Designs and methods for the readout of thermal contrast amplification signals

Abstract

A thermal contrast amplification assay reader includes a light emitting diode (LED) source element, a sensor, and I / O circuitry and an opening to receive sample. The reader is configured to compare changes in temperature of a test region on the sample to changes in temperature of background regions surrounding the test region.

Description

DESIGNS AND METHODS FOR THE READOUT OF THERMAL CONTRASTAMPLIFICATION SIGNALSFIELD

[0001] The present disclosure relates to diagnostics, and in particular to thermal contrast amplification, which can be applied to colorimetric samples such as lateral flow assays.BACKGROUND

[0002] LFA (lateral flow assay, or lateral flow immunoassay, also called rapid diagnostic test (RDT), or bioassays) technology has found widespread use both in and out of laboratory settings. In a typical assay, a fluid sample from a patient is applied to a test strip. The sample interacts with chemicals on the test strip causing the strip to optically change characteristics. The visual indicator may be observed by a person, for example, using a home pregnancy test.

[0003] Thermal contrast amplification (TCA) is a method that can be applied to colorimetric lateral flow assays (LFAs) and microfluidic (MF) samples to quantify and improve sensitivity and specificity. In particular, visual readouts like the color of a test line or fluorescence (in fluorescence immunoassays) are directly proportional to the incoming light. In contrast, the temperature signal in TCA builds up over time since it is an integrative signal of heat generation. While cooling effects or burning effects eventually limit the signal to be generated, and heat flow in the system across time can blur signals, integrative signals are still powerful in creating signals that rise above noise. In addition, these thermal signals on a dry sample are highly stable in contrast to fluorescence which typically decays due to photobleaching. Thus, at its core, TCA can be used for multiple readings to increase signaL to-noise ratios of dilute samples.

[0004] LFAs and MFs often come with colorimetric (visible color and optical density changes to the eye) indicators. Molecules in an LFA (often gold nanoparticles (GNPs)), may have a strong interaction with visible light, producing color changes that may be used for thermal contrast detection. These color changes are caused by changes in absorption and reflection of incident light. Such tests, especially LFAs, are commonly used for non-critical diseases for rapid diagnosis such as flu, strep, or COVID- 19. While they do not require any additional equipment for readout, they suffer from low sensitivity and a lack of quantitative readout.

[0005] Such samples could be made to have a visual readout with a more sensitive extra tag like fluorescence or magnetic particles. However, quality control typically reduces yields in manufacturing.

[0006] The core concept for TCA is that light is used to heat up those same molecules that are naturally used for the colorimetric readout.

[0007] Traditional TCA implementation uses temperature readouts based on infrared thermometry. The sensors in such implementations use changes in emission of long-infrared (7 to ~14um) electromagnetic radiation caused by temperature changes, per the Stefan- Boltzmann law (commonly known via blackbody radiation). However, such methods suffer from limited sources offering such sensors, costs, size, and export restrictions. While singlepixel infrared detectors may be used, either the sample must be moved along with a motor, hence increasing cost and size while decreasing robustness, or an array of such detectors must be used along with infrared optics, significantly increasing the costs.SUMMARY

[0008] In one aspect, a thermal contrast amplification assay reader includes a light emitting diode (LED) source element; a sensor; and I / O circuitry and an opening to receive sample; wherein the reader is configured to compare changes in temperature of a test region on the sample to changes in temperature of background regions surrounding the test region.

[0009] Implementation of further aspects may include one or more of the following features:

[0010] 2. The thermal contrast amplification assay reader of aspect 1, wherein changes in temperature of at least one of the test region and the background regions are measured by a proxy for temperature.

[0011] 3. The thermal contrast amplification assay reader of aspect 2, wherein changes in temperature of at least one of the test region and the background regions are inferred or measured by changes in electrical properties in the sample.

[0012] 4. The thermal contrast amplification assay reader of aspect 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in voltages across various pairs or sets of points in the sample.

[0013] 5. The thermal contrast amplification assay reader of aspect 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in resistance across various pairs or sets of points in the sample.

[0014] 6. The thermal contrast amplification assay reader of aspect 1, wherein the sample includes an array of electrical contact pins and voltage and / or resistance measurement components.

[0015] 7. The thermal contrast amplification assay reader of aspect 2, wherein changes in temperature of at least one of the test region and the background regions are inferred or measured by changes in optical properties in the sample.

[0016] 8. The thermal contrast amplification assay reader of aspect 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in color or optical density in the sample.

[0017] 9. The thermal contrast amplification assay reader of aspect 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in luminescence such as changes in brightness, color, and / or afterglow duration.

[0018] 10. The thermal contrast amplification assay reader of aspect 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in fluorescence or phosphorescence.

[0019] 11. The thermal contrast amplification assay reader of aspect 1, wherein the sensor is outside of an area of LED projection.

[0020] 12. The thermal contrast amplification assay reader of aspect 1, wherein the sensor is transparent or reflecting of thermal contrast assay LED light.

[0021] 13. The thermal contrast amplification assay reader of aspect 1, and further comprising a reflective coating between readout components and TCA-LED light configured to reflect away the TCA-LED light.

[0022] 14. The thermal contrast amplification assay reader of aspect 1, wherein reader components are transparent or reflecting to TCA-LED light.

[0023] In another aspect, a thermal contrast amplification assay reader includes a light emitting diode (LED) source element; a camera; and a lateral flow assay (LFA) tray comprising input / output (I / O) circuitry and an opening to receive an assay strip; wherein the camera is configured to capture images of the sample for color measurements and / or fluorescence changes in a test region on the sample and background regions surrounding the test region the sample.

[0024] In another aspect, a method of reducing heating of components of a thermal contrast amplification assay reader as shown and described herein is provided.

[0025] In yet another aspect, a method of reading temperature change in a sample for the analysis of thermal contrast, for the detection of antigens, drugs, and other molecules that aretargets of detection using the heating of colorimetric nanoparticles such as gold, silver, and carbon nanoparticles as shown and described herein is provided.

[0026] In still another aspect, a thermal contrast amplification assay reader includes a light emitting diode (LED) source element; a lateral flow assay (LFA) including I / O circuitry and an opening to receive an assay strip; and a sensor configured to detect compare changes in temperature of a test region on the sample to changes in temperature of background regions surrounding the test region.

