Method and apparatus for measuring biomarker molecules or other target analytes of interest through periodic readouts of a lateral flow test

Abstract

The present invention pertains to a reader that periodically reads the test line of a lateral flow test strip to aid in the determination of a final endpoint measurement.

Description

METHOD AND APPARATUS FOR MEASURING BIOMARKER MOLECULES OROTHER TARGET ANALYTES OF INTEREST THROUGH PERIODIC READOUTS OFA LATERAL FLOW TESTCROSS-REFERENCE TO RELATED APPLICATIONS:

[0001] This application claims the benefit of and claims priority to provisional patent application Serial Number 63 / 582,906, Attorney Docket Number TRT-025 with a filing date of September 15, 2023, which is hereby incorporated by reference in their entirety.

[0002] The present invention pertains to a method and apparatus for measuring biomarker molecules and other target analytes of interest through periodic readouts of a lateral flow test.BACKGROUND

[0003] In light of the SARS-COV-2 pandemic, telemedicine and at-home testing has expanded significantly, with significant drivers towards repeated measurements of clinically- relevant biomarkers for disease diagnosis, prognosis, and treatment management.

[0004] Lateral flow immunoassays (also known as “dipstick” assays, “test strips,” or LFIAs) stand as one of the most commercially viable platforms for low-cost, easy-to-use, at-home testing in a CLIA-waived environment. Widespread adoption of and familiarity with both hCG pregnancy testing (a $1.3 billion market in 2022, MarketWatch) and COVID-19 lateral flow immunoassays ($6 billion in 2022, GlobalNewsWire) has enabled the opportunity for expanded at-home testing. Commercial manufacturing of lateral flow test strips is easily performed at-scale and reproducible, with the appropriate selection of reporter molecules. However, current commercially-available platforms typically report the presence or absence of a single biomarker by relying on the user visually identifying a test line, which can be challenging for blind and low-vision individuals and offer limited to no quantitation. Applications that require precise quantitation or semi-quantitation of biomarker concentrations and changes over time in concentration requires the involvement of calibrated instruments.

[0005] In parallel, some biomarkers (including but not limited to glucose and lactate) are primarily detectable not by lateral flow immunoassays, but rather by enzymatic electrochemical (EEC) sensors. Such platforms require the involvement of instrumentation for data analysis and reporting.

[0006] For cost-effective expansion of at-home biomarker detection beyond the binary detection of a single biomarker, there is a need for quantitative, precise, sensitive, accurate,and reproducible assessment of LFIAtest line intensity. Pairing an LFIA with an EEC sensor can further expand the capabilities for at-home test.

[0007] What is needed is a lateral flow immunoassay that enables a low-cost, rapid, quantitative or semi-quantitative readout in the home, clinic, or other locations.SUMMARY

[0008] The present invention pertains to measuring target molecules collected from life forms, wastewater, environmental water sources, soil or other sources. Biomarkers and other molecules can be measured using Lateral Flow Immunoassay (LFIA) or enzymatic electrochemical (EEC) sensors among other methods.

[0009] A method for LFIA involves collecting a sample from a human, other life form, or other source. Such a sample typically is substantially a liquid such as saliva, mucus, blood, or urine or is converted to liquid by addition of a buffer such as a nasal swab into buffer for an at-home Covid test. The sample may be applied directly to a disposable test device. Alternatively, the sample may be processed with additional reagents and / or thermal processing and subsequently applied to the test device such as in the case of lysis and / or LAMP / PCR amplification prior to detection and / or the swirling of a sample swab into buffer. The test device comprises one or multiple zones of sensitivity, such as a test line, each of which is sensitive to the quantity of a target, such as a protein, that could be present in the sample. The test device is either inserted into or placed in contact with or in proximity to a reader.

[0010] The reader is arranged to have at least one sensor that can detect each of the test lines, or other analyte binding sites, on the test strip. Each sensor provides a measure of the degree of response of each of the test zones on the lateral flow assay test strip. Such measure is related to the concentration of the target that is present in the sample. Configurations of the reader may include separate sensors for each test line, or other analyte binding site, one or more sensors that scans all of the test lines, or multiple sensors for each test line. A processor is used to control the reader such that it periodically measures the same test line multiple times over the duration of the testing process until an endpoint measurement is taken. This data sampled and collected over time can be used to corroborate, verify, adjust, or otherwise modify the final result corresponding to the endpoint data. For example, based on the data sampled over the duration of the testing process, a hook effect can be determined. By readingthe same test strip multiple times during the testing process, a more accurate and / or confident result can be calculated as compared to that of a single readout at the endpoint.

