Tricorder reflectometer for lateral flow immunoassays

Abstract

A reflectometer and method for use in measuring the amount of analyte in a sample are provided. The reflectometer can include multiple readheads for obtaining reflectance information from discrete areas on a lateral flow test strip. The reflectance information is processed to obtain digital values ​​for the reflectometer. The discrete areas can include a test zone and at least two control zones, each having a different amount of detection moiety-binding substance. The digital values ​​for each control zone can be used to establish a high-end standard and a unique low-end standard. Extrapolating the digital values ​​for the test zones to the standards provides an accurate quantitative value of the analyte in the sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 071,464, filed August 28, 2020, the contents of which are incorporated by reference. [Background technology]

[0002] Lateral flow immunochemical tests are well known for detecting the presence or absence of analytes in samples of blood, urine, or other bodily fluids. These tests typically utilize test strips that immobilize or stabilize an analyte binding zone and a detection moiety binding zone. The bodily fluid sample can be treated, either beforehand or on the test strip, with a detection moiety or analyte label that binds to the analyte. This creates a conjugated analyte that provides a visual or other detectable signal indicating the presence of the analyte in the bodily fluid sample. When the bodily fluid sample is placed on the test strip, it migrates through the strip until it reaches the control zone and analyte binding zone. The conjugated analyte with the attached detection moiety binds to the analyte binding zone, thereby capturing the analyte and the attached detection moiety, indicating the presence of the analyte in the bodily fluid. Excess detection moiety that does not bind to the analyte in the sample will only bind to the control zone, indicating the test strip has functioned. In some cases, the darkness or intensity of the conjugated analyte bound to the analyte binding zone provides a basic qualitative indication of the analyte concentration in the bodily fluid sample. In other cases, a control zone contains a known concentration of a detection moiety binding substance and can be used for comparison with conjugated analyte bound to the test zone.

[0003] A recent development is a lateral flow immunochemical test that utilizes two control zones in which different concentrations of detection moiety-binding material are immobilized or stabilized. During the test, different amounts of detection moiety are bound or captured in each control zone. Several detection moieties are known in the art, with colloidal gold being the most commonly used. Detection moieties, which use reflected light as a measure of concentration bound to each zone, produce a color and / or intensity that can be detected and measured using a reflectometer. By comparing the reflectance and / or color of each control zone to a predetermined extrapolation curve, a unique high-end and a unique low-end specification can be established for a particular test strip. The amount of conjugated analyte bound to the test zone can then be determined by comparing the reflectance measurements to high-end and low-end specifications calculated for the test strip or a manufacturing "lot" of test strips. The accuracy of this test depends on the reflectance meter's ability to accurately detect the amount of reflectance from the detection moiety bound to the control zone and the conjugated analyte-binding zone. U.S. Patent No. 5,929,494 describes a reflectometer that uses a feedback mechanism and temperature to calibrate for external factors that adversely affect the measurement of reflectance from the detection moiety bound to the test strip. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 6,574,425 [Patent Document 2] U.S. Patent Application Publication No. 2021 / 0063390 Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need for a reflectometer that can more accurately measure reflectance from test strips. It would be beneficial if the reflectometer utilized multiple control zones that could establish a baseline to facilitate interpolation of test results. Ideally, the reflectometer would be able to compensate for the same external factors in the control and test zones, as well as variations that may exist between tests and test strips. [Means for solving the problem]

[0006] According to embodiments of the present invention, the problem of accurately measuring the amount of detection moiety bound to a discrete area on a test strip is solved by a reflectometer embodiment that includes a dedicated read head for each discrete area on the test strip. The read head may include a light source directed at the discrete area. Light is reflected from the discrete area at a wavelength that produces a color that can be captured, analyzed, and compared to programmed information within the reflectometer to provide an accurate measurement of the amount of analyte present in the sample of bodily fluid. Advantageously, the reflectometer can be calibrated for use with a particular test strip or manufactured "lot" of test strips, reducing the effect of test strip-to-test strip variability on the results.

[0007] A reflectometer may be particularly advantageous for test strips comprised of two or more distinct control zones, each with a different amount of detection moiety-binding material. After a test is performed, the reflectometer can measure the different, known amounts of detection moiety bound to the two or more control zones. The reflectometer can also measure the amount of conjugated analyte bound to the distinct test zones. Each control zone can have a dedicated read head for independently emitting light and receiving reflected light from the control zone. The test zone can also have a dedicated read head for emitting light and receiving reflected light from the test zone. Each read head can transmit its respective reflectance information to a microprocessor. Additionally, background reflectance from the test strip can be measured and transmitted to the microprocessor. Temperature can also be measured and used to compensate for the effect of reflectance on the reading.

[0008] The microprocessor can be programmed with an algorithm that utilizes a model extrapolation or "standard" curve that can fit reflectance measurements from two or more control zones. Background reflectance can also be subtracted from each reflectance measurement from an individual zone to provide a more accurate measure of the detection moiety bound to each individual zone. Reflectance measurements from the test zones can then be interpolated against the fitted curve to determine the reflectance value from the test zone. The microprocessor can also be programmed with one or more look-up tables (LOTs) that can compare test zone reflectance values ​​to determine the exact amount of analyte in the sample. This process can eliminate the need to attempt to compensate or calibrate for external factors by assuming that each individual region is similarly affected and then compensated for by fitting the extrapolation curve.

[0009] In one embodiment, the reflectometer may include a case, a first readhead having first scanning optics configured to illuminate a first control zone of the test strip and receive first light from the first control zone, a second readhead having second scanning optics configured to illuminate a test zone of the test strip and receive second light from the test zone, a third readhead having third scanning optics configured to illuminate a second control zone of the test strip and receive third light from the second control zone, and a microprocessor in operative communication with the first readhead, the second readhead, and the third readhead. The microprocessor can be configured to receive and analyze the first analog signal, the second analog signal, and the third analog signal corresponding to the first light, the second light, and the third light, respectively, and determine a first level of analyte in the first control zone based on the first light, a second level of analyte in the test zone based on the second light, a third level of analyte in the second control zone based on the third light, and a total level of analyte in the test solution based on the first level, the second level, and the third level.

[0010] In other embodiments, a kit for measuring the total level of an analyte in a test solution can include a test strip as described herein, a cassette as described herein configured to receive the test strip, and a reflectometer as described herein.

[0011] In other embodiments, a method for measuring the total level of an analyte in a test solution can include providing a kit or reflectometer as described herein, providing a test strip to the reflectometer, and providing the test solution to the test strip to receive the total level of analyte in the test solution from the reflectometer.

