thermal cycler system and related methods of the present invention are used to facilitate chemical, biological, or medical assays or reactions. In some embodiments, the reaction requires temperature changes. In some embodiments, the reaction requires, or preferably requires, rapid temperature changes to avoid nonspecific reactions and / or reduce waiting times. In some embodiments, the systems and methods of the present invention are used to facilitate reactions that require cyclic temperature changes to amplify nucleotides in fluid samples; these reactions include, but are not limited to, polymerase chain reaction (PCR). The following description uses PCR as an example to illustrate the performance and use of the thermal cycler system and methods of the present invention. However, it should be noted that some embodiments of the apparatus, systems, and methods described herein are also applicable to other assays and / or reactions that require temperature control and changes.

[0916] In some embodiments, the assay (e.g., PCR) can be performed using an untreated sample. For example, the template for the PCR reaction can be provided by a sample obtained directly from the subject without additional processing. In some embodiments, the sample can be whole blood from an individual. In some embodiments, this "one-step" approach would allow for more convenient use of the apparatus described herein.

[0917] In some embodiments, sample 90 is a premixed reaction medium for polymerase chain reaction (PCR). For example, in some embodiments, the reaction medium includes components such as, but not limited to: DNA template, two primers, DNA polymerase (e.g., Taq polymerase), deoxynucleoside triphosphate (dNTP), divalent cation (e.g., Mg2+), monovalent cation (e.g., K+), and buffer solution. The specific components, the concentration of each component, and the total volume vary depending on the rational design of the reaction. In some embodiments, the PCR assay requires several variations / changes in sample temperature between the following steps: (i) an optional initialization step that requires heating the sample to 92-98°C; (2) a denaturation step that requires heating the sample to 92-98°C; (3) an annealing step that requires cooling the sample temperature to 50-65°C; (4) an extension (or elongation) step that requires heating the sample to 75-80°C; (5) repeating steps (2)-(4) approximately 20-40 times; and (6) completing the assay and cooling the sample temperature to ambient temperature (e.g., room temperature) or cooling to approximately 4°C. The specific temperature and time period for each step vary and depend on many factors, including but not limited to the length of the target sequence, the length of the primers, the cation concentration, and / or the GC percentage.

[0918] The thermal cycling system of the present invention enables rapid temperature changes for PCR assays. For example, referring to Figures (A) and (B) of Figure 3 and Figure (B) of Figure 4, in some embodiments, a sample 90 (e.g., a premixed reaction medium) is added to one or both plates 10 and 20 in an open configuration, and the plates are switched to a closed configuration to compress the sample 90 into a thin layer with a thickness 102, which is adjusted by a spacer mechanism (not shown); a heating source 202 projects electromagnetic waves 210 onto the first plate 10 (e.g., specifically onto a heating / cooling layer 112); the heating / cooling layer 112 is configured to absorb the electromagnetic waves 210 and convert at least a majority of the electromagnetic waves 210 into heat, which raises the temperature of the sample; removal of the electromagnetic waves 210 causes a decrease in the temperature of the sample 90.

[0919] In some embodiments, the thermal cycler system provides rapid heating (temperature increase) for any or all of the initialization step, denaturation step, and / or extension / stretching step by projecting electromagnetic waves 210 onto the heating / cooling layer 112 or increasing the intensity of the electromagnetic waves; in some embodiments, cooling and / or final cooling steps in the annealing step are rapidly achieved by removing the electromagnetic waves projected by the heating source 202 or reducing the intensity of the electromagnetic waves. In some embodiments, an increase in the intensity of electromagnetic wave 210 produces a temperature rise rate of at least 80°C / s, 70°C / s, 60°C / s, 50°C / s, 45°C / s, 40°C / s, 35°C / s, 30°C / s, 25°C / s, 20°C / s, 18°C / s, 16°C / s, 14°C / s, 12°C / s, 10°C / s, 9°C / s, 8°C / s, 7°C / s, 6°C / s, 5°C / s, 4°C / s, 3°C / s, or 2°C / s, or within any two values. In some embodiments, the average temperature rise rate in the PCR assay is 10°C / s or higher. In some embodiments, the removal of electromagnetic wave 210 or a reduction in the intensity of electromagnetic wave 210 results in a temperature decrease rate of at least 80°C / s, 70°C / s, 60°C / s, 50°C / s, 45°C / s, 40°C / s, 35°C / s, 30°C / s, 25°C / s, 20°C / s, 18°C / s, 16°C / s, 14°C / s, 12°C / s, 10°C / s, 9°C / s, 8°C / s, 7°C / s, 6°C / s, 5°C / s, 4°C / s, 3°C / s, or 2°C / s, or within any two values. In some embodiments, the average temperature decrease rate in the PCR assay is 5°C / s or higher. As used herein, the term "heating / cooling rate" refers to the rate of temperature change between two preset temperatures. In some embodiments, the average temperature rise or fall for each step is different.