[0011] These and other objects and advantages of the various embodiments of the invention will be recognized by those of ordinary skill in the art after reading the following detailed description of the embodiments that are illustrated in the various drawing figures.

[0012] BRIEF DESCRIPTION OF DRAWINGS

[0013] The accompanying drawings, which are incorporated in and form a part of this specification and in which like numerals depict like elements, illustrate embodiments of the present disclosure and, together with the detailed description, serve to explain the principles of the disclosure.

[0014] Figure l is a one embodiment of the device, including embodiments of the device’s LFIA illumination and detection module with an AFE (analog front end) and slit.

[0015] Figure 2 is a diagram illustrating an embodiment of a combined enzymatic electrochemical sensor and lateral flow assay.

[0016] Figure 3 is an example of one configuration of optical reader.

[0017] Figure 4 is a diagram illustrating a PCB slit with mirrored surfaces configuration.This enables a longer path length for the LED than the PD, while keeping the COGS low by mounting both on the same PCB.

[0018] Figure 5 shows an example of an LFIA kinetic readout with an exemplary hook effect.

[0019] Figure 6 illustrates a low cost assay reader that uses a DC motor to rotate the lead screw to translate the optical stage along the lateral flow assay strip.

[0020] Figure 7 shows a DC motor with three motor windings and a three-segment commutator.

[0021] Figure 8 shows an example of current pulses as a pulse width modulated (PWM) voltage is applied to a DC motor.DETAILED DESCRIPTION

[0022] Reference will now be made in detail to the various embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. While described in conjunction with these embodiments, it will be understood that they are not intended to limit the disclosure to these embodiments. On the contrary, the disclosure is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the disclosure as defined by the appended claims. Furthermore, in the following detailed description of the present disclosure, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be understood that the present disclosure may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present disclosure.

[0023] The descriptions may refer to antibodies, as they are the typical binder used in lateral flow immunoassays, for ease of communication, but the invention is also applicable to other binders that may be used in lateral flow immunoassays including, but not limited to, molecularly imprinted polymers, streptavidin / biotin, protein receptors, aptamers, complementary RNA or DNA strands and other structures that may bind to a particular target of interest.

[0024] The descriptions may refer to reflectance measurements with the Photodiode / Light Emitting Diode (PD / LED) pair for ease of communication but the invention is also applicable to fluorescence measurements, chemiluminescent measurement, capacitive measurement, magnetic measurements, and other methods of detecting a reporter (gold, fluorophore, enzyme, magnet, other etc.) attached to a detection antibody or other binder that is typically stored on a conjugate pad upstream of the test lines on a lateral flow immunoassay strip. The descriptions are also applicable to optical detectors beyond photodiodes including photodiode arrays, CMOS sensors, CCD sensors, cameras, pin-hole cameras and other methods of detecting light hitting a detector. The descriptions are also applicable to other light sources beyond LEDs, but LEDs are often used due to availability, compact size, and low cost.

[0025] One improvement on existing lateral flow assay readers, which rely on a single endpoint measurement, is the use of kinetic test data to allow for improved quantitative performance, better lower limit of detection, and expanded range of detectable concentrations. With kinetic test data, the lateral flow assay reader measures the change in intensity over time of a test line as that test line develops over the course of the lateral flow assay. As an example, the line absorbance may be measured every 1-30 seconds over the course of a 5-15 minute assay.