[0012] So that the above-described invention may be more accurately understood, a more particular description of the invention briefly described above will be given by reference to specific embodiments illustrated in the accompanying drawings. The drawings shown herein may not be drawn to scale, and references to dimensions in the drawings or the following description are specific to the disclosed embodiments. Any variations in these dimensions that enable the invention to function for its intended purpose are considered to be within the scope of the invention. As such, these drawings are not to be considered limiting in scope, as they depict only representative embodiments of the invention, and the invention will be described and explained with additional specificity and detail using the accompanying drawings. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows an embodiment of a reflectometer having a case and a cassette in which lateral flow test strips are secured for insertion into the case. [Figure 2] This shows how the cassette of Figure 1 is inserted into the case of the reflectometer. [Figure 3] Figure 1 is a cross-sectional view of a readhead according to one embodiment of the present invention, shown engaged with a cassette to receive reflectance information from a lateral flow test strip; [Figure 4A] FIG. 4A shows a circuit diagram of a first read head according to one embodiment of the present invention. [Figure 4B]FIG. 4B shows a circuit diagram of a second read head according to one embodiment of the present invention. [Figure 4C] FIG. 4C shows a circuit diagram of a third read head according to one embodiment of the present invention. [Figure 5] 1 shows a circuit diagram of a microchip having a microprocessor for controlling the operation of a reflectometer, according to an embodiment of the present invention, and also shows circuit diagrams for a temperature sensor and a background reflectance sensor. [Figure 6] FIG. 1 shows a circuit diagram of a microprocessor that can be used to control the operation of a reflectometer, according to one embodiment of the present invention. [Figure 7] 1 shows a circuit diagram of an interface connector operatively connected to a microchip according to an embodiment of the present invention, and also shows a voltage converter for converting analog signals from a read head into digital signals that can be sent to a microprocessor for analysis. [Figure 8] 1 shows an example of a look-up table (LOT) that can be used by a microprocessor to determine analyte concentration. [Figure 9] 1 illustrates a test strip that can be used with a reflectometer according to an embodiment of the present invention. [Figure 10] A schematic diagram of the lower part of the reflectometer case is shown. [Figure 11] An image of the bottom of the reflectometer case with the printed circuit board (PCB) and baffle plate is shown. [Figure 12] FIG. 1 shows a schematic diagram of a baffle plate that can be used in a reflectometer to help hold the cassette in place. [Figure 13] 1 shows a cross-sectional schematic of a cassette that can be used with the reflectometer. [Figure 14] 1 shows a cross-sectional schematic of a cassette that can be used with the reflectometer. [Figure 15] 1 shows a cross-sectional view of a cassette that can be used with a reflectometer. [Figure 16] 1 shows a schematic diagram of a cassette that can be used with the reflectometer. [Figure 17]FIG. 1 shows a cutaway schematic of the base of a cassette that can be used with the reflectometer. [Figure 18] 1 shows a cutaway schematic view of the cover of a cassette that can be used with the reflectometer. [Figure 19] FIG. 1 shows a side view of a cassette that can be used with the reflectometer. [Figure 20] 1 shows a circuit diagram of an interface connector operatively connected to a microchip according to an embodiment of the present invention, and also shows a voltage converter for converting analog signals from a read head into digital signals that can be sent to a microprocessor for analysis. DETAILED DESCRIPTION OF THE INVENTION

[0014] Embodiments of the present invention provide methods and devices for measuring an analyte in a bodily fluid sample. More specifically, the embodiments provide a reflectometer or similar device for detecting and analyzing the amount of detection moiety bound to a discrete region on a lateral flow test strip. Embodiments of the present invention can be used to accurately measure the amount of analyte in a sample by conjugating it to a detection moiety, measuring the amount of analyte bound to a test zone, and comparing the result to the amount of detection moiety bound to two or more control zones. The reflectometer can be programmed to extrapolate or "fit" the results obtained from two or more control zones with an extrapolation curve, such that measurements obtained from the test zones can be interpolated to obtain a digital value that can be used in a look-up table (LOT) or algorithm to accurately quantify the analyte concentration in the sample.

[0015] A recent development is a lateral flow immunochemical test that utilizes two control zones in which different concentrations of detection moiety-binding material are immobilized or stabilized. During the test, different amounts of detection moiety are bound or captured in each control zone. Several detection moieties are known in the art, with colloidal gold being the most commonly used. Detection moieties, which use reflected light as a measure of concentration bound to each zone, produce a color and / or intensity that can be detected and measured using a reflectometer. By comparing the reflectance and / or color of each control zone to a predetermined extrapolation curve, a unique high-end and a unique low-end specification can be established for a particular test strip. The amount of conjugated analyte bound to the test zone can then be determined by comparing the reflectance measurements to high-end and low-end specifications calculated for the test strip or a manufacturing "lot" of test strips. The accuracy of this test may depend on the reflectance meter's ability to accurately detect the amount of reflectance from the detection moiety bound to the control zone and the conjugated analyte-binding zone. U.S. Patent No. 5,929,494 describes a reflectometer that uses a feedback mechanism and temperature to calibrate for external factors that adversely affect the measurement of reflectance from the detection moiety bound to the test strip.

[0016] There is a need for a reflectometer that can more accurately measure reflectance from test strips. It would be beneficial if the reflectometer utilized multiple control zones that could establish a baseline to facilitate interpolation of test results. Ideally, the reflectometer would be able to compensate for the same external factors in the control and test zones, as well as variations that may exist between tests and test strips.

[0017] The following description discloses that embodiments of the present invention are particularly useful in the field of lateral flow immunoassays that utilize conjugated analytes with detection moieties capable of reflecting light. While this application describes its use for measuring reflected light from discrete areas on a lateral flow test strip, and much of the terminology herein refers to that, other variations that are apparent to those skilled in the art with the benefit of this disclosure are contemplated as being within the scope of the present invention.

[0018] In the following description, a number of terms will be used. In order to provide a clear and consistent understanding of the specification and claims, including the scope of such terms, the following definitions are provided:

[0019] First, the use of "and / or" is inclusive, and the term "A and / or B" is intended to be read as including the configurations "A and B," "A or B," and "A" and "B."

[0020] When the terms "approximately" or "about" are used herein in conjunction with a numerical value, it is understood that the value can range from 95% to 105% of that value, i.e., the value can be + / - 5% of the stated value. For example, "about 1 kg" means 0.95 kg to 1.05 kg.

[0021] As used herein, terms indicating relative directions or orientations, including but not limited to "top," "bottom," "up," "below," "vertical," "horizontal," "outside," "inside," "front," "rear," etc., are intended to facilitate the description of embodiments of the invention by indicating relative directions or orientations in accordance with their ordinary usage and understanding, and do not limit the scope of embodiments of the invention to such directions or orientations.

[0022] Also, as used herein, unless otherwise specified, the terms "operable communication," "operably connected," "operably connected," "cooperatively engaged," and grammatical variations thereof mean that particular elements are connected in a cooperative manner to achieve their intended functionality. "Connected" or "engaged" may be direct or indirect, physically or remotely.