[0920] In the PCR process, once the target temperature has been reached at any step, the sample needs to be maintained at the target temperature for a period of time. The thermal cycler system of the present invention provides temperature maintenance by: (1) adjusting the intensity of electromagnetic wave 210, decreasing the intensity of electromagnetic wave 210 if the temperature has risen to the target temperature, or increasing the intensity of electromagnetic wave 210 if the temperature has fallen to the target temperature, and / or (2) maintaining the target temperature by balancing the heat supplied to the sample and the heat removed from the sample.

[0921] Figure 9A cross-sectional view is shown of an exemplary process for nucleic acid amplification using a device according to some embodiments. Examples of the steps include (A) introducing a sample containing nucleic acid into the inside of a first plate (substrate); (B) pressing a second plate (QMAX card) onto the inner surface of the first plate to form a closed configuration of the device, wherein necessary reagents for nucleic acid amplification are dried on the inner surface of the second plate; and (C) accumulating nucleic acid amplification products in a chamber sealed by the first and second plates.

[0922] If necessary, the sample can be introduced onto the first or second plate, or even both. This article... Figure 9 An example of introducing a sample onto the inner surface of the first plate is provided.

[0923] More specifically, in step (B), the second plate is pressed against the inner surface of the first plate, making contact with the sample to form a closed structure of the device. The "second plate" may refer to a plate with periodic spacers on the inner surface in contact with the sample.

[0924] More specifically, in step (C), when the device is in a closed configuration, the heating source projects electromagnetic waves onto a heating / cooling layer on the inner or outer surface of the first or second plate, or both. The heating / cooling layer is configured to absorb the electromagnetic waves and convert at least a majority of the energy from the electromagnetic waves into heat, which is transferred to the sample in the closed chamber. In some embodiments, the heating source is programmed to regulate the temperature of the sample within a range from ambient temperature to 98°C. In some embodiments, such as for conventional PCR, the sample is first heated to 98°C and then subjected to repeated cycles of 94°C, 50-65°C, and 72°C for 15 to 40 times. In some embodiments, such as for isothermal amplification, the temperature of the sample is maintained at a constant temperature. In some embodiments, such as when performing isothermal amplification via LAMP, the sample is heated to 60-65°C for approximately 1-70 minutes.

[0925] Figure 10 A cross-sectional view is shown of an exemplary assay method using a card device to combine nucleic acid extraction and amplification according to some embodiments. Examples of the steps include (A) fixing a capture probe to the inner surface of a first plate (substrate); (B) introducing a sample onto the inner surface of the first plate; (C) pressing a second plate onto the inner surface of the first plate to form a closed configuration of the device, wherein a necessary reagent 1 for promoting the release and capture of nucleic acid is dried on the inner surface of the second plate; (D) capturing nucleic acid from the sample onto the inner surface of the first plate; (E) removing the second plate and cleaning the inner surface of the first plate with a sponge; (F) pressing a third plate onto the inner surface of the first plate, wherein a necessary reagent 2 for nucleic acid amplification is dried on the inner surface of the third plate; and (G) accumulating nucleic acid amplification products in a chamber enclosed by the first and third plates.