[0026] The intensity of a line on a lateral flow immunoassay test strip will depend on the concentration of the analyte in the sample fluid up until the quantity of the analyte in that sample fluid is enough to fully saturate the test line (the binding sites of all / almost all of the Ab (antibody ) / binders on the test line are already bound to the detection Ab / binder). By utilizing a kinetic readout instead of just an end point read out, the detectable concentration range of a test strip can be expanded. By taking continuous measurements over time the reader can identify not just the final intensity of the test line, but also how long it takes the test line to saturate or the rate of change of the test line intensity. The time to saturate the test line will typically decrease and the initial rate of change of line intensity will typically increase with higher concentrations of target analyte in the sample. This quantification may involve measuring the slope of the signal on the line over time, the endpoint signal on the line, and other features of the change in signal intensity of the line over time. The addition of the kinetic readout allows one to detect a higher range of analyte concentrations that would not be detectable with a single end point readout on a given lateral flow assay test strip as the end point signal may be similar across those higher concentrations that all eventually fully saturate the test line. In particular, utilizing a kinetic readout enables one to detect the hook effect. The hook effect is when an excess of target antigens in the sample binds to both the immobilized and labelled antibodies leading to a lower signal on the test line by preventing the antibody-antigen-antibody sandwich from forming. With the kinetic readout these hook effect samples can be correctly classified as high concentration and quantified vs reading as falsely low with an endpoint readout alone.

[0027] A kinetic readout also provides value at lower concentrations, especially when using a PD / LED pair or other detection method that does not provide a 2D image of the test strip. At lower concentrations, the light absorption, or other signal, from background antibodies on the test strip (Ab that are flowing through during the assay) may be similar to the light absorption,or other signal, from the line. This can be particularly difficult to distinguish with a PD / LED pair as spatial information is not available as it would be in a camera, photodetector array, or other 2D imaging source. When viewing the kinetic data, one can see that background antibodies will typically slowly decrease over time as fewer antibodies are released from the conjugate pad resulting in a reflectance increase and conversely the reflectance from a faint test line will slowly decrease over time as detection antibodies continue to bind to the surface. This can allow for the accurate detection of faint test lines that would otherwise fall within the range of background signal seen by the LED / PD pair in a single end point measurement.

[0028] To further improve accuracy of concentration measurements in the sample, the system may include a readout of a background region of the test strip as well as of the control line. By comparing these values to the signal intensity of the test line, the overall accuracy of the measurement can be improved.

[0029] PD / LED lateral flow assay readers are appealing because they are very low cost. One drawback is the lack of spatial information which can limit the sensitivity of the system. Incorporating a scanning mechanism into a reader with a PD / LED pair can greatly improve the SNR (signal to noise ratio) of a PD / LED pair and provide spatial information in what would otherwise be a single point measurement device. Combining scanning with a slit, lens, mirror, diffractive element, or other optical beam shaping technique(s) to further narrow the illumination area of the LED further improves performance by improving SNR. In the case of a fixed LED / PD pair the field of view needs to be large enough to measure the test line as well as + / - l-2mm to handle variability in where the line may be located from test strip to test strip from manufacturing tolerances. This extra field of view width to account for variability in line placement is not needed in a scanning system as the full test strip can be read, capturing the line intensity regardless of position on the test strip. By scanning, a much narrower field of view can be utilized on the PD / LED pair to allow measurement of just the test line followed be measurement of the background region of the test strip near the test line. This allows the ability to distinguish between Ab bound to the test line and Ab flowing past the test line. The narrower field of view with a scanning system also results in a larger drop in PD signal for a given line intensity than a wider field of view that includes both the line and non-line portions of the test strip as typically occurs with a fixed PD / LED pair.

[0030] The scanning mechanism slides the lateral flow assay position relative to the LED / PD, or other readout mechanism, or the LED / PD pair relative to the lateral flow assay as shown in Fig 1. This scanning may include a stage, a motor, a pulley system, a solenoid, a linear shape-memory alloy (SMA) actuator, a wind up spring, and / or other movement mechanism. The scanning allows for easier detection of a low concentration line vs. the background signal and for easier relative quantification of the different test lines on a strip. It also allows a given reader to read an arbitrary number of test lines at different locations without having to define in advance when building the reader the exact position and number of test lines, as one would need to do when using the traditional method of fixed PD / LED pairs at each line. This allows for building a generic reader that can be used with a variety of test cartridges. These test cartridges may measure multiple analytes for assessing health such as various biomarker concentration levels, detect the presence / absence of multiple targets for different infectious diseases, or other single or multiplex applications.

[0031] The reader may include a universal mount to enable reading lateral flow assays from a variety of vendors or it may include a specific mount that only fits a specific test cartridge. The reader may be paired with a cloud infrastructure to conduct analysis to determine biomarker concentration, provided diagnoses, share information with medical providers, and / or monitor large populations for more effective infectious disease tracking. This cloud infrastructure also enables the adding of future diagnostic or other health or environmental monitoring tests after the initial reader product is launched.