[0023] Additionally, throughout this specification, reference is made to the "proximal end" and the "distal end." As used herein, the proximal end is the end closest to where the lateral flow test strip is received or inserted. Conversely, the distal end of the device is the end furthest from where the lateral flow test strip is received or inserted.

[0024] The drawings and descriptions of the embodiments of the present invention are simplified to show elements relevant to a clear understanding of the present invention and may exclude other well-known elements for the sake of clarity. Those skilled in the art will recognize that other elements are desirable and / or required to implement the present invention. However, because such elements are well-known in the art and do not facilitate a better understanding of the present invention, such elements will not be described herein.

[0025] The present invention is more particularly described in the following examples, which are intended to be illustrative only, since numerous modifications and variations thereon will be apparent to those skilled in the art. As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0026] The transitional phrase "comprising" is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any component, step, or ingredient not specified in the claim. The phrases "consisting of" or "essentially consisting of" indicate that the claim includes embodiments that include the specified materials or steps and that do not materially affect the basic and novel characteristics of the claim. Use of the term "comprising" contemplates other embodiments that "consist of" or "consist essentially of" the recited components.

[0027] Any references herein to "one embodiment," "embodiment," "exemplary embodiment," "further embodiment," "alternative embodiment," etc. are for literal convenience. The meaning is that any particular feature, structure, or characteristic described in connection with such embodiment is included in at least one embodiment of the present invention. The appearance of such phrases in various places in the specification does not necessarily refer to the same embodiment. Furthermore, any element or limitation of any invention or embodiment thereof disclosed herein may be combined with any and / or all other elements or limitations (individually or in any combination) disclosed herein or with other inventions or embodiments thereof, and all such combinations are contemplated within the scope of the present invention, without limitation thereto.

[0028] It should be noted that Patent Document 2 is related to certain aspects of the present application and is incorporated herein by reference in its entirety.

[0029] Reference is now made to the accompanying drawings. Like reference numerals are used throughout to indicate like or similar components. Referring to the accompanying drawings illustrating specific embodiments of the present invention, a reflectometer 50 can be seen to have a case 100 with a slot 110 into which a lateral flow test strip 10 having a plurality of discrete regions 30 can be inserted. The case can house circuitry for multiple readheads 200 having scanning optics 210 including a light source 212 and a light receiver 214. The circuitry can incorporate a microprocessor 300 programmed with information that can be used to analyze and interpret the wavelengths of light from the scanning optics. The case can have a display screen 120 for viewing the results of the test. There can also be one or more interface connectors 400 incorporated into the case and circuitry to allow connection to an external device 450 for receiving information from and / or transmitting information to the microchip. Each of these general components can have one or more subcomponents, which are described in detail below.

[0030] The components of the reflectometer 50 can be enclosed within a case 100. The case can be handheld and contain the circuitry, scanning optics, and other components for acquiring and analyzing information from the lateral flow test strips, or similar devices, inserted therein. The case can be constructed from any suitable material, such as plastic, nylon, metal, other materials, and / or combinations thereof. Ideally, the material should be inexpensive and suitable for disposal. Preferably, the material is non-reflective and opaque with respect to the wavelength of the light source, so that background signals are blocked from stray reflections of light from other discrete areas on the test strip. The case can also be configured to house a battery with sufficient power to analyze a predetermined number of test strips, or to be connected to another energy source, after which the reflectometer can be deployed in an appropriate manner.

[0031] The case can be configured with a slot 110 at the proximal end 2 through which the distal end 7 of the test strip 10 can be inserted to engage a channel 112 that precisely aligns the test strip within the housing. Once aligned, the scanning optics 210 in each readhead 200 in the case can receive light at and reflect light from a discrete area 30 on the test strip. FIGS. 1 and 2 show examples of cases with slots and channels for receiving test strips (FIG. 1 shows the bottom of the case, and FIG. 2 shows the top of the case). In certain embodiments, the test strip has three discrete areas, including a test zone 32 and two control zones 31 and 33. The control zones can be located to the side of the test zone, or there can be a control zone on each side of the test zone. The control zones can each have a different, known amount of immobilized detection moiety-binding substance. The test zones can have an amount of immobilized analyte-binding substance that is greater than the amount of analyte expected in the sample. The immobilized analyte-binding substance in the test zone can be different from the immobilized detection moiety-binding substance in the control zone.

[0032] 9 illustrates a test strip 10 that can be used with a reflectometer, according to an embodiment of the present invention. The test strip 10 can include a substrate 11, a test sample line 14, low and high test control lines 13, and, optionally, an absorber 15 that can be in direct physical contact with the substrate 11. The arrows on the left indicate the proximal end 5 and distal end 7 of the test strip 10. The cassette 150 can be inserted into the slot 110 of the reflectometer 50 such that the distal end 7 of the test strip 10 enters first and the proximal end 5 of the test strip 10 is exposed and / or visible not through the case 100 of the reflectometer 50 but instead through the well 152 of the cassette 150.

[0033] In one embodiment, the test strip 10 is mounted in a cassette 150 that can be slidably inserted into slots and channels toward the distal end 4 of the case. FIG. 1 shows an example of a cassette into which the test strip can be mounted. As is often the case, the cassette can comprise a rigid or semi-rigid material with a non-reflective surface that is opaque to the wavelength of the light source to block background signals from stray light reflections from other discrete areas. In one embodiment, the cassette is suitable for disposal in a standard biohazard container.

[0034] The cassette can be configured to allow insertion to a specific depth, for example, where there may be one or more stops 156 that prevent the cassette from being inserted beyond a certain predetermined point. FIG. 1 shows an example of a cassette with stops that limit cassette insertion, as shown in FIG. 2. When inserted to the full extent permitted by the cassette, the discrete regions can be aligned with the scanning optics 210 in each readhead 200 within the case. The cassette can have at least one well 152 into which sample can be applied to the attached test strip. In further embodiments, the cassette can have walled openings or observation windows / slots 154 over the discrete regions 30 on the test strip, for example, as shown in FIG. 1. In one embodiment, the readheads each engage with the walled openings, thereby forming at least partially sealed chambers through which the readheads emit and receive light directed at their respective discrete regions. The engagement need not provide a light-tight fit with the walled openings.