[0027] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a simplified diagram showing a lateral flow assay or microfluidic test strip and traditional implementation of a TCA reader system using an infrared camera;

[0029] FIGS. 2-3 shows example sample designs where TCA-light is minimally absorbed by the components converting thermal changes into electrical signals;

[0030] FIGS. 4-5 show examples of sample designs where TC A-light is reflected away from the components converting thermal changes into electrical signals;

[0031] FIGS. 6-7 illustrate a simplified spatial readout scheme of electrical property changes in the reader and a sample readout circuit using (de)multiplexers to reduce component counts;

[0032] FIGS. 8A-8B illustrate how a simple thermochromic material, combined with illumination LEDs and a camera may be used to readout temperature changes in a spatial manner;

[0033] FIGS. 9A-9D show examples of where and how different layers of luminescent materials, fluorophores, or phosphors may be applied;

[0034] FIGS. 10A-10B show examples of how a phosphor with a reasonable delay between excitation light absorption and emission light release may be analyzed;

[0035] FIGS. 11A-1 IB show how spectral separation between TCA and optical readout may be performed;

[0036] FIGS. 12A-12E illustrate different optical configurations to perform TCA and optical readouts in the same system; and

[0037] FIGS. 13A-13B illustrate two example methods in which various components of the alternate readout methods may be shielded from the TCA light.DETAILED DESCRIPTION

[0038] Embodiments of the present disclosure describe systems and methods of reading out proxies of temperature changes in a thermal contrast amplification assay sample using a customized sample and reader.

[0039] TCA light refers herein to light used to generate thermal contrast. It is typically a laser or light emitting diode (LED). TCA-LED is defined as the LED used for generating the thermal contrast. The color, power, and size of LEDs are chosen to generate larger temperature changes in the test region than the background due to the absorption spectrum of the colorimetric particle.

[0040] A colorimetric particle that also contributes to the generation of thermal contrast is referred to herein as a Thermal-Contrast-Generating-Particle (TCGP). TCGPs are traditionally things like gold, silver, or platinum nanoparticles, but can also be based on latex, carbon or any number of other molecules that can be easily functionalized for molecular binding and can absorb visible light.

[0041] Readout-LED is used herein as an LED (or set of LEDs) used for reading out proxies of temperature changes that use optical readouts. For example, these may be used as the excitation light for phosphors and fluorophores., Alternatively, for thermochromic dyes and other color or optical density readout, these LEDs could be used to provide a broadband white light for color cameras or to assess color and / or optical density at specific wavelengths by providing narrowband colored light and used with monochrome cameras.

[0042] Readout-component is used herein as the part or parts built into the sample that will experience a temperature change due to the TCA-LED heating of colorimetric particles in the test region or the background material. Readout components can themselves be heated by the TCA-LED (component-self-heating). If this heating amount is low, it is not an issue. If it is high, design considerations to reduce or eliminate this effect are discussed.

[0043] Reader-sensor(s) is used herein as a part of the reader that interfaces or interacts with a sample to read out a proxy of temperature changes. Reader sensors themselves can be heated by the TCA-LED (sensor- self-heating). If this heating amount is low, it is not an issue. If it is high, design considerations to reduce or eliminate this effect are discussed.

[0044] Embodiments of the disclosure describe reflecting or transmitting of light. However, these terms can apply broadly. At the simplest, it may refer to something like clear glass that transmits a broad spectrum of visible light. However, for example, if green light is used, whether it is from TCA-LEDs or as emission spectra from a fluorophore, a bandpass ordichroic filter that may pass green light but not blue light would be considered transmitting as well. The context and meaning should be clear from the optical considerations of the designs, and a few examples are discussed further herein below.

[0045] A standard LFA or microfluidic chip is typically a membrane (LFA) or channels (MF) through which the sample solution liquid travels. To simplify, this layer is referred to as the channel (since membranes are microporous channels). These are on a backing material, called backing for short, often a clear flexible plastic (Mylar) whose primary necessary property is to prevent sample liquid from dropping out the bottom of the LFA or MF chip and to provide a mechanical support layer. Part of the function of the backing is that the membrane or channels also adhere quite well to the backing to prevent leakage from the sides.

[0046] A typical TCA reader design 100 is shown in FIG. 1. A typical sample for TCA includes a porous membrane layer (for LFAs) or channels (for MFs) 101 through which a sample solution will flow. The layer(s) 101 are built on top of a backing layer 102, typically a transparent plastic or glass layer. A typical colorimetric sample will have one or more test regions 103 which are specially coated and treated regions with fixed antibodies or similar specific binding molecules for a target analyte. As the solution flows through, the target analyte will be captured. In addition, upstream of this location, typically in solution or in a special location of the sample itself, the sample solution will have mixed with colorimetric particles (which also act as a TCGP) that are bonded to antibodies or similar binding molecules that attach to the target analyte. Thus, after the solution has gone through, there are a large number of TCGPs that will have bound to the fixed antibodies or similar in the test region, leading to the visible lines with sufficiently high concentration of target analytes. The control region, 104, is similarly designed, but made to directly bind the TCGP-antibody combination that flows through. Thus, there will be a noticeable color and TCGP accumulation in the control regions 104 regardless of the presence of the target analyte. In TCA, light 105, usually from lasers or LEDs, is applied to the sample. At a minimum, the test region 103 is illuminated, however better signals may be obtained if the membrane / channel regions surrounding the test region 103 are also illuminated. Control region 104 may or may not be illuminated. Upon illumination, the TCGPs in the test region 103, when combined with the background heating of the membrane / channel 101 there, generate more heat at the test region than in the surrounding membrane / channel regions with heating from only layer 101 which is typically white, translucent, or transparent. The heat generated is linear to the incoming TCA light intensity. The temperature change, ignoring cooling effects and heatdiffusion, is the time integral of the heat generated. This time integration further creates contrast in the resulting temperature. This contrast in temperature is the basis of TCA.

[0047] TCA has traditionally been implemented with the use of a thermal camera 107. This is typically the lowest cost approach as the infrared optics used for non-camera IR detectors to obtain good signal-to-noise ratios in such implementations are quite expensive. Contact methods such as attempting to apply thermocouples or thermistors at these regions often have significant drawbacks such as inconsistencies, lack of accurate measurements from the surrounding regions for a good “contrast” measurement, or heating of such sensors by the TCA light itself. To date, no reasonable alternative has been described that could provide the cost-effectiveness and signal-to-noise ratios that an economical thermal camera provides. Modifications of samples with tailored readers to perform TCA using alternative methods of temperature or proxy-of-temperature readouts are described further herein according to embodiments of the present disclosure.