[0032] By incorporating a slit, lens, mirror, diffractive element, a combination of several of these elements, or other optical beam shaping technique that results in an illumination area on the lateral flow immunoassay test strip narrower than the test line, one can maximize the change in reflectance between the strip background and the test line of interest resulting in better SNR and ability to quantify the concentration of the target analyte of interest. Even without a narrow slit, the scanning mechanism can increase SNR by detecting subtle changes between background and line and allowing for normalizing to the variable amount of background Ab, or other binder, that may be present from test strip to test strip. Similar scanning mechanisms would have utility in the case of fluorescent, chemiluminescent, magnetic, capacitive or other readout mechanisms of reporters that may be attached to antibodies or other binders in a lateral flow assay.

[0033] Several methods exist for determining which test line of a multiplexed lateral flow assay is being read at any given point in time during the scanning mechanism. In addition to utilizing motors with encoders and homing mechanisms to determine spatial position, which may be expensive, one can also use the optical data itself to determine position. By bouncing light off of the edges of the cartridge body at each side of the cartridge opening into the lateral flow assay window one can see a change in signal due to the cartridge body. The signal may increase or decrease depending on the relative position of the PD and LED, the angle of the cartridge body, and the position of the PD / LED mask to the cartridge body. If the PD and LED are arranged along the long axis of the test strip then one can optically differentiate one side of the cartridge from the other even if both sides of the cartridge body are identical. The signal will change based on whether it is the PED or the LED that first crosses over the cartridge body. Using this optical location method enables the pairing of a very low-cost DC motor with a gearing mechanism to continuously scan back and forth as the motor runs continuously in one direction. One can precisely determine position for each scanned data point based on time since crossing either side of the cartridge opening without the use of precise motor control, homing mechanisms, or encoders. Scanning the two ends of the cartridge body also helps facilitate automatic alignment checks prior to running the assay. The system could scan the cartridge when it is first inserted and only after confirming adequate alignment, instruct the user to load sample. If the alignment check fails, the system may instruct the user to remove and reinsert the cartridge prior to loading sample. Scanning the two ends of the cartridge body opening of the lateral flow assay window can also help facilitate automatic checks on PD and LED performance and allow for calibration of the signal prior to running the assay.

[0034] Several methods exist for determining which test line of a multiplexed lateral flow assay is being read at any given point in time during the scanning mechanism. In addition to utilizing motors with encoders and homing mechanisms to determine spatial position, which may be expensive, one can also use the optical data itself to determine position. By bouncing light off of the edges of the cartridge body at each side of the cartridge opening into the lateral flow assay window one can see a change in signal due to the cartridge body. The signal may increase or decrease depending on the relative position of the PD and LED, the angle of the cartridge body, and the position of the PD / LED mask to the cartridge body. If the PD and LED are arranged along the long axis of the test strip then one can optically differentiate one side of the cartridge from the other even if both sides of the cartridge bodyare identical. The signal will change based on whether it is the PED or the LED that first crosses over the cartridge body. Using this optical location method enables the pairing of a very low-cost DC motor with a gearing mechanism to continuously scan back and forth as the motor runs continuously in one direction. One can precisely determine position for each scanned data point based on time since crossing either side of the cartridge opening without the use of precise motor control, homing mechanisms, or encoders. Scanning the two ends of the cartridge body also helps facilitate automatic alignment checks prior to running the assay. The system could scan the cartridge when it is first inserted and only after confirming adequate alignment, instruct the user to load sample. If the alignment check fails, the system may instruct the user to remove and reinsert the cartridge prior to loading sample. Scanning the two ends of the cartridge body opening of the lateral flow assay window can also help facilitate automatic checks on PD and LED performance and allow for calibration of the signal prior to running the assay.

[0035] Another mechanism to aid in determining which test line of a multiplexed lateral flow assay is being read at any given point in time during scanning is printing fiducial markers on the test strip itself. This may be done in parallel with the plotting of the test and control lines on the strip. These fiducials open up the tolerances allowable in the placement of the test strip relative to the cartridge body. The fiducials can help facilitate automatic alignment checks and can also be utilized for calibrating the signal from the PD. Lastly, the fiducials could indicate to the reader which test strip is being read to enable automatic correct processing of the data without additional user input, barcodes, or other test identification methods. Fiducials could also be placed on the cartridge body instead of the test strip to identify what test is being run and / or what lot of test is being run in the case that there are lot by lot calibration factors.