[0926] In some embodiments, in step (a), the capture probe is fixed to the inner surface of the first plate. As used herein, “capture probe” refers to an oligonucleotide with a length of 1-200 bp, preferably 5-50 bp, more preferably 10-20 bp. The capture probe may have a sequence complementary to the nucleic acid sequence associated with the sample. In some embodiments, the same capture probe may be fixed to the surface of the first plate. In some other embodiments, different capture probes with different base pair compositions are fixed to the surface of the first plate. The capture probe may be DNA, or RNA, or both, but preferably single-stranded DNA. As used herein, “fixation” refers to the process of anchoring the capture probe to the plate surface. In some embodiments, the capture probe is covalently anchored, wherein, for example, the 5' or 3' end of the capture probe is modified to promote coating on the plate surface. Commonly used 3' end modifications include, but are not limited to, thiols, dithiols, amines, biotin, etc. In some other embodiments, the capture probe may be passively absorbed onto the substrate surface.

[0927] After immobilization with the capture probe, the plate surface is sealed using a sealing agent solution. Suitable sealing agents include, but are not limited to, 6-mercaptohexanol and bovine serum albumin.

[0928] like Figure 10 As shown in step (B), the “sample” can be any nucleic acid, including or not containing a sample, including but not limited to human fluids such as whole blood, plasma, serum, urine, saliva, and sweat, as well as cell cultures (mammals, plants, bacteria, fungi). The sample can be freshly obtained or stored or processed in any desired or convenient manner, such as by dilution or the addition of buffers or other solutions or solvents. Cellular structures such as human cells, animal cells, plant cells, bacterial cells, fungal cells, and viral particles can be present in the sample.

[0929] If necessary, the sample can be introduced onto the first or second plate, or even both. This article... Figure 10 An example of introducing a sample onto the inner surface of the first plate is provided.

[0930] In some embodiments, in step (C), the second plate is pressed against the inner surface of the first plate (substrate) to contact the sample, thereby forming a closed configuration of the device. The necessary reagent 1 for nucleic acid amplification is either in a dry form on the inner surface of the first or second plate or both, or in a liquid form encapsulated, embedded, or surrounded in a material (e.g., paraffin) that melts with increasing temperature.

[0931] In some embodiments, in step (D), after contact with the sample described above, the dried necessary reagent 1 is dissolved in the sample. The target nucleic acid, released from the disrupted cellular structure or present as cell-free nucleic acid, or a combination thereof, hybridizes with complementary capture probes on the plate surface. The hybridization time varies considerably depending on the specifications of the spacers on the inner surface of the plate. In some embodiments, for example, when using a plate with spacers of 30 μm height, experimental data are indicated after 2 minutes, indicating that hybridization between the relevant nucleic acid and the immobilized capture probe has reached equilibrium. As used herein, “unhybridized nucleic acid” refers to nucleic acid that has not been captured by the immobilized capture probe.

[0932] In some embodiments, Figure 10 In step (E), the second plate is separated from the first plate (substrate), and the surface of the first plate (substrate) is cleaned using a sponge. As used herein, "sponge" refers to a type of flexible porous material whose pore size changes under different pressures. The sponge containing a washing buffer is brought into contact with the surface of the first plate to remove contaminants. In some embodiments, the sponge is brought into contact with the surface of the first plate once. In some other embodiments, the sponge is brought into contact with the surface of the first plate two or more times. As used herein, "contaminant" refers to compounds that are detrimental to nucleic acid amplification reactions, including but not limited to cell debris, proteins, and nonspecific nucleic acids.

[0933] In some embodiments, Figure 10 In step (F), the third plate (QMAX card 2) is pressed onto the inner surface of the first plate to contact the sample, thereby forming a closed structure of the device. The necessary reagent 2 for nucleic acid amplification may be in a dry form on the inner surface of the first or third plate or both, or in a liquid form encapsulated, embedded, or surrounded in a material that melts with increasing temperature (e.g., paraffin).

[0934] In some embodiments, Figure 10 In step (G), when the device is in a closed configuration, a heating source projects electromagnetic waves onto a heating / cooling layer on the inner or outer surface of the first or third plate, or both. The heating / cooling layer is configured to absorb the electromagnetic waves and convert at least a majority of the energy from the electromagnetic waves into heat, which is transferred to the sample in the closed chamber. In some embodiments, the heating source is programmed to regulate the temperature of the sample within a range from ambient temperature to 98°C. In some embodiments, such as for conventional PCR, the sample is first heated to 98°C and then subjected to repeated cycles of 94°C, 50-65°C, and 72°C for 15-40 times. In some embodiments, such as for isothermal amplification, the temperature of the sample is maintained at a constant temperature. In some embodiments, such as when performing isothermal amplification via LAMP, the sample is heated to 60-65°C for approximately 1-70 minutes.