[0035] FIGS. 13-19 show various views of a cassette 150 that can be used with a reflectometer 50 according to an embodiment of the present invention. The cassette 150 of FIGS. 13 and 14 has a slightly different shape than that of FIGS. 15-19. Referring to FIGS. 15-19, the cassette 150 can include a base 157 and a cover 158. The cover 158 can include a well 152 and one or more stops 156 that can prevent the cassette from being inserted beyond a certain predetermined point. The cover 158 can further include slots / separate regions 30 (e.g., including a first control zone or one or more first control slots 31, a test zone or one or more test slots 32, and / or a second control zone or one or more second control slots 33). The cover can also include one or more holes 159 to allow air to escape from the cassette 150 through the test strip 10 as a test fluid chromatograph. 13-19 depict various components of the cassette 150 and test strip 10 in particular colors, but these are for illustrative purposes only and to help highlight differences between the components; these colors are not to be construed as limiting. For example, the cover 158 and / or base 157 of the cassette 150 may be black (e.g., matte non-reflective (or low-reflectance) black) to reduce reflections. The absorber 15 may be white, for example. Also, while the test sample line 14 and low and high test control lines 13, 14 are shown in FIG. 17 for illustrative purposes, these lines 13, 14 are often colorless and / or invisible (or nearly invisible) before a test is performed.

[0036] 13 and 14, in one embodiment, cassette 150 can have a first well 152a and a second well 152b, where first well 152a can be located closer to distal end 4 of cassette 150 (i.e., first well 152a can be located on cassette 150 such that when cassette 150 is inserted into case 100, first well 152a is closer to case 100 than second well 152b). First well 152a can be narrower than second well 152b, although embodiments are not limited thereto. An embodiment having two wells 152a, 152b, such as those shown in FIGS. 13 and 14, can include any or all of the features of cassette 150 described and illustrated herein (e.g., in FIGS. 15-19). The cassette 150 with two wells 152a, 152b can be used, for example, to test fluids having components that need to be filtered out (e.g., whole blood, sputum). The test strip 10 can include a filter thereon at a position corresponding to the first well 152a, which can be configured to filter out any components desired to be removed from the particular test fluid. For example, the cassette 150 with two wells 152a, 152b can be used to test whole blood, and the test strip 10 can have a red blood cell filter at a position corresponding to the first well 152a. During testing, whole blood can be supplied to the first well 152a, and after absorption onto the test strip, a buffer solution (e.g., 50 microliters (μL), approximately 50 μL, or at least 50 μL) can be supplied to the second well 152b. Chromatography can then be initiated with the buffer to move the colorless portion of the blood (e.g., plasma or serum) to the control zones 31, 33 and the test zone 32 of the test strip 10. Because the color produced by colloidal gold (a common detection moiety) tends to be red or reddish, removing red blood cells is very important when testing whole blood. Cassettes 150 with one well 152 (e.g., as shown in Figures 15-19) can be used for fluids (e.g., serum, plasma, saliva, urine) where filtering of the material is not required.

[0037] In many embodiments, it is important to slide each cassette 150 into the same position within the case 100 of the reflectometer 50 to keep the distance from the surface of the test strip 10 to the scanning optics 210 the same (or nearly the same) for each test. To accomplish this, the case 100 of the reflectometer 50 can include spring clips 191 and / or a baffle plate 195 to hold each cassette 150 in place. FIG. 10 shows a cutaway schematic of the lower portion of the case 100 of the reflectometer 50 with certain elements cut away to reveal the inside of the lower portion of the case 100, and FIG. 11 shows an image of the lower portion of the case 100, including the baffle plate 195 and the printed circuit board (PCB) 130, which may include the scanning optics 210. The lower portion of the case 100 can include spring clips 191 that apply pressure to the cassette 150 to help hold it in place as it slides through the slot 110. The baffle plate 195 can be mounted within the case 100 (e.g., using one or more posts 192 (e.g., screw posts)). That is, the baffle plate 195 can include one or more legs 197 that can each be attached to one or more posts 192. The PCB 130 can also be mounted within the case using one or more posts 192 (e.g., screw posts), which can be the same posts 192 used to mount the baffle plate 195. The baffle plate 195 can include one or more stopper slots 196 that can respectively receive one or more stops 156 of the cassettes 150 when inserted into the case 100 through the slots 110, thereby ensuring that each cassette 150 is inserted to the same depth. This, in combination with the spring clips 191, locks each cassette 150 into a precise, predetermined position so that the test strips 10 for each test are the same distance from the scanning optics 210 (this distance is also determined by the thickness of the cover 158 of the cassette 150, so that the distance will be the same as long as the thickness of the cover 158 of the cassette 150 is the same). The baffle plate 195 may also include a number of viewing slots 198 which may allow viewing of the slots / individual areas 30 of the cassette 150 during testing.

[0038] Within the reflectometer 50 is a readhead 200 that may include scanning optics 210 for measuring the amount of detection moiety bound to each discrete region 30 on the test strip 10. To facilitate understanding of embodiments of the reflectometer of the present invention, it is helpful to understand the basic concepts of lateral flow immunoassays. To use a lateral flow test strip, a liquid test sample is applied to the proximal end 5 of the test strip 10, such as well 152. To facilitate chromatography, a buffer solution (e.g., 2-3 drops, e.g., 100-200 microliters) may also be added along with the sample. The buffer solution may optionally be treated with other chemicals (e.g., antibodies, chemicals to impart a specific pH, or chemicals to activate the antibodies on the test strip) to drive the reaction. The sample containing or suspected of containing the analyte of interest (along with the buffer solution, if present) migrates toward the distal end 4, 7 and contacts the conjugate release region on the test strip, where the detection moiety (e.g., colloidal gold, latex beads, etc.) immobilized on the conjugate release region becomes conjugated to the analyte of interest in the sample. The conjugated analyte, along with any unbound detection moieties, then migrates to the first discrete region 30. In one embodiment, this first discrete region is a first control zone 31, where unbound detection moieties can bind to the immobilization material in the first control zone while the conjugated analyte passes through. The sample continues to migrate to the next discrete region. In one embodiment, the next discrete region is a test zone 32 having immobilized analyte-binding material to which the conjugated analyte can bind and capture the bound detection moiety. The remaining conjugated analyte and unbound detection moiety then migrate to the next discrete region. In one embodiment, the next discrete region is a second control zone 33 having a different amount of immobilized detection moiety-binding material than the first control zone, to which unbound detection moieties can bind. Conjugated analytes in the sample do not bind to the second control zone. The amount of detection moiety bound to the immobilization material in each discrete region can be detected and measured using a reflectometer 50. For example, if the detection moiety is colloidal gold, each discrete region 30 will have a color that reflects at a particular measurable wavelength according to the amount of detection moiety bound to that particular zone.The distal ends 4, 7 may also be provided with absorbent pads 17 to absorb sample migrating to the distal ends, which may act to drive the test sample along the test strip and continue the reaction to completion. Typically, it takes at least a few seconds (or at least a few minutes) for the result to complete or stabilize after the sample is applied to the test strip.