[0048] It should be noted that the same or like reference numerals are used in different figures for same or similar elements. It should also be understood that the terminology used herein is for the purpose of describing embodiments, and the terminology is not intended to be limiting. Unless indicated otherwise, ordinal numbers (e.g., first, second, third, etc.) are used to distinguish or identify different elements or steps in a group of elements or steps, and do not supply a serial or numerical limitation on the elements or steps of the embodiments thereof. For example, "first," "second," and "third" elements or steps need not necessarily appear in that order, and the embodiments thereof need not necessarily be limited to three elements or steps. It should also be understood that, unless indicated otherwise, any labels such as "left," "right," "front," "back," "top," "bottom," "forward," "reverse," "clockwise," "counter clockwise," "up," "down," or other similar terms such as "upper," "lower," "aft," "fore," "vertical," "horizontal," "proximal," "distal," "intermediate" and the like are used for convenience and are not intended to imply, for example, any particular fixed location, orientation, or direction. Instead, such labels are used to reflect, for example, relative location, orientation, or directions. It should also be understood that the singular forms of "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0049] It will be understood that, when an element is referred to as being "connected," "coupled," or “attached” to another element, it can be directly connected, coupled or attached to the other element, or it can be indirectly connected, coupled, or attached to the other element where intervening or intermediate elements may be present. In contrast, if an element is referred to as being "directly connected," "directly coupled" or "directly attached" toanother element, there are no intervening elements present. Drawings illustrating direct connections, couplings or attachments between elements also include embodiments, in which the elements are indirectly connected, coupled or attached to each other.Alternate Method of Reading Out Temperature - Electrical

[0050] A number of implementations of reading out a proxy of the temperature, including the contrast between the test regions and the areas surrounding it, may be used. Such implementations include using changes in electrical properties induced by changes in temperature. In one embodiment, a reader for such samples includes pairs of contact pins (such as pogo pins) that contact the sample either directly or through electrical contact pads that are built or deposited onto the underside of the sample. Electrical circuits for accurate measurements of voltages and resistances are very economical, and many of the materials and manufacturing methods are well established.

[0051] It should be understood that any material that generates a certain voltage at a specific temperature, such as an array of thermocouples, possibly cross-linked with resistors, may be used without departing from the scope of the disclosure. Similar electrical potential generating methods may also be used.

[0052] Nickel-alloy thermocouples, platinum / rhodium-alloy thermocouples, tungsten / rhenium- alloy and other thermocouple type materials may also be used, with the contact joint for the two differing metals in the sample itself, for example embedded in the backing layer. The joints from an array of thermocouples may span the test region and surrounding regions.

[0053] It should be further understood that any material with resistivity that is temperature-sensitive may also be used. Thermistors are typically built by using metal oxides. Carbon-based resistor elements and thin-film elements can also be used. All of these methods are well established. Such materials can be placed in the backing layer of the sample.

[0054] Three exemplary designs utilizing the temperature-dependence of resistance in three materials are discussed below: Indium Tin Oxide (ITO)-coated (and similar transparent coating) glass and plastics; liquid crystals; and polished silicon.

[0055] ITO-films are used for touchscreens and a number of related technologies. They are transparent as a coating layer, with resistances that can be adjusted per manufacturing parameters. ITO-coated glass and plastics have very low heating from the TCA-LED due to the transparency of the materials, and thus can more accurately measure change in temperature in the test regions. In contrast, with a translucent or opaque film, any TCA-LED light coming through may lead to significant heating of the film itself, thus hiding thetemperature change signals due to the test region heating. Thus, the use of an ITO-film backing for an LFA or MF has high signal contrast. Manufacturing, processing, and handling of such films is also well established and economical. Similar conducting films and coatings that do not lead to absorption of the TCA light may be used. Thus, a coating may be colored as long as it does not lead to absorption of the TCA light.

[0056] FIG. 2 illustrates an example sample design in which TCA-light is minimally absorbed by the components converting thermal changes into electrical signals. In this implementation, a relatively standard membrane / channel 201 is used. The backing 202 may be a transparent plastic or glass substrate. Test region(s) 203, control region 204, and TCA light 205 are also as in a typical system. The difference in this implementation is that the temperature increases that conduct 206 through the system are measured. Backing 202 is coated with a thin-film conductive layer 207, such as ITO. Heat traveling down at arrows 206 then changes the conductivity of layer 207 in a spatially dependent manner. In this embodiment, the thermal conductivity through the membrane / channel layer 201 and backing layer 202 is controlled. For example, plastics typically conduct heat better than glass, and would likely be better materials for backing layer 202. With membranes, their density and material can affect thermal conductivity as well. Overall, with this implementation, a large amount of light 208 illuminating regions outside the test or control regions is either scattered, reflected, or transmitted as the components tend to be white, translucent or transparent, and thus not generating a large background thermal signal.

[0057] Another material that is relatively affordable with temperature-dependent physical characteristics are liquid crystals (LCs). LCs have properties similar to both liquids and crystals. Further, LCs can exist in various phases, and their optical and electrical properties are often tied to their temperature. LCs are typically known from LCD monitors (where electricity can orient LCs to adjust light transmission) and from economical forehead strip thermometers (where LCs that are temperature sensitive and change in color are used).

[0058] LCs may be designed to have temperature-dependent electrical resistivity, and this property, similar to the ITO film, may be leveraged to read out the temperature. For this use of liquid crystals, it is desirable for them to stay transparent and not change optical density (such as in LCD displays) or color (strip thermometers) since it could lead to the liquid crystals being heated by the TCA light. If the LC does change optical properties, it should not be in a way that increases absorption of TCA light or alternate mitigating factors such as a reflective coating above them (discussed later) should be used to block TCA light from reaching the LCs. With LCs remaining transparent, similar to ITO film, their transparencyalso reduces their heating by the TCA-LED. Any similarly transparent material that is conductive, with resistivity dependent on temperature may be used as the backing layer if it can provide sufficient mechanical robustness. Specially modified plastics (to have good electrical, and often also thermal conductivity) and conductive polymers are widely available. Alternatively, if the material itself is not sufficiently robust mechanically, an additional layer may be added. This layer may be a transparent, non-conductive layer with electrical contact pads, matching the reader’s contact posts, that will electrically connect to the liquid crystal or similar material in a manner allowing spatial readout of resistance changes. At the simplest, these could just be vias that reach through electrically in the same spatial pattern as the reader’s posts.