[0036] Combining strip scanning with a kinetic readout further builds on the advantages of both (high SNR, improved quantitation, improved LLOD (lower limit of detection) and increased range of concentrations that can be detected on a given lateral flow immunoassay test line). This enables for easier distinguishing between background Ab and Ab bound to the test line while allowing for the measurement of the change in intensity of that test line over time. This leads to further improvements over a kinetic readout alone by eliminating the contribution of variability in background Ab, or other binder, in introducing error to the accurate quantification of target molecule concentration. This error can be significant at lowanalyte concentrations when the signal from the background Ab, or other binder, is of the same order of magnitude as the signal from the bound Ab, or other binder, on the line.

[0037] Readers can include devices where the lateral flow strip is inserted into the reader or devices where the lateral flow strip is placed external to the reader. It can include standalone dedicated readers as well as fixtures to allow reading by a cell phone camera in a fixed kinetic format and / or scanning method with incorporated optical beam shaping.

[0038] One configuration utilizes an unlensed LED and an optical aperture, in the form of a rectangular slit with large aspect ratio (such as a long and narrow slit). The optimal spacing between the LED and rectangular slit is dependent on the size of the slit in each dimension. It is desired that the optical beam projected onto the test line of the test strip is smaller than the test line, and possess a uniform optical profile, which limits the distance between the LED and slit as determined by the relationship between the optical uniformity of the beam, the size of the slit in each dimension, and the divergence angle of the chosen LED. One configuration keeps the slit height from the test strip constant, and changes the distance of the LED relative to the optical beam-shaping slit. In turn, this changes the height of the LED relative to the test strip. One embodiment utilizes a smaller slit compared to a generally larger one, while avoiding significant diffractive effects. Another embodiment places the photodiode as close as possible to the test line, as this maximizes the number of received photons in the event the test strip base material is a strong optical scatterer, such as nitrocellulose or other. An example of an embodiment is shown below in Figure 3 below. Table 1 provides some examples of illuminated FOV on the test strip based on slit size and LED distance as well as the power transfer from the LED to the PD. As the LED is brought closer to the slit, the power transfer increases (more photons transfer from the LED to PD), but a narrower slit is required, which can impact fabrication costs. The non-uniformity of illumination also increases on the test strip as the LED is brought closer to the slit, which may not be desirable for assay quantification.

[0039] One configuration may involve the PD placed closer to the test strip than the LED as shown in Figure 3. This enables a higher power transfer from the LED to the PD while maintaining illumination uniformity and a narrower field of view for a given slit size by keeping the LED at a further distance from the test strip For low-cost embodiments, simplifying the components and part count is critical for meeting COGS (cost of goods sold)targets including reducing the number of PCBs. The PD and LED are two components on the PCB. One embodiment can utilize low-cost mirrored surfaces to enable placing the LED on the same PCB as the PD, while providing the needed extra pathlength for the LED to fully illuminate the slit and enable a wider slit as shown in Table 1. Figure 4 four shows one embodiment of a PD / LED pair mounted on a PCB with multiple reflected surfaces. Other embodiments may involve the PD and LED on the same side of the PCB with a separate slit mask. Spacing the LED further from the test strip enables a more uniform illumination of the test strip through the slit. When the PD is placed at the same farther working distance as the LED, the photon recovery decreases resulting in a lower SNR. By placing the PD much closer to the test line, one can achieve the optimal combination of a uniform LED illumination and higher recover of photons to improve SNR. This described embodiment allows this to be done with a single PCB, to reduce COGS.