[0935] In some embodiments, the sample contact area of ​​one or both plates includes a squeeze open flow monitoring surface structure (MSS) configured to monitor how much flow has occurred after COF. For example, in some embodiments, the MSS comprises a shallow square array that will induce friction against components in the sample (e.g., 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.

[0936] The depth of the MSS can be 1 / 1000, 1 / 100, 1 / 100, 1 / 5, 1 / 2 of the height of the spacer, or within any two values, and it can be either protruding or hole-shaped.

[0937] Multiplexing

[0938] Figure 8A and 8B It shows an open construction ( Figure 8A ) and closed structures ( Figure 8B A perspective view of a sample holder 100, wherein multiple sample contact areas exist on the plate, allowing for the handling and analysis of multiple samples. Figure 8A and 8B As shown, the thermal cycler system of the present invention includes a sample holder 100 and a thermal control unit 200; the sample holder 100 includes a first plate 10, a plurality of second plates 20 and a plurality of spacing mechanisms (not shown); the thermal control unit 200 includes a heating source 202 and a controller 204.

[0939] refer to Figure 8A One or two plates (e.g., the second plate 20) contain multiple sample contact areas (unmarked). In some embodiments, one or two plates (e.g., the second plate 20) contain multiple heating / cooling layers 112. Figure 8A A sample holder 100 in an open configuration is shown, wherein a first plate 10 and a second plate 20 are partially or completely spaced apart, allowing one or more samples to be deposited on one or both plates. In the open configuration, the spacing between the first plate 10 and the second plate 20 is not adjusted by a spacer mechanism.

[0940] Figure 8B A sample holder 100 in a closed configuration is shown, wherein the inner surfaces of two plates face each other, and the spacing 102 between the two plates is adjusted by a spacer mechanism (not shown). If one or more samples have been deposited on the plates, the plates are configured to press each sample into a layer, the thickness of which is adjusted by the spacer mechanism.

[0941] like Figure 8BAs shown, multiple first plates 10 are used to cover a portion of a second plate 20. For example, each first plate 10 covers a single sample contact area on which a sample is deposited. A spacer mechanism exists for each sample contact area, and the spacer mechanism has a different height, thereby creating different spacing 102 for each sample contact area and for different thicknesses of each sample layer. For example, the spacer mechanism is a columnar spacer; each sample contact area has a set of very uniform spacers; different sets of spacers may have the same or different heights, resulting in sample layer thicknesses that are the same or different for different samples.

[0942] See Figure 8A and 8B In some embodiments, controller 204 directs heating source 202 to project electromagnetic waves 210 onto second plate 20 (and thus onto heating / cooling layer 112), wherein the electromagnetic waves 210 are absorbed by heating / cooling layer 112 and converted into heat, causing a temperature change in the sample. In some embodiments, multiple samples are processed and analyzed when multiple sample contact areas are present. For example, in some embodiments, each sample is a premixed PCR reaction medium with different components. A sample holder 100 is used to test different conditions for amplifying the same nucleotide and / or different nucleotides under the same or different conditions.

[0943] Other exemplary embodiments

[0944] AAA-1.1. A device for rapidly changing the temperature of a fluid sample, comprising:

[0945] The structure comprises a first plate (10), a second plate (20), a heating layer (112-1), and a cooling layer (112-2), wherein:

[0946] Each of the first and second plates has a sample contact area on its respective inner surface for contacting the fluid sample; wherein the sample contact areas face each other, the average spacing between them is 200 μm or less, and are capable of contacting and holding the sample between them;

[0947] Heating layer:

[0948] Positioned on the inner surface, outer surface, or inner side of one of the plates, and

[0949] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0950] Cooling layer:

[0951] Located on the inner surface, outer surface, or inner side of one of the plates;

[0952] Configured for cooling-related sample volumes; and

[0953] It includes a material layer with a thermal conductivity and heat capacity ratio of 0.6 cm² / sec or greater;

[0954] Wherein the distance between the cooling layer and the surface of the relevant sample volume is zero or less than a distance configured such that the thermal conductivity per unit area between the cooling layer and the surface of the relevant sample volume is equal to 70 W / (m2·K) or greater; and

[0955] In some embodiments, the heating layer and the cooling layer are layers of the same material having heating and cooling zones, and the heating and cooling zones may have the same area or different areas.