[0059] FIG. 3 illustrates another example sample design in which TCA-light is minimally absorbed by the components converting thermal changes into electrical signals. In this implementation, membrane / channel 301, test region 303, control region 304, and TCA light 305 remain the same as in a typical TCA. However, a special backing 302 is used. The backing 302 is either doped 307, or is made from specially-modified plastics, polymers, or other bulk material that is electrically conductive and optically transparent, with the conductivity changing with temperature. As in the implementation of FIG. 2, the implementation of FIG. 3 also uses differential heating between test region 303 and surrounding regions. A resultant spatial signal in the heat conducting to backing 302 may be measured as electrical conductivity changes that differ in space. As before, the optical properties of this implementation are such that a large portion of the light 308 illuminating outside the test or control regions will be scattered, reflected, or transmitted, and thus not generating a large background thermal signal.

[0060] In both implementations of FIG. 2 and FIG. 3, a transparent plastic backing and layer (202+207 or 302) would be advantageous. However, as an example, if TCA light that is green is being used, these layers could also be colored as long as the absorption of green light is low. Hence, these layers upon inspection under light may appear green if they transmit green light or selectively reflect green light and transmit the rest, as example. The backing layer and the doping or film coating do not absorb the TCA-light significantly, in one embodiment, since otherwise they would become a high source of heat generation in this system that would reduce detection of the desired heat signals from test regions.

[0061] In another implementation, instead of transparent, electrically conducting materials, reflective electrically conducting materials may be used. Silicon or germanium, orother semiconducting materials, particularly polished ones, are another exemplary material on which these samples may be based.

[0062] Semiconductors are known to have temperature-dependent resistances. In addition, many can be polished such that they would be heated less by the TCA-LED, and in fact reflect back some of the illumination into the test region, thereby increasing the amount of light available for generating the thermal contrast.

[0063] FIG. 4 illustrates a sample design in which TCA-light is reflected away from the components converting thermal changes into electrical signals. As before, membrane / channel 401, test region 403, control region 404, and TCA light 405 remain typical. However, the backing 402 used is a reflective conductive material, such as polished silicon. As before, the spatially distinct heat generation and resulting heat conduction 406 down to the electrically conductive material is used for the readout. However, in contrast to the implementations of FIGS. 2-3, light 408 is now transmitted back toward the membrane / channel 401 as well as toward test region 404. Thus, there is significantly enhanced illumination from the same light source that may be used for thermal contrast generation.

[0064] While a polished metal backing may be used as well for this purpose, the resistances and resistance changes of such a polished metal backing are typically much lower than that of semiconductors. Thus, while such polished metal backings may be used, more accurate and complex measurement approaches like a 4-wire measurement configuration are also often required in such a configuration.

[0065] Alternatively, any mildly conducting material of the correct thickness (such that the bulk resistance when measured from edge to edge of the sample width is in the range of 0.1 Ohms to IGOhms) may be measured with reasonably low cost electronics at high accuracy. If such a material is not or cannot be made transparent or highly reflective by polishing, a highly reflecting layer may be applied between the membrane / channel region and the backing made by this mildly conducting material.

[0066] FIG. 5 illustrates another sample design in which TCA-light is reflected away from the components converting thermal changes into electrical signals. As before, membrane / channel 501, test region 503, control region 504, and TCA light 505 remain typical. In this embodiment, backing material 502 may be made of any number of materials, including unpolished silicon (a more economical material than polished silicon); an opaque but electrically conductive plastic; a bulk layer made of materials typically used for thermistors; or the like. One factor for choosing the material is that the material should be amenable to a highly TCA-light-reflecting coating 507 such that layer 502 is not heated bythe TCA light directly, and is simply reporting the spatial pattern of temperature from heat conductance 506. Reflective layer 507 may be generated by a number of techniques, including electroplating or a number of chemical or vacuum deposition methods. A separate physical layer of noticeable and measurable thickness, with a mirror coating may alternatively be used, provided that the spatial heat map is transferred well through it and that its electrical conductivity properties does not prevent the accurate measurement of layer 502.

[0067] In FIG. 4 and FIG. 5, the reflection of light (408 or 508) need not be broadband (across a wide swath of the visible spectrum or beyond). Merely having high reflection of the TCA light is sufficient. For example, if the TCA light is blue, a longpass filter coating that reflects anything in the blue range would suffice as well. In such a case, the backing material 402 or 502 will not be exposed to the TCA light and thus will not generate unwanted background heat and temperature signals.Reader for sample with material with voltage or resistance changes

[0068] Using an array of thermocouples or thermistors as part of a reader does not typically work well, because it requires having a consistent and reliable transfer of heat to these temperature sensors. This is much more difficult than obtaining a reliable electrical contact. In addition, the size and fragility of such sensors could be an issue, and many (such as thermistors) tend to be opaque and would absorb the TCA-LED light.

[0069] With electrical contacts, there are several options. One, the contacts could be outside of the illumination area, as long as the electrical path whose resistance change is of interest is in between. Second, as most such electrical contacts are metallic or coated, they can be made highly reflecting and thus minimally absorbing of TCA-LED light.

[0070] FIG. 6 illustrates a simplified spatial readout scheme of electrical property changes in a reader and a sample readout circuit using (de)multiplexers to reduce component counts. Consider a sample 603 with test region 604 and control region 605. An illustrative reader comprises a circuit board 601 with electrical contact posts 602, such as spring-loaded pogo pins (not shown). The posts or pogo pins contact the underside of the sample 603. Optionally, the underside of sample 603 may further have metalized contact pads built into the sample as well. While multiple measurements may be made, at the most basic, consider the pairs of posts Al-Bl, A2-B2, A5-B5, and A6-B6 contacting the background regions surrounding the test region 604 and not contacting control region 605. Upon TCA light illumination, of the areas in between, a measurable but small change in resistance may be expected between Al and Bl, or A2 and B2, and so on due to background heating. For some instances, the changes may not be measurable due to the signal size being below the detectionlimit. In contrast, if the sample contains the target analyte, test region 604 will heat up more, resulting in a larger change in resistance between A3 and B3 and A4 and B4 than that seen outside of the test regions 604. It is also possible, but unnecessary, to obtain estimates of true temperature and temperature changes by simple calibration measurements of the resistances by using alternate temperature measurement methods during production or quality control, for example.