[0039] Advantageously, reflectometers according to embodiments of the present invention are configured to analyze the test and control zones to provide highly accurate measurements of analyte concentration in a sample. Once the reaction in the individual regions 30 is sufficiently stable, a readhead within the case 200 can analyze each individual region. In one embodiment, there is a dedicated readhead 200 for each individual region 30. Thus, in the example described above, the test zone, first control zone, and second control zone each have their own dedicated readhead. The readheads can include the necessary scanning optics 210 to measure the amount of conjugated analyte and unbound detection moiety captured in each individual region. The reflectometer 50 can be activated to detect and measure the wavelengths of color present in the individual regions on the test strip. The readhead acquires wavelength information, which is converted into a signal voltage level that correlates to the detected color according to the detection moiety bound to the individual region. The signal voltage level can be used in conjunction with an algorithm and / or look-up table (LOT) to correlate the signal voltage level to the analyte concentration. The analyte concentration can be output on the display screen 120 on the case. Alternatively or additionally, the information may be output via one or more interface connectors 400 to an external device 450, such as a computer, phone, tablet, or other device for processing and analysis.

[0040] Referring to FIG. 3, a cross-sectional view of the readhead 200 in the handheld reflectometer 50 shown in FIGS. 1 and 2 is shown. The readhead 200 can include scanning optics 210 capable of analyzing the individual regions 30. The scanning optics can include a light source 212 that emits light to illuminate the individual regions 30 to which the detection moieties are attached. In one embodiment, the light source is a light-emitting diode (LED) mounted in the support 205 and is at a particular angle θ relative to the light-receiving portion 214, also mounted in the support. The light source can emit light of any suitable color relative to the color wavelengths to be detected. As an example, a green LED has been found to produce the most beneficial results in detecting the color tints that occur in the individual regions when gold colloid is used as the detection moiety. Other light source colors, such as LEDs of other colors (e.g., red or blue), can be used depending on the detection moiety selected. The support 205 can be constructed of the same or similar materials as those used for the housing and / or cassette, as described above. The angle θ can also be any angle that minimizes detection of specular reflections. In one embodiment, angle θ is approximately 35°, 36°, 37°, 38°, 39°, 40°, 41°, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, 50°, 51°, 52°, 53°, 54°, 55°, or an angle between any two of the listed values. In certain embodiments, angle θ is 45° or approximately 45°.

[0041] The light source 212 in the readhead 200 can have a relatively narrow projection angle for its emitted light output. For example, an LED can have a projection angle of 15° or approximately 15°. To further focus the light toward the individual regions 30, the light output from the light source 212 can be directed along or through a light focusing device, such as a collimator or a light pipe, as shown in FIG. 3. The focused light can be directed through an opening 207 in the support 205 to illuminate the individual regions 30, as shown in FIG. 3. The position of the light source relative to the light focusing device can be adjusted to change the intensity of the illumination on the individual regions 30, as well as the effects and circumstances of background signal and stray light reflections. In the example shown in FIG. 3, if the light source 212 is a green LED and the light focusing device 216 is a light pipe, the LED can be adjusted within the light pipe to achieve the desired illumination on the individual regions and minimize undesired interference.

[0042] In one embodiment, the light receiving element 214 is mounted to the read head 200 with the support 205 and oriented at an angle θ relative to the light source 212 and the individual regions 30. In one embodiment, the light receiving element has a narrow field of view of 10° (or approximately 10°) to 20° (or approximately 20°). For example, the light receiving element may be a phototransistor with a field of view of 15° or approximately 15°. Focused light from the light source 212 may pass through an opening 207 in the support 205 of the read head 200 and be directed toward each individual region 30. Light reflected from the surface of the individual regions 30 on the test strip 10 may be directed toward the light receiving element along a second light focusing device 216, such as a light pipe or collimator. The second light focusing device 216 may be the same type of device as the first light focusing device 216 or a different type of device. 3, if the light receiving element 214 is a phototransistor and the second light focusing device 216 is a light pipe, the phototransistor can be adjusted within the light pipe to change the sensitivity and tolerance of the phototransistor in receiving light reflected from the surface of the individual areas, and thus the reflectometer's ability to read the wavelength and interpret the results. Ideally, the angle θ of the light source, e.g., an LED, relative to the light receiving element, e.g., the phototransistor, is such that detection of specular reflections from the individual surfaces of the test strip is minimized.

[0043] 4A, 4B, and 4C show example, non-limiting configurations of scanning optics 210 that can be used in first readhead 201, second readhead 202, and third readhead 203. In these examples, a green LED acts as the light source 212 that is detected by an NPN phototransistor acting as a light receiver 214. The wavelength detected by the phototransistor in each readhead 201, 202, and 203 generates an analog signal 217 that is sent to analog-to-digital converters 218A, 218B, and 218C, respectively, as shown in FIG. 5. Exemplary circuitry 213 for display 120 is also shown.

[0044] As mentioned above, the case 100 and support 205 of the readhead 200 can be fabricated from one or more materials that are non-reflective and opaque to the wavelengths utilized by the light source. This can suppress interference, such as stray reflectance from adjacent discrete regions. The test strip material can also reflect light, which can combine with and interfere with the reflectance from the discrete regions. This reflectance from the test strip material can also interfere and obscure the detectable wavelengths from the discrete regions 30, potentially introducing inaccuracies. One option is to pre-determine the reflected wavelength of the test strip material and calibrate that wavelength from the final measurement. While useful, this method may not account for variations between test strips or between test strip production lots. One embodiment of the present invention utilizes a background light receiver 220 that receives reflected light from regions of the test strip material. The background light receiver can be paired with one of the light sources 212 in the readhead to provide reflectance measurements from the test strip regions.

[0045] FIG. 5 illustrates an embodiment of a partial circuit of a reflectometer having first, second, and third readheads 201, 202, and 203, each including a light source 212 and a light receiver 214, as shown in FIGS. 4A, 4B, and 4C, respectively. Paired with one or more of the light sources (e.g., the light source of the third readhead) is a background light receiver 220. Wavelengths detected by the background light receiver, which may include a phototransistor, generate an analog signal 222 that can be sent to each readhead 201, 202, 203. As shown in FIG. 5, the background light analog signal 222 can be analyzed by a microprocessor 300 along with the analog signal 217 from the readhead. The microprocessor can be programmed to compensate for the background light analog signal to more accurately read the individual areas 31, 32, and 33.