[0956] AAA-1.2. A device for rapidly changing the temperature of a fluid sample, comprising:

[0957] The structure comprises a first plate (10), a second plate (20), a heating layer (112-1), and a cooling layer (112-2), wherein:

[0958] Each of the first and second plates has a sample contact area on its respective inner surface for contacting the fluid sample; wherein the sample contact areas face each other, are spaced apart by an average separation distance of 200 μm or less, and are capable of contacting and holding the sample between them;

[0959] Heating layer:

[0960] It is positioned on the inner surface, outer surface, or inner side of one of the plates.

[0961] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0962] Cooling layer:

[0963] Located on the inner surface, outer surface, or inner side of one of the plates;

[0964] Configured for cooling-related sample volumes; and

[0965] It includes a material layer with a thermal conductivity to heat capacity ratio of 0.6 cm² / sec or greater, wherein the layer with a high thermal conductivity to heat capacity ratio has an area greater than the lateral area of ​​the sample volume;

[0966] Wherein the distance between the cooling layer and the surface of the relevant sample volume is zero or less than a distance configured such that the thermal conductivity per unit area between the cooling layer and the surface of the relevant sample volume is equal to 70 W / (m2·K) or greater; and

[0967] In some embodiments, the heating layer and the cooling layer are layers of the same material having heating and cooling zones, and the heating and cooling zones may have the same area or different areas.

[0968] AAA-1.3. A device for rapidly changing the temperature of a fluid sample, comprising:

[0969] The structure comprises a first plate (10), a second plate (20), a heating layer (112-1), and a cooling layer (112-2), wherein:

[0970] The first and second plates can be moved relative to each other to form different configurations;

[0971] The first plate and the second plate each have a sample contact area on their respective inner surfaces for contacting the fluid sample; wherein the sample contact areas face each other, are separated by an average spacing of 200 μm or less, and are capable of clamping the sample between them;

[0972] Heating layer:

[0973] It is positioned on the inner surface, outer surface, or inner side of one of the plates.

[0974] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0975] Cooling layer:

[0976] Located on the inner surface, outer surface, or inner side of one of the plates;

[0977] Configured for cooling-related sample volumes; and

[0978] It includes a material layer with a thermal conductivity and heat capacity ratio of 0.6 cm² / sec or greater;

[0979] The distance between the cooling layer and the surface of the relevant sample volume is zero or less than a distance configured such that the thermal conductivity per unit area between the cooling layer and the surface of the relevant sample volume is equal to 70 W / (m2·K) or greater.

[0980] One of the constructions is an open construction, wherein the two plates are partially or completely separated and the average spacing between the plates is at least 300 μm;

[0981] Another type of configuration among these is a closed configuration, which is configured after a fluid sample has been deposited onto one or two sample contact areas of an open configuration; and in the closed configuration: at least a portion of the sample is defined as a layer by two plates, wherein the average sample thickness is 200 μm or less; and

[0982] In some embodiments, the heating layer and the cooling layer are layers of the same material having heating and cooling zones, and the heating and cooling zones may have the same area or different areas.

[0983] AAA-1.4. A device for rapidly changing the temperature of a fluid sample, comprising:

[0984] The components include a first plate (10), a second plate (20), a spacer, a heating layer (112-1), and a cooling layer (112-2), wherein:

[0985] The first and second plates can be moved relative to each other to form different configurations;

[0986] Each of the first and second plates has a sample contact area on its respective inner surface for contacting the fluid sample; wherein the sample contact areas face each other, the average separation distance between them is 200 μm or less, and are capable of contacting the sample and clamping the sample between them;

[0987] One or two plates include spacers, and the spacers are fixed to the inner surface of the respective plates;

[0988] The spacers have a predetermined substantially uniform height of 200 micrometers or less, and a predetermined spacer spacing.