[0071] Variations of the design for the sample or the reader include conductive leads leading out from the test regions and surrounding regions such that the contact points could be placed safely out of the TCA-LED illumination range. These leads could connect to a pad on the edges, or the bottom side, or connect through the thickness of the sample. While a simple 2 column, n row array of contacts is shown for the reader side, any number of arrangements providing spatial sampling of electrical properties in which pairs of points can be accessed for measurement could be used without departing from the scope of the disclosure.

[0072] FIG. 7 illustrates another simplified spatial readout scheme of electrical property changes in the reader and a sample readout circuit using (de)multiplexers to reduce component counts. A test voltage at 701 is applied. The electrical signals (voltage or resistance) across pairs of pins 702 may be measured with relatively simple circuitry. As the components for such measurements are cheap, it is quite economically reasonable to have a reader with many parallel circuits to do this measurement (not shown). However, alternatively, analog multiplexers / demultiplexers 703 and 704, relays, or any number of methods to reduce the number of circuits used in measurement while retaining the ability to address the individual pins may be used. At the simplest, for performing a resistance measurement, a high-tolerance resistor of suitable resistance 706, a voltmeter 705 or a voltage analog-to-digital converter, possibly equipped with an external amplifier such as an instrumentation amplifier, and stable test 701 and reference 707 voltages may be used.Alternate Method of Reading Out Temperature - Optical

[0073] In other embodiments, a number of implementations of reading out a proxy of the temperature, including the contrast between the test regions and the areas surrounding it, using changes in optical properties induced by changes in temperature, may be used. A reader for such samples is described later, but at the simplest, such a reader includes a standard camera or one equipped with at most some optical filters and LEDs of specifications that are more common than the ones used for TCA-LEDs. Some implementations may lead to readers that are still cheaper than those based on infrared cameras. Others may be of similar cost, butmarket forces from the smartphone and drone industries and the ever advancing technologies can help drive future costs down or improve signal-to-noise ratios. In addition, the likelihood of relying on sole sources for such cameras is extremely low given the competitive field.Thermochromic dyes and liquid crystals (LCs)

[0074] As discussed before, LCs can have a multitude of electrical and optical sensitivities to temperature. Thermochromic dyes and LCs change color upon changes in temperature. They are low cost as well. A well-known example is the simple forehead thermometer that changes color. One implementation is to use such a strip as the backing of the LFA. The color may be simply readout by a color camera with a white LED illumination. Alternatively, a monochrome camera with multiple colors of Readout-LED may be used to determine the color. Such thermochromic LCs may be further manipulated by electrical fields depending on construction to enhance color changes or to shift the colors. In other words, electrical fields can also be used to help bias a thermochromic LC. One example uses a thermochromic LC that has a small temperature range in which it changes from a red color to a blue color. The electricity applied may be used to bias the thermochromic LC such that this temperature range is shifted according to ambient temperature. One example shown in FIG. 8A is to use two rails of different voltages 801 and 802 in a reader that contact the two edges of the sample strip’s thermochromic layer 800. Note that while the conversion process is from TCA-light to heat back to color, the time-integrative nature of TCA may help in creating stronger signals.

[0075] FIG. 8B illustrates one embodiment in which a thermochromic material, combined with illumination LEDs and a camera, may be used to readout temperature changes in a spatial manner. In this implementation, a relatively standard membrane / channel 811 is used. The backing 812 may be any number of materials that can transmit temperature changes 816 from the membrane / channel well. Test region(s) 813, control region 814, and TCA light 815 are also as in a typical system. One different component is a mirror-ized reflective layer 817 that reflects back a large portion of the TCA light 818, increasing effective light intensity for a given LED and collection optics. Furthermore, this layer 817 prevents the TCA light from heating the thermochromic layer 819. The layer 819 will change color or amount of light absorption over reflection depending on the temperature. An external light source 820, such as a low-cost white LED (for use with a color camera) or strobed LEDs of different colors (for use with a monochrome camera) may be used in conjunction with a camera 821 to obtain a spatial map of the temperature changes. The camera 821 may be considered a “regular” or “standard” camera that operates in the visible light spectrum, but different cameras may beused for different light spectra if those different light spectra are used, without departing from the scope of the disclosure.Phosphor thermometry, thermoluminescence, fluorescence thermography

[0076] A number of materials show a temperature dependence of emitted light, including brightness, color, and afterglow duration. Note that this is light emitted by the material, as opposed to differentially reflected as in the thermochromic dye case. A Readout-LED is not needed in this case of luminescence. Instead, the color or intensity of the emitted light is observed. The luminescent material may substitute the fluorophores and phosphors in subsequent descriptions.

[0077] In addition, a number of materials show a temperature dependence of light that is re-emitted (emission light) upon absorption of an excitation light. Depending on the time scales and mechanisms, these may be called fluorescence (typically sub-nanosecond reemission of light) or phosphorescence (microseconds and beyond, such as in glow-in-the- dark toys). A Readout-LED is used in these cases to provide the “excitation” light that is absorbed for the re-emission.

[0078] For fluorescence, due to the near immediate time response of the emitted light, one method to detect temperature change is the change in intensity of emitted light. Typically, an increase in temperature of a fluorescence molecule leads to a drop in emission light as well as a slight shift in the emission spectra. Note that this approach is still distinct from fluorescence immunoassays that are commonly used. First is that compared to most fluorescence immunoassays which require a special reader for any result, embodiments of the present disclosure using fluorescence still retain a colorimetric visual component. Second, compared to those with double-labeling of antibodies or other complex organic molecules, for example with a gold nanoparticle with attached fluorophores, a separate approach is used in the present disclosure. Such double-labeling can be expensive with low yields passing quality control. In contrast, applying a fluorophore to a sheet material can often easily be done in high volume or surface areas with manufacturing methods based more in physics and chemistry, making consistent quality and high yields more possible. Furthermore, changes in fluorescence signals in samples according to embodiments of the present disclosure still rely on the time-integrated signals of heating, in contrast to the simple momentary and immediate re-emission of absorbed light by fluorophores. Thus, the readout, or any proxy of heat buildup over time, has a strong natural mechanism for stronger signals.