[0046] The brightness and intensity of LED light can be affected by temperature relative to light output. Therefore, it is important to measure the temperature near the LED light source and compensate for temperature-driven changes that may occur in the reflected signal. In one embodiment, the reflectometer of the present invention includes at least one temperature sensor 500. The temperature sensor can generate an output signal 510 indicative of the temperature at or near the LED light source. The temperature sensor can be located near each light source (or near only one or several of the light sources). The temperature sensor can be located on or in the case 100 (e.g., on or near the support 205 for each light source 212). As briefly discussed above, the intensity of light output from an LED is affected by ambient temperature. As temperature increases, the intensity of light output decreases. Conversely, as temperature decreases, the intensity of light output increases. Therefore, it is essential to account for temperature changes in the light source so that the detected steady-state DC voltage can accurately represent color and hue. In one embodiment, the temperature sensor in the reflectometer comprises a thermistor. In a further embodiment, the thermistor is in operative communication with a microprocessor 300, as shown in FIG. 5. The temperature analog signal 510 is sent to a microprocessor which is programmed to convert the analog signal into a value that can be used to compensate for temperature interference with the analog signal 217 obtained from the read head.

[0047] In one embodiment, operation of the reflectometer 50 is controlled by a microprocessor 300. In a more specific embodiment, operation of the reflectometer is controlled by a microprocessor on a microchip 350. Each of the first readhead 201, second readhead 202, and third readhead 203 can also be operably attached to the microchip 350 so as to be controlled by the microprocessor. FIG. 6 illustrates an example of a microprocessor 300 that can be utilized on the microchip 350 in the reflectometer 50, for example, but not limited to, as shown in FIG. 5. As shown in FIGS. 5 and 6, the microprocessor can be used to control operation of the light source 212, and each of the analog-to-digital converters 218A-218C can send information to the microchip for processing. In one embodiment, the microprocessor also receives the analog temperature signal 217 and the background signal 222 and analyzes them to compensate for their respective interference with the readhead analog signal 217.

[0048] In one embodiment, the microprocessor 300 is functionalized for analog-to-digital conversion to convert the analog signal 217 from each of the readheads 201, 202, 203 into a digital value. The digital value can be further processed by the microprocessor into a detected analyte concentration. FIGS. 7 and 20 show two examples of the analog-to-digital circuit 375. The detected analyte concentration information generated by the analog-to-digital circuit can then be displayed on the reflectometer's display screen 120 (which can be in operative communication with the microprocessor). The analyte concentration can also be stored in the microprocessor for later retrieval, review, and / or transfer. The microchip 350 can further have an operative connection to one or more interface connectors 400, e.g., a universal serial bus (USB), for transmitting the analyte concentration information to an external device 450, such as, but not limited to, a personal computer, a cell phone, a tablet, and other devices. Additional calculations can be performed on the information by the external device, if necessary or desired.

[0049] The analog signals 217A, 217B, 217C from each light receiver 214 in each readhead 200 are processed by a microprocessor 300 to generate a digital value 219 of the wavelength received by the light receiver. The digital value can be compared by the microprocessor 300 to a stored LOT 310 that correlates the digital value to an analyte concentration 225. FIG. 8 shows a non-limiting example of a LOT that can correlate the digital value, converted to millivolts (mV), to an analyte concentration (mg / dL). Alternatively, the microprocessor can be programmed with an appropriate algorithm that utilizes the digital value to calculate the analyte concentration. In one embodiment, the reflectometer data (such as the manufacturing batch code of the test strip 10) and the type of test being performed (e.g., vitamin D concentration versus D-dimer concentration in serum) identify which of multiple stored LOTs or algorithms should be used by the processor when evaluating the digital value to determine the corresponding analyte concentration.

[0050] In one embodiment, a lateral flow test strip having a test zone, a first control zone having a first amount of detection moiety binding material, and a second control zone having a second amount (different from the first amount) of detection moiety binding material is analyzed by a reflectometer 50. Using analog signals generated by reflection from the zones, a microprocessor can determine the analyte concentration in the test zone 32 by first processing the analog signals from the control zones to establish digital values ​​representing the high-end and low-end standards for the particular test strip. Digital values ​​can also be calculated for the analog signals generated by the background receiver 222, and these values ​​can be subtracted from the digital values ​​calculated for the high-end and low-end standards. Each standard can be extrapolated to a model curve. The digital values ​​calculated for the test zone 32 can then be interpolated based on the extrapolated high-end and low-end standards. The interpolated test zone digital values, along with the LOT, can then be used to determine the analyte concentration of the sample. As mentioned above, digital values ​​of the background reflectance of the test strip material can also be used in the analysis to improve the accuracy of the results.

[0051] Lateral flow immunochemical tests are easy to administer and can provide relatively rapid results. A past limitation of such tests has been the inability to accurately quantify results. Embodiments of the present invention provide advantageous devices and methods for accurately quantifying results from lateral flow immunoassay strips by utilizing multiple controls that establish high-end and low-end specifications that allow for more accurate interpolation of test results. Background interference is also compensated for to further improve accuracy.