[0989] Heating layer:

[0990] It is positioned on the inner surface, outer surface, or inner side of one of the plates.

[0991] The relevant volume of the sample is configured to be heated, wherein the relevant volume of the sample is a portion or all of the sample being heated to the desired temperature; and

[0992] Cooling layer:

[0993] Located on the inner surface, outer surface, or inner side of one of the plates;

[0994] Configured for cooling-related sample volumes; and

[0995] It includes a material layer with a thermal conductivity and heat capacity ratio of 0.6 cm² / sec or greater;

[0996] The distance between the cooling layer and the surface of the relevant sample volume is zero or less than a distance configured such that the thermal conductivity per unit area between the cooling layer and the surface of the relevant sample volume is equal to 70 W / (m2·K) or greater.

[0997] 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 plates; and

[0998] Another configuration is a closed configuration, which is formed after the sample is deposited in the open configuration; and in this closed configuration: at least a portion of the sample is pressed into a layer of very uniform thickness by two plates, wherein the uniform thickness of the layer is defined by the sample contact surfaces of the plates and adjusted by the plates and spacers; and

[0999] In some embodiments, the heating layer and the cooling layer are layers of the same material having heating and cooling zones, and the heating and cooling zones may have the same area or different areas.

[1000] AAA-1.5. A device for rapidly changing the temperature of a fluid sample, comprising:

[1001] The first plate (10), the second plate (20), and the heating / cooling layer (112), wherein:

[1002] The first board (10) and the second board (20) face each other and are separated by a certain distance;

[1003] Each plate has a sample contact area on its respective inner surface (11, 21) for contacting the fluid sample; wherein these sample contact areas face each other, contact the sample, sandwich the sample between them, and have an average spacing (102) between them.

[1004] The heating / cooling layer (112) is located on the outer surface (22) of the second plate (20); and

[1005] The heating / cooling layer is configured to include a heating zone and a cooling zone; wherein the heating zone is configured to heat the fluid sample, and the cooling zone is configured to intensely cool the sample by thermal radiation.

[1006] The heating zone is configured to receive heating energy from a heating source and has an area smaller than the total area of ​​the heating / cooling layer; and

[1007] At least a portion of the heating zone of the heating layer overlaps with the sample area.

[1008] AAA-1.6. A device for rapidly changing the temperature of a fluid sample, comprising:

[1009] The first plate (10), the second plate (20), and the heating / cooling layer (112), wherein:

[1010] Each of the first plate (10) and the second plate (20) has a sample contact area on its respective inner surface (11, 21) for contacting a fluid sample; wherein the sample contact areas face each other, are separated from each other by an average spacing (102), and are able to contact the sample and clamp the sample between them.

[1011] The heating / cooling layer (112) has a thermal conductivity of 50 W / (m·K) or greater and is located on the outer surface (22), inner surface, or inner upper surface of the second plate (20); and

[1012] The heating / cooling layer is configured to include a heating zone and a cooling zone; wherein the heating zone is configured to heat a portion of the sample and has an area smaller than the total area of ​​the heating / cooling layer, and wherein the cooling zone is configured to cool the sample;

[1013] The heating zone, the second plate, and a portion of the sample are configured to have a scaling thermal conductivity ratio (STC ratio) of 2 or greater.

[1014] The heating zone is configured to receive heating energy from a heating source; and at least a portion of the heating zone of the heating layer overlaps with the sample area.

[1015] AAA-1.7. A device for rapidly changing the temperature of a fluid sample, comprising:

[1016] The first plate (10), the second plate (20), and the heating / cooling layer (112), wherein:

[1017] Each plate has a sample contact area on its respective inner surface (11, 21) for contacting the fluid sample; wherein the sample contact areas face each other, contact the sample, sandwich the sample between them, and have an average spacing (102) between them;

[1018] The heating / cooling layer (112) has a thermal conductivity of 50 W / (m·K) or greater and is located on the outer surface (22), inner surface, or inner upper surface of the second plate (20); and

[1019] The heating / cooling layer is configured to include a heating zone and a cooling zone; wherein the heating zone is configured to heat a portion of the sample and has an area smaller than the total area of ​​the heating / cooling layer, and wherein the cooling zone is configured to cool the sample;

[1020] The heating zone, the second plate, and a portion of the sample are configured to have a scaling thermal conductivity ratio (STC ratio) of 2 or greater.