[0079] A number of configurations and variations may be used for making fluorescence or phosphorescence compatible with the sample by applying thin layers, films, or coatings or doping materials with fluorophore or phosphor. For example:

[0080] 1. Channel doping - Read from top or bottom. If from the bottom, the layers below(backing, etc.) must be transparent to the excitation and emission light of the fluorophore or phosphor.

[0081] 2. Backing, top-layer coating - A coating of a fluorophore or phosphor is applied to the topside of the backing. Can be read from top or bottom. With LFAs, signals will be better from the bottom as the membrane is somewhat light-scattering. If reading from the bottom, an optional first top-layer mirror above the doping layer or film may be used, and such a mirror may further increase both TCA-light that is effectively collected and the optical signals read below.

[0082] 3. Backing doping - this is similar to above, but the body of the backing layer is doped instead of having a film or coating layer. Again, an optional first top-layer mirror may greatly improve signals.

[0083] 4. Backing, bottom-layer coating - Again, similar to above, but with the bottommost layer coated.

[0084] FIGS. 9A-9D illustrate examples of where and how different layers of luminescent materials, fluorophores, or phosphors may be applied.

[0085] FIG. 9A illustrates the case where the membrane or channel 901 on a backing layer 902 is doped with a fluorophore or phosphor 907. In such a case, the heat 906 generated by differential absorption of the TCA light 905 at test region 903 or control region 904 will lead to differential changes in fluorescence or phosphorescence through the body of this layer 901 due to the dopant 907.

[0086] FIG. 9B illustrates a case where fluorophore or phosphor is applied to the top of the backing. If the optional reflective layer 918 is not used, for the case of a microfluidic channel, this approach in some embodiments may be identical to the case in Fig9A depending on the channel construction. This reflective layer 918, however, can be useful for increasing signals and simplifying the choice of TCA and optical readout colors, though with other costbenefit considerations such as manufacturing cost, including reduced yields or additional quality control measures. In FIG. 9B, instead of doping the channel or membrane layer 911, a doped layer or film 917 is added below the channel or membrane layer 911. The backing layer 912 lies below. As before, TCA light 915 can cause a differential heating pattern 916due to the test region 913 or control region 914. The heat 916 travels through the body of the sample, affecting phosphorescence or fluorescence properties of dopants in 917.

[0087] FIG. 9C illustrates a case where this fluorophore or phosphor is applied to the bottom surface of the backing layer. If the reflective layer 928 is used, the backing 922need not be transparent. In addition, reflective layer 928 will allow more flexible selection of the part of the electromagnetic spectrum used for both TCA light and the fluorophore / phosphor excitation and emission light. Membrane / channel layer 921, backing layer 922, test region 923, control region 924, and TCA light 925 remain typical. The heat 926 travels through the body of the sample, affecting phosphorescence or fluorescence properties of dopants in 927.

[0088] FIG. 9D illustrates a case where this fluorophore or phosphor 937 is embedded through the thickness or substantial portion of the backing layer 932. If the reflective layer 928 is used, the backing 922need not be transparent. Membrane / channel layer 931, test region 933, control region 934, and TCA light 935 remain typical. The heat 936 travels through the body of the sample, affecting phosphorescence or fluorescence properties of dopants in 937.

[0089] For phosphorescence, due to the time-delay between excitation light absorption and emission light release, a number of factors of the emitted light may he analyzed. These factors may include amplitude; phase or time delays between excitation light and emitted light; and changes in decay time, shape of emitted light, and frequency components. With a microsecond-to-millisecond time-scale phosphor, these can easily be done at various points in the TCA-LED heating of the test regions. Control and measurements at such timescales are simple and low-cost with modern electronics.

[0090] FIGS. 10A-10B illustrate examples of how a phosphor with a reasonable delay between excitation light absorption and emission light release can be analyzed. Shown are two example illumination patterns in time for a phosphor with a delay between excitation light absorption and emission light release, particularly in the range of lus to 100ms. In FIG. 10A, the excitation light may be sinusoidally modulated 1001. In FIG. 10B, the excitation light may be in square pulses 1003. The resulting time-delayed light 1002 (FIG. 10A) or 1004 (FIG. 10B) can be analyzed for a number of parameters, such as phase offsets of the troughs or peaks for sinusoidally-modulated light or the delay from the start or end of illumination pulses to peaks or troughs. In addition, the amplitude of the emitted light, as well as measurements of distortions, shapes, and frequency components can also be analyzed. The examples shown and described are exemplary, but are not meant to be limiting on the modulation shape of the excitation light or the parameters analyzed from the emitted light. Inaddition, while the emission delay of on the order of about 1 microsecond (ps) to about 100 milliseconds (ms) is chosen due to the relative simplicity and cost of electronic components necessary and to obtain a timely measurement of temperature changes which is a highly dynamic process, other emission delays may also be utilized.

[0091] If the TCA-LED light will reach the fluorophore or phosphor and the Readout- LED light reaches the TCGPs in the test regions, additional considerations may be taken. First, it should be noted that the intensity and duration of light used for the readout is typically an order or two of magnitude lower than for TCA. Thus, even with a broadly- absorbing TCGP, the contributions to heating from the Readout-LED will be small. Temporal separation is useful as well. This could be a very simple scheme such as having TCA-LED light on for a certain duration, then having the Readout-LED on for a certain duration during which correlates of temperature are measured. This cycle could be repeated. However, the largest concern typically would come from the photobleaching of the fluorophore or phosphor with the TCA-LED. In those cases, using fluorophores and phosphors with excitation and emission spectra far from the TCA-LED spectra, with the option of using a TCGP with spectra far from the fhiorophore / phosphor spectra, would be desirable.

[0092] If using silver-NPs, typically use blue LEDs for TCA. Use yellow excitation, red emission or longer wavelengths for the fluorosphere or phosphor.

[0093] If using gold-NPs, typically green LEDs can be used for TCA. For some larger or specialty shapes of gold-NPs, reddish LEDs can be used for TCA. Use near-UV / blue or red / near IR fluorophores and phosphors.