[0052] The present invention includes, but is not limited to, the following exemplary embodiments. Embodiment 1 1. A reflectometer (e.g., for a lateral flow immunoassay), comprising: Case and a first readhead including first scanning optics configured to illuminate a first control zone of the test strip and to receive first light from said first control zone; a second readhead including second scanning optics configured to illuminate a test zone of the test strip and receive second light from the test zone; a third readhead including third scanning optics configured to illuminate a second control zone of the test strip and receive third light from the second control zone; a microprocessor in operative communication with the first read head, the second read head, and the third read head; the microprocessor receives and analyzes first, second, and third analog signals corresponding to the first light, the second light, and the third light, respectively; a reflectometer configured to determine a first level of analyte in the first control zone based on the first light, a second level of analyte in the test zone based on the second light, a third level of analyte in the second control zone based on the third light, and a total level of analyte in the test solution based on the first level, the second level, and the third level. Embodiment 2 the first readhead includes a first support disposed in the case, a first light source disposed on the support, and a first light receiving unit disposed on the support; the second readhead includes a second support disposed within the case, a second light source disposed on the support, and a second light receiving unit disposed on the support; 2. The reflectometer of claim 1, wherein the third readhead includes a third support, a third light source disposed on the support, and a third light receiving unit disposed on the support. Embodiment 3 the first light source and the first light receiving unit are arranged on the first support body so that a first angle between a path of light emitted from the first light source and a path of light received by the first light receiving unit is in a range of 35° to 55°; the second light source and the second light receiving unit are arranged on the second support body so that a second angle between a path of light emitted from the second light source and a path of light received by the second light receiving unit is in a range of 35° to 55°; The reflectometer of embodiment 2, wherein the third light source and the third light receiving unit are arranged on the third support such that a third angle between the path of light emitted from the third light source and the path of light received by the third light receiving unit is in the range of 35° to 55°. Embodiment 4 the first angle is about 45 degrees; the second angle is about 45°; 4. The reflectometer of embodiment 3, wherein the third angle is about 45°. Embodiment 5 the first light source is a light emitting diode (LED); the second light source is an LED; 5. The reflectometer according to any one of embodiments 2 to 4, wherein the third light source is an LED. Embodiment 6 the first light receiving unit is a phototransistor, the second light receiving unit is a phototransistor, 6. The reflectometer according to any one of embodiments 2 to 5, wherein the third light receiving unit is a phototransistor. Embodiment 7 the first readhead further includes at least one first light collection device disposed on the first support; the second readhead further includes at least one second light collection device disposed on the second support; 7. The reflectometer of any one of embodiments 2 to 6, wherein the third readhead further comprises at least one third light collecting device disposed on the third support. Embodiment 8 the at least one first light collecting device includes at least one of a light pipe and a collimator; the at least one second light collecting device includes at least one of a light pipe and a collimator; 8. The reflectometer of embodiment 7, wherein the at least one third light collecting device comprises at least one of a light pipe and a collimator. Embodiment 9 9. The reflectometer of any one of claims 1 to 8, wherein the microprocessor converts the first analog signal, the second analog signal, and the third analog signal into a first digital signal, a second digital signal, and a third digital signal, respectively, and compares the first digital signal, the second digital signal, and the third digital signal with at least one lookup table to determine the first level of the analyte, the second level of the analyte, and the third level of the analyte based on the first digital signal, the second digital signal, and the third digital signal, respectively. Embodiment 10 10. A reflectometer as described in any one of claims 1 to 9, wherein the case includes a slot at a first end thereof, the slot configured to receive a cassette containing the test strips. Embodiment 11 11. The reflectometer according to any one of claims 1 to 10, wherein the case includes a baffle plate attached thereto so that the distances from the first read head to the test paper, the distances from the second read head to the test paper, and the distances from the third read head to the test paper are constant for different test papers. Embodiment 12 The baffle plate is a plurality of legs attached to a plurality of posts within the case; at least one stop slot configured to receive at least one stop of a cassette containing said test strip; 12. The reflectometer of claim 11, comprising a plurality of observation slots configured to align with the first control zone, the test zone, the second control zone, the first readhead, the second readhead, and the third readhead. Embodiment 13 13. The reflectometer of any one of claims 1 to 12, further comprising a spring clip disposed within the case, the spring clip configured to hold a cassette containing the test strip in place. Embodiment 14 14. The reflectometer according to any one of embodiments 1 to 13, further comprising a circuit board disposed within the case, the microprocessor being disposed on the circuit board. Embodiment 15 15. The reflectometer according to any one of embodiments 1 to 14, wherein the test solution is a biological test solution. Embodiment 16 16. A reflectometer as described in any one of claims 1 to 15, wherein the case includes a display screen in operative communication with the microprocessor and configured to display the total level of the analyte. Embodiment 17 17. A reflectometer according to any one of embodiments 1 to 16, further comprising at least one interface connector integrated into the case and configured to connect to at least one external device. Embodiment 18 further comprising a temperature sensor in operative communication with the microprocessor and configured to measure a temperature within the case and generate an output signal indicative of a temperature of at least one of the first read head, the second read head, and the third read head; 18. The reflectometer of any one of claims 1-17, wherein the microprocessor is configured to utilize the output signal to compensate for temperature interference when determining total analyte level. Embodiment 19 a background light receiving section in operative communication with the microprocessor and configured to receive reflected light from an area of ​​the test strip and generate a reflected light analog signal; 19. The reflectometer of any of embodiments 1-18, wherein the microprocessor is configured to utilize the reflected light analog signal to compensate for background light when determining the total analyte level. Embodiment 20 20. A reflectometer as described in any one of claims 1 to 19, wherein the first level of the analyte in the first control zone and the third level of the analyte in the second control zone are utilized to establish high-end and low-end standards, respectively, to which the second level of the analyte in the test zone is compared. Embodiment 21 1. A method for determining the total level of an analyte in a test solution, comprising: Provided is a reflectometer according to any one of embodiments 1 to 20, providing a test strip to the reflectometer; providing the test solution to the test strip and receiving from the reflectometer a total level of analyte in the test solution. Embodiment 22 1. A kit for determining the total level of an analyte in a test solution, comprising: test strips (e.g., at least one test strip); cassettes configured to receive the test strips (e.g., at least one cassette, each of which may be disposable); A kit comprising the reflectometer according to any one of embodiments 1 to 19. Embodiment 23 23. The kit of embodiment 22, wherein the test strip comprises at least one of an absorbent body at its proximal end and an absorbent pad at its distal end. Embodiment 24 24. A kit according to any one of embodiments 22 to 23, wherein the cassette comprises at least one well on its upper surface, the at least one well comprising an opening configured to receive the test solution (and / or buffer solution) so as to expose and allow access to the test strip. Embodiment 25 24. The kit of any of embodiments 22-23, wherein the cassette comprises a first well and a second well on its upper surface, the first well comprising a first opening configured to expose the test strip (e.g., a portion of the test strip having or near a filter (e.g., a red blood cell filter)) and receive the test solution to reach the test strip, and the second well comprising a second opening (which may be wider than the first opening) configured to expose the test strip and receive a buffer solution (e.g., to initiate chromatography and / or to help move the test solution to the test zone of the test strip). Embodiment 26 26. A kit as described in any one of embodiments 22 to 25, wherein the cassette includes at least one stop on an upper surface of the cassette configured to prevent the cassette from being inserted beyond a predetermined point within the reflectometer. Embodiment 27 27. A kit according to any one of embodiments 22 to 26, wherein the cassette comprises a plurality of holes on its top surface configured to allow air to escape from within the cassette. Embodiment 28 A kit described in any of embodiments 22 to 27, wherein the cassette includes a plurality of observation slots on its upper surface configured to be aligned with the first control zone, the test zone, the second control zone, the first read head, the second read head, and the third read head. Embodiment 29 1. A method for determining the total level of an analyte in a test solution, comprising: A kit according to any one of embodiments 22 to 28 is provided, providing the test strip to the reflectometer; providing the test solution to the test strip and receiving from the reflectometer the total level of analyte in the test solution. Embodiment 30 The reflectometer of any one of embodiments 1 to 20, the method of embodiment 21, the kit of any one of embodiments 22 to 28, or the method of embodiment 29, wherein the test strip has any or all of the characteristics described herein for the test strip.

[0053] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in light thereof will be suggested to those skilled in the art, which are intended to be included within the spirit and scope of the present specification.

[0054] All patents, patent applications, provisional applications, and other documents mentioned or cited in this specification are incorporated herein by reference in their entirety, including any drawings and tables, unless they contradict the express disclosure of this specification. In addition, the entire contents of the references cited within the references cited herein are also incorporated by reference in their entirety. In particular, U.S. Patent Nos. 6,275,999 and 6,275,999 are incorporated herein by reference in their entirety.