[1021] The thermal conductivity of the heating / cooling layer multiplied by its thickness is in the range of 6×10⁻⁵ W / K to 3×10⁻⁴ W / K.

[1022] The heating zone is configured to receive heating energy from a heating source; and

[1023] At least a portion of the heating zone of the heating layer overlaps with the sample area.

[1024] AAA-2.1. The apparatus according to any of the foregoing embodiments, wherein the heating layer is configured to be heated by a heating source.

[1025] AAA-2.2. The apparatus according to any of the foregoing embodiments, wherein the heating layer is the same layer as the cooling layer, and the same layer includes a heating area and a cooling area.

[1026] AAA-2.3. The apparatus according to any of th...

Claims

1. An apparatus comprising: The first plate comprises a polymer or glass material and has a thickness of less than or equal to 100 μm; A second plate, comprising a polymer or glass material and having a thickness of less than or equal to 100 μm; and A heating / cooling layer is disposed on the first plate or the second plate, and the heating / cooling layer has a thermal conductivity between 6 × 10⁻⁵ W / K multiplied by the thickness of the heating / cooling layer and 1.5 × 10⁻⁴ W / K multiplied by the thickness of the heating / cooling layer. The heating / cooling layer is 15 μm or thinner and has a surface thermal radiation capacity of at least 50% that of a blackbody. The first plate and the second plate are arranged in parallel facing each other and spaced apart by a distance of 150 μm or less, and the first plate and the second plate are configured to receive a fluid sample sandwiched between the first plate and the second plate.

2. A method for rapidly changing the temperature of a sample, comprising: i. Providing an apparatus, the apparatus comprising a first plate, a second plate, and a heating / cooling layer, wherein: a. The first plate comprises a polymer or glass material and has a thickness of less than or equal to 100 μm; b. The second plate comprises a polymer or glass material and has a thickness of less than or equal to 100 μm; c. The heating / cooling layer is disposed on the first plate or the second plate, and the thermal conductivity of the heating / cooling layer is between 6×10-5W / K multiplied by the thickness of the heating / cooling layer and 1.5×10-4W / K multiplied by the thickness of the heating / cooling layer; as well as d. A clamp that presses the first plate and the second plate together to secure the two plates together, wherein the pressing applies pressure to the plates; ii. Deposit a fluid sample on one or both of the sample contact areas of the first plate or the second plate; iii. Press the plate down by hand so that the contact areas of the samples face each other. The first plate and the second plate are arranged in parallel facing each other and spaced apart by a distance of 150 μm or less, and the first plate and the second plate are configured to receive a fluid sample sandwiched between the first plate and the second plate. The pressure is applied to the periphery of the area where the sample is clamped, thereby reducing the amount of sample flowing out of the area.

3. A method for amplifying nucleic acids, comprising: A fluid sample containing nucleic acid is deposited on the first plate of the fluid device; The second plate is placed on the first plate such that the fluid sample is sandwiched between the first plate and the second plate, wherein the reagent for nucleic acid amplification is present on the inner surface of the second plate; Activate a heat source, which is configured to radiate electromagnetic radiation onto a heating layer located on the first plate or the second plate; Using at least the heating layer, at least a portion of the fluid sample is heated at a rate of at least 30°C / sec; as well as Nucleic acid amplification products accumulate in at least a portion of the fluid sample sandwiched between the first and second plates.

4. A method for detecting the presence or absence of a target nucleic acid sequence in a sample, comprising: A fluid sample containing nucleic acid is deposited on the first plate of the fluid device; A second plate is placed on the first plate such that the fluid sample is sandwiched between the first plate and the second plate, wherein reagents for nucleic acid amplification are present on the inner surface of the second plate, and wherein the reagents contain primers capable of hybridizing with the target nucleic acid; Activate a heat source, which is configured to radiate electromagnetic radiation onto a heating layer located on the first plate or the second plate; Using at least the heating layer, at least a portion of the fluid sample is heated at a rate of at least 30°C / sec; as well as The fluid sample is tested to determine whether it contains the amplification product of the target nucleic acid sequence.