[0094] For black- NPs (many carbon-based nanoparticles), typically any color LED can be used for TCA. While more colors could be combined for TCA illumination, the optics cost increase with some diminishing returns on total power. In addition, spectral separation limits how much this can be done. Still, with such a broad-absorbing nanoparticle a TCA-LED color and fluorescence-Ex / Em color may be chosen relatively freely.

[0095] FIGS. 11A-11B illustrate how spectral separation between TCA and optical readout may be performed. In FIGS. 11A-11B, spectral separation is described with respect to two realistic examples. In the first, shown in FIG. HA, the TCA-LED spectrum 1101 is in the 600 nanometer (nm) range, of amberish color. The TCGP may have an absorption spectrum 1102 A that is narrow but in that range, such as a large or specialty shape gold nanoparticle, or could have a broad excitation spectrum 1102B such as those seen in black (commonly carbon-based) nanoparticles. The fluorophore or phosphor chosen may have a much lower excitation 1103 and emission 1104 wavelength. A blue / green excitation andgreen / yellow emission fluorophore, for example, is quite common. The absorption by the TCGP in the broad spectrum case (1102B) of the excitation light 1103 may be reduced as the power and durations for such measurements may be made orders of magnitude less than that for TCA light.

[0096] Similarly, as shown in FIG. 11B, a TCA-LED spectrum 1111 is shown in the 400nm range, of blueish color. The TCGP may have an absorption spectrum 1112A that is narrow but in that range, such as a silver or platinum nanoparticle, or could have a broad excitation spectrum 1112B such as those seen in black (commonly carbon-based) nanoparticles. The fluorophore or phosphor chosen may have a much higher excitation 1113 and emission 1114 wavelength. A yellow / red excitation and an orange / near-IR emission fluorophore, for example, may be used. The absorption by the TCGP in the broad spectrum case 1112B of the excitation light 1 113 may be reduced as the power and durations for such measurements may be made orders of magnitude less than that for TCA light.Reader examples for sample with materials with changes in optical properties

[0097] In various embodiments, example readers include a CMOS or CCD camera. There may be additional illumination LEDs (Readout-LED). For fluorescence or luminescence, there may be optical filters selecting for narrower bands of the electromagnetic spectrum placed in front of the Readout-LEDs or camera. Dichroic mirrors may be used to compact or simplify the imaging pathway.

[0098] FIGS. 12A-12E illustrate some possible configurations for readers:

[0099] 1. Read from top, with TCA and readout paths separate in Fig 12A

[0100] 2. Read from top, with TCA and the camera paths merging in Fig 12B

[0101] 3. Read from top, with TCA, Readout-LED, and camera paths merging in Fig 12C

[0102] 4. Read from bottom, with TCA and readout paths separate in Fig 12D

[0103] 5. Read from bottom, with readout- LED, and camera paths merging in Fig 12E

[0104] Discussions of some considerations for implementation are described below. Also, note that the “top” and “bottom” sides may be flipped depending on the construction and optical properties of the sample.

[0105] 1. As shown in FIG. 12 A, when the TCA and readout paths 1200 are separate, it is likely one or both pathways will not quite be “head-on” or orthogonal to the sample 1202A. In this case, since the heating process is more complex, one design has the TCA-LED 1210 projected head on to reduce or eliminate inhomogeneity of the TCA-LED light 1212 on the sample 1202 A. Angling of light, for example, may create inhomogenous heat generation that may be more complicated to account for, and the spread of the heat in time may play a largerpart and should be carefully considered. Such designs would require more calibrations on blank samples, and would benefit even more from heat generation and flow models and data from actual samples. In contrast, thermal-proxy readout by light can be done relatively simply since the processes are typically linear with light intensity at the moment, and simpler field corrections for any inhomogeneity of excitation light done with calibration measurements and in software. At the simplest, a normalization for each pixel is just done once as part of calibration. Thus, angled illumination and imaging may be performed with fewer corrections needed.

[0106] 2. As shown in FIG. 12B, when luminescence is used, the light emitted from a sample 1202B as its temperature changes is all that is used in one embodiment. For this, if the wavelength of this emitted light is different from the TCA-LED light 1212, the implementation is trivial. One uses a dichroic filter mirror, and optionally additionally a spectral filter on the camera to perform measurements. In addition, strobing or other ways of alternating times of heating by turning on the TCA-LED 1210 and measurement times may be easily performed with simple circuits and software control.

[0107] 3. As shown in FIG. 12C, when fluorescence or phosphorescence is used for a top-side measurement, and the TCA-LED is on the same side, two or more dichroic filters may be used. Again, spectral separation tends to provide the simplest solution, though temporal separation as described before may be applied alternatively or in conjunction.

[0108] 4. and 5. As shown in FIGS. 12D and 12E, for cases in which the TCA-LED light comes through the sample, spectral separation is recommended, though temporal separation can also be used alternatively or in conjunction. However, if the TCA-LED light does not go through the sample 1202D, 1202E, for example due to a reflective layer in the sample, spectral and temporal separation are not required, and the problem is greatly simplified from both sample design and reader design perspectives.Preventing Heating of the Readout-Component or Reader-Sensors (sensor-self-heating) by the TCA-LED Illumination

[0109] For macroscopic materials on the sample, such as thermocouple wires and junctions, or opaque materials (especially those that would absorb the TCA-LED light well at room temperatures), additional construction considerations to reduce the heating of the sensor materials themselves are useful to enhance sensitivity. Similarly, if Readout-Sensor components absorb the TCA-LED light, they must be similarly protected in certain cases. The following discussions apply primarily to the electrical readouts, but can also apply to optical readouts, especially readouts of temperature based on color, fluorescence, orphosphorescence, where the TCA light may actively interfere with the readout if proper wavelength separation is not done.

[0110] To the first approximation, the following general design principles should be considered:

[0111] 1. If the TCA light can be prevented from reaching any components of the sample or reader that may absorb light, excluding the test region and the surrounding background, and without heating up itself, that method is preferred. At the simplest, this is a highly- reflecting mirror layer just below the channel layer. At the cheapest, one can think of polished aluminum foil or the mirror-reflective plastic films from part and hobby shops. At the high end, one could have high quality mirror surfaces that use more advanced silvering or dielectric coating methods. Since these layers are thin and / or highly thermally conductive, they will allow the thermal signatures to travel down below where the TCA light does not reach. Considerations would be factors such as cost-benefit ratio and quality control considerations.