Claims

1. A case, a first readhead including first scanning optics configured to illuminate a first control zone of a test strip in which a test solution is provided and to receive first light from said first control zone; a second readhead including second scanning optics configured to illuminate a test zone of the test strip and receive second light from the test zone; a third readhead including third scanning optics configured to illuminate a second control zone of the test strip and receive third light from the second control zone; a microprocessor in operative communication with the first read head, the second read head, and the third read head; the microprocessor receives and analyzes first, second, and third analog signals corresponding to the first light, the second light, and the third light, respectively; configured to determine a first level of analyte in the first control zone based on the first light, a second level of analyte in the test zone based on the second light, a third level of analyte in the second control zone based on the third light, and a total level of analyte in the test solution based on the first level, the second level, and the third level; the first readhead includes a first support disposed within the case, a first light source disposed on the support, and a first light receiving unit disposed on the support; the second readhead includes a second support disposed within the case, a second light source disposed on the support, and a second light receiving unit disposed on the support; the third readhead includes a third support, a third light source disposed on the support, and a third light receiving unit disposed on the support; the first light source, the second light source, and the third light source are all configured to provide light of the same wavelength as one another and are calibrated for use with a particular lot of test strips.

2. the first light source and the first light receiving unit are arranged on the first support such that a first angle between a path of light emitted from the first light source and a path of light received by the first light receiving unit is in a range of 35° to 55°; the second light source and the second light receiving unit are arranged on the second support such that a second angle between a path of light emitted from the second light source and a path of light received by the second light receiving unit is in a range of 35° to 55°; 2. The reflectometer of claim 1, wherein the third light source and the third light receiving unit are arranged on the third support such that a third angle between a path of light emitted from the third light source and a path of light received by the third light receiving unit is in a range of 35° to 55°.

3. the first angle is approximately 45 degrees; the second angle is approximately 45 degrees; The reflectometer of claim 2 , wherein the third angle is approximately 45°.

4. the first light source is a light emitting diode (LED); the second light source is an LED; The reflectometer of any one of claims 1 to 3, wherein the third light source is an LED.

5. the first light receiving unit is a phototransistor, the second light receiving unit is a phototransistor, 5. The reflectometer according to claim 1, wherein the third light receiving section is a phototransistor.

6. the first readhead further includes at least one first light focusing device disposed on the first support; the second readhead further includes at least one second light focusing device disposed on the second support; The reflectometer of any one of claims 1 to 5, wherein the third readhead further comprises at least one third light collecting device disposed on the third support.

7. the at least one first light collecting device includes at least one of a light pipe and a collimator; the at least one second light collecting device includes at least one of a light pipe and a collimator; The reflectometer of claim 6 , wherein the at least one third light collection device comprises at least one of a light pipe and a collimator.

8. 8. The reflectometer of claim 1, wherein the microprocessor converts the first analog signal, the second analog signal, and the third analog signal into a first digital signal, a second digital signal, and a third digital signal, respectively, compares the first digital signal, the second digital signal, and the third digital signal to at least one lookup table, and determines the first level of the analyte, the second level of the analyte, and the third level of the analyte based on the first digital signal, the second digital signal, and the third digital signal, respectively.

9. A reflectometer according to any preceding claim, wherein the case includes a slot at a first end thereof, the slot configured to receive a cassette containing the test strips.

10. 10. The reflectometer of claim 1, wherein the case includes a baffle plate mounted within the case and configured to keep a distance from the first read head to the test paper, a distance from the second read head to the test paper, and a distance from the third read head to the test paper constant for different test papers.

11. The baffle plate is a plurality of legs attached to a plurality of posts within the case; at least one stop slot configured to receive at least one stop of a cassette containing said test strip; a plurality of observation slots configured to align with the first control zone, the test zone, the second control zone, the first readhead, the second readhead, and the third readhead.

12. The reflectometer of any preceding claim, further comprising a spring clip disposed within the case, the spring clip configured to hold a cassette containing the test strip in place.

13. The reflectometer of any one of claims 1 to 12, further comprising a circuit board disposed within the case, the microprocessor being disposed on the circuit board.

14. A reflectometer according to any preceding claim, wherein the test solution is a biological test solution.

15. The reflectometer of any preceding claim, wherein the case includes a display screen in operative communication with the microprocessor and configured to display the total level of the analyte.

16. A reflectometer according to any preceding claim, further comprising at least one interface connector integrated into the case and configured to connect to at least one external device.

17. further comprising a temperature sensor in operative communication with the microprocessor and configured to measure a temperature within the case and generate an output signal indicative of a temperature of at least one of the first read head, the second read head, and the third read head; A reflectometer according to any preceding claim, wherein the microprocessor is configured to utilize the output signal to compensate for temperature interference when determining total analyte level.

18. a background light receiving section in operative communication with the microprocessor and configured to receive reflected light from an area of ​​the test strip and generate a reflected light analog signal; A reflectometer according to any preceding claim, wherein the microprocessor is configured to utilize the reflected light analog signal to compensate for background light when determining total analyte level.

19. 19. The reflectometer of any preceding claim, wherein the first level of the analyte in the first control zone and the third level of the analyte in the second control zone are utilized to establish high-end and low-end standards, respectively, to which the second level of analyte in the test zone is compared.

20. 1. A method for determining the total level of an analyte in a test solution, comprising: Providing a reflectometer according to any one of claims 1 to 19, providing a test strip to the reflectometer; providing the test solution to the test strip and receiving from the reflectometer a total level of analyte in the test solution.

21. 1. A kit for determining the total level of an analyte in a test solution, comprising: Test paper, a cassette configured to receive the test strip; A kit comprising a reflectometer according to any one of claims 1 to 18.

22. 22. The kit of claim 21, wherein the test strip includes at least one of an absorbent body at its proximal end and an absorbent pad at its distal end.

23. 23. The kit of any of claims 21 to 22, wherein the cassette includes at least one well on its top surface, the at least one well including an opening configured to receive the test solution to expose and access the test strip.

24. 24. The kit of any of claims 21 to 23, wherein the cassette includes at least one stop on a top surface of the cassette configured to prevent the cassette from being inserted beyond a predetermined point in the reflectometer.

25. 25. The kit of any of claims 21 to 24, wherein the cassette includes a plurality of holes on a top surface thereof configured to allow air to escape from within the cassette.

26. 26. The kit of any of claims 21 to 25, wherein the cassette includes a plurality of viewing slots on its top surface configured to be aligned with the first control zone, the test zone, the second control zone, the first readhead, the second readhead, and the third readhead.

27. 1. A method for determining the total level of an analyte in a test solution, comprising: A kit according to any one of claims 21 to 26, providing the test strip to the reflectometer; providing the test solution to the test strip and receiving from the reflectometer the total level of analyte in the test solution.

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