5. A method for diagnosing a condition in a subject, comprising: A fluid sample from the subject is deposited on the first plate of the fluid device; The second plate is placed on the first plate such that the fluid sample is sandwiched between the first plate and the second plate, wherein the reagent for detecting the analyte is present on the inner surface of the second plate; Activate a heat source, which is configured to radiate electromagnetic radiation onto a heating layer located on the first plate or the second plate; Using at least the heating layer, at least a portion of the fluid sample is heated at a rate of at least 30°C / sec; as well as The fluid sample is tested to determine whether it contains the analyte. The presence or absence of the analyte indicates whether the subject has a disease.

6. A method for diagnosing a condition in a subject, comprising: A fluid sample from the subject is deposited on the first plate of the fluid device; The second plate is placed on the first plate such that the fluid sample is sandwiched between the first plate and the second plate, wherein the reagent for detecting the analyte is present on the inner surface of the second plate; A heat source is activated within a given time period, the heat source being configured to radiate electromagnetic radiation onto a heating / cooling layer located on the first plate or the second plate; The heat source is deactivated after the given time period, wherein at least a portion of the fluid sample adjacent to the heating / cooling layer is cooled at a rate of at least 30°C / sec after the deactivation; as well as The fluid sample is tested to determine whether it contains the analyte. The presence or absence of the analyte indicates whether the subject has a disease.

7. A method for diagnosing a condition in a subject, comprising: A fluid sample from the subject is deposited on the first plate of the fluid device; The second plate is placed on the first plate such that the fluid sample is sandwiched between the first plate and the second plate, wherein the reagent for detecting the analyte is present on the inner surface of the second plate; Activate a heat source, which is configured to radiate electromagnetic radiation to a heating layer located on the first plate or the second plate, wherein the heat source consumes less than 500mW of power. At least a portion of the fluid sample is heated using at least the heating layer; as well as The fluid sample is tested to determine whether it contains the analyte. The presence or absence of the analyte indicates whether the subject has a disease.

8. A method for diagnosing a condition in a subject, comprising: A fluid sample from the subject is deposited on the first plate of the fluid device; The second plate is placed on the first plate such that the fluid sample is sandwiched between the first plate and the second plate, wherein the reagent for detecting the analyte is present on the inner surface of the second plate; Activate a heat source, which is configured to radiate electromagnetic radiation onto a heating layer located on the first plate or the second plate; Using at least the heating layer, at least a portion of the fluid sample is heated at a rate of at least 30°C / sec; Quantify the amount of the analyte in the fluid sample; as well as Compare the amount with a control or reference amount of the analyte; A higher or lower amount of the analyte in the sample compared to the control or reference amount indicates that the subject has a condition.

9. A method for diagnosing a condition in a subject, comprising: A fluid sample from the subject is deposited on the first plate of the fluid device; The second plate is placed on the first plate such that the fluid sample is sandwiched between the first plate and the second plate, wherein the reagent for detecting the analyte is present on the inner surface of the second plate; A heat source is activated within a given time period, the heat source being configured to radiate electromagnetic radiation onto a heating / cooling layer located on the first plate or the second plate; The heat source is deactivated after the given time period, wherein at least a portion of the fluid sample adjacent to the heating / cooling layer is cooled at a rate of at least 30°C / sec after the deactivation; Quantify the amount of the analyte in the fluid sample; as well as Compare the amount with a control or reference amount of the analyte; A higher or lower amount of the analyte in the sample compared to the control or reference amount indicates that the subject has a condition.

10. A method for diagnosing a condition in a subject, comprising: A fluid sample from the subject is deposited on the first plate of the fluid device; The second plate is placed on the first plate such that the fluid sample is sandwiched between the first plate and the second plate, wherein the reagent for detecting the analyte is present on the inner surface of the second plate; Activate a heat source, which is configured to radiate electromagnetic radiation to a heating layer located on the first plate or the second plate, wherein the heat source consumes less than 500mW of power. At least a portion of the fluid sample is heated using at least the heating layer; as well as Quantify the amount of the analyte in the fluid sample; as well as Compare the amount with a control or reference amount of the analyte; A higher or lower amount of the analyte in the sample compared to the control or reference amount indicates that the subject has a condition.

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