[0112] 2. Consider the electrical Readout-Sensor scenario with pogo pins. First, these pogo pins tend to be highly reflective. They could be made further reflective. In addition, even if they do heat up, the changes in resistance of these contacts with already negligible resistance is small compared to the resistance changes across, for example, ITO film or silicon.

[0113] 3. Simple optical techniques like limiting the TCA light, such as projective TCA-LED light to just in between the pogo pin locations may also be used.

[0114] FIGS. 13A-13B illustrate two example methods in which various components of the alternate readout methods may be shielded from the TCA light.

[0115] In FIG. 13 A, it is shown that reflective layers can be quite helpful. For a generic membrane / channel 1301, with test region 1304, control region 1305, and TCA light 1306, a very broadly applicable method is to apply a reflective mirror layer 1302 to prevent the TCA light from reaching the backing layer 1303. In addition, the layer 1302 helps reflect TCA light upwards 1307, including to the test region 1304, thereby increasing effective light collection and signal amplitude. Note that layer 1302 need only reflect TCA light, and thus could be a bandpass, notch, longpass, or shortpass filter as well.

[0116] In addition, the LED projection area can be limited, as shown in FIG. 13B. In FIG. 13B, sample 1310 is amenable to TCA readout via electrical means. Test region 1311 and control region 1312, and some surrounding regions, may be contacted via electrical contact posts 1313 on a circuit board 1314. If the TCA-LED light 1315 reaches the posts 1313, andthe posts are absorbing of the light, then they will possibly heat up and alter the heating pattern and electrical changes. However, the TCA-LED may be projected to an area in between the posts, resulting in temperature and hence electrical property changes within the sample 1310, which can be detected while avoiding heating of posts 1313.

[0117] The illustrations of the embodiments described herein are intended to provide a general understanding of the structure of the various embodiments. The illustrations are not intended to serve as a complete description of all of the elements and features of apparatus and systems that utilize the structures or methods described herein. Many other embodiments may be apparent to those of skill in the art upon reviewing the disclosure. Other embodiments may be utilized and derived from the disclosure, such that structural and logical substitutions and changes may be made without departing from the scope of the disclosure. Additionally, the illustrations are merely representational and may not be drawn to scale. Certain proportions within the illustrations may be exaggerated, while other proportions may be reduced. Accordingly, the disclosure and the figures are to be regarded as illustrative rather than restrictive.

[0118] One or more embodiments of the disclosure may be referred to herein, individually and / or collectively, by the term “invention” merely for convenience and without intending to limit the scope of this application to any particular invention or inventive concept. Moreover, although specific embodiments have been illustrated and described herein, it should be appreciated that any subsequent arrangement designed to achieve the same or similar purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all subsequent adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the description.

[0119] The Abstract is provided to comply with 37 C.F.R. § 1.72(b) and is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, various features may be grouped together or described in a single embodiment for the purpose of streamlining the disclosure. This disclosure is not to be interpreted as reflecting an intention that the claimed embodiments employ more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter may be directed to less than all of the features of any of the disclosed embodiments.

[0120] The above-disclosed subject matter is to be considered illustrative, and not restrictive, and the appended claims are intended to cover all such modifications, enhancements, and other embodiments, which fall within the true spirit and scope of the present disclosure. Thus, to the maximum extent allowed by law, the scope of the present disclosure is to be determined by the broadest permissible interpretation of the following claims and their equivalents, and shall not be restricted or limited by the foregoing detailed description.

Claims

WHAT IS CLAIMED IS:

1. A thermal contrast amplification assay reader, comprising: a light emitting diode (LED) source element; a sensor; andI / O circuitry and an opening to receive sample; wherein the reader is configured to compare changes in temperature of a test region on the sample to changes in temperature of background regions surrounding the test region.

2. The thermal contrast amplification assay reader of claim 1, wherein changes in temperature of at least one of the test region and the background regions are measured by a proxy for temperature.

3. The thermal contrast amplification assay reader of claim 2, wherein changes in temperature of at least one of the test region and the background regions are inferred or measured by changes in electrical properties in the sample.

4. The thermal contrast amplification assay reader of claim 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in voltages across various pairs or sets of points in the sample.

5. The thermal contrast amplification assay reader of claim 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in resistance across various pairs or sets of points in the sample.

6. The thermal contrast amplification assay reader of claim 1, wherein the sample includes an array of electrical contact pins and voltage and / or resistance measurement components.

7. The thermal contrast amplification assay reader of claim 2, wherein changes in temperature of at least one of the test region and the background regions are inferred or measured by changes in optical properties in the sample.

8. The thermal contrast amplification assay reader of claim 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in color or optical density in the sample.

9. The thermal contrast amplification assay reader of claim 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in luminescence such as changes in brightness, color, and / or afterglow duration.

10. The thermal contrast amplification assay reader of claim 2, wherein changes in temperature of at least one of the test region and the background regions are measured by changes in fluorescence or phosphorescence.

11. The thermal contrast amplification assay reader of claim 1, wherein the sensor is outside of an area of LED projection.

12. The thermal contrast amplification assay reader of claim 1, wherein the sensor is transparent or reflecting of thermal contrast assay LED light.

13. The thermal contrast amplification assay reader of claim 1, and further comprising a reflective coating between readout components and TCA-LED light configured to reflect away the TCA-LED light.

14. The thermal contrast amplification assay reader of claim 1, wherein reader components are transparent or reflecting to TCA-LED light.

15. A thermal contrast amplification assay reader, comprising: a light emitting diode (LED) source element; a camera; and a lateral flow assay (LFA) tray comprising input / output (I / O) circuitry and an opening to receive an assay strip; wherein the camera is configured to capture images of the sample for color measurements and / or fluorescence changes in a test region on the sample and background regions surrounding the test region the sample.

16. A method of reducing heating of components of a thermal contrast amplification assay reader as shown and described herein.

17. A method of reading temperature change in a sample for the analysis of thermal contrast for the detection of antigens, dmgs, and other molecules that are targets of detection using the heating of colorimetric nanoparticles such as gold, silver, and carbon nanoparticles as shown and described herein.

18. A thermal contrast amplification assay reader, comprising: a light emitting diode (LED) source element; a lateral flow assay (LFA) including TO circuitry and an opening to receive an assay strip; and a sensor configured to detect compare changes in temperature of a test region on the sample to changes in temperature of background regions surrounding the test region.

Images