[0040] Some target analytes are compatible with enzymatic electrochemical readouts, such as glucose, lactate, and alcohol. In these cases, better quantitative performance can be achieved by using an enzymatic electrochemical sensor instead of a lateral flow assay. For certain medical applications, it may be advantageous to measure a number of biomarkers in parallel from a given sample. In this case, overall better performance can be achieved by combining a lateral flow assay test strip with one or more enzymatic electrochemical sensors. The system can be designed to allow a given sample to flow over both the enzymatic electrochemical sensors and the lateral flow assay utilizing capillary action or another motive mechanism. Figure 2 shows an example of one possible layout for a combined EEC and multiplexed lateral flow assay. A enzymatic electrochemical sensor may include an insulating substrate, one or more sensor electrodes, one or more counter electrodes, one or more reference electrodes, one or more types of enzymes, and one or more physical, chemical, composite layers for the immobilization and / or preservation of enzymes within the sensor architecture.

[0041] Automatically detecting the specific test utilized by the reader is valuable to ensure correct readout. This information may include the specific targets of the test as well as lot to lot information that may impact interpretation of results or calibration factors. Methods of automatically tracking the test include incorporating a separate scanner or a companion cell phone app to read RFID tags, barcodes, QR codes, or other features. In another embodiment the specific assay inserted into the reader may be identified directly from the scanning testreadout based on the position of visual markers on the test strip itself. The inclusion of automated test detection allows for documentation for medical professionals, potential improved assay performance by taking into account any lot-to-lot variability, and reduced risk from a user incorrectly identifying the test being utilized with the reader. The system can also be used to automatically detect fraudulent tests or tests from other manufacturers.

[0042] Some embodiments of the device include a combination of modules, which may include all or some of the following: a module for LFIA illumination and detection, a module for cartridge movement, a module for thermal control (for example to enable a LAMP assay), a module for data analysis, a module for data input and reporting, a module for data storage, and a module for power. Combinations of modules allow for specific features of the device to match user needs, biomarker detection needs, and low-COGS designs for wide adoption across many markets and users, especially those who canonically cannot access existing concepts due to cost or usability.

[0043] The device’s thermal control module may include a heat conductor that may actively or passively transfers heat into or away from the device. That heat conductor may be comprised of a metal of high thermal conductivity, a fan, a pipe with pumped liquid such as water or silicone, or another mechanism by which to passively or actively conduct or transfer heat. The heat from the environment may be transferred into the device. The heat from the device may be transferred to the environment. The thermal control unit may include a thermometer, a thermistor, a thermocouple, a thermopile, a thermoelectric cooler or Peltier device, or another device to measure the temperature within and / or external to the device. Temperature irregularities or inconsistencies affects many aspects of these assays, including but not limited to antibody-antigen binding. Such embodiments enable the measurement and control, either passive or active, of temperature for improved analytical sensitivity. This thermal control module may also be used to enable LAMP, RPA or PCR for DNA / RNA amplification prior to detection.

[0044] The device’s data storage module may include digital memory, including on-board EEPROM or flash memory. Such a module would enable the storage of repeated measurements and, in combination with the data reporting module, enable reporting of said repeated measurements back to the user. This module may interface with the data analysis module and / or the data input and reporting module. The device’s storage module may alsoreport to the user whether biomarker values are increasing or decreasing over time over 2 or more uses of the system to read a given biomarker. The device itself or a companion cell phone app may display a graph with analyte concentration values to date to allow for tracking over time or between different sample sources.

[0045] The reader may include automated transmission of data via wifi or Bluetooth or USB- C or other cable to a computer, website, and / or a smartphone app. Such embodiments would enable the reporting of biomarker data to users, caregivers, medical professionals, and other relevant individuals. Such embodiments could also include data encryption for HIPAA compliance.

[0046] Figure 1 shows an embodiment of the device, including embodiments of the device’s LFIA illumination and detection module with an AFE and slit. This also includes embodiments of the device’s cartridge movement module including a scrolling mechanism, and the device’s data analysis module including a microprocessor. Because the slit is narrow, the LED 101 provides LED illumination focused more narrowly on the test lines. The reflected light from one or more test line and control line is detected by the photodiode 102. The signal from the photodiode 102 is then processed by an analog front end 103. A microprocessor 104 interprets the results and displays the results on readout 105. A chart of the photodiode signal as a function of current vs. time is shown with alternating background illuminated and test lines illuminated as the strip is moved relative to the slit and LED. The general measurement architecture comprises quantitative kinetic or static LFA and a reconfigurable firmware allowing modular design. In one embodiment the test strip is actuated relative to the LED 101 and photodiode 102. An improved signal to noise ratio and lower detection limit is achieved through limiting the illumination to the test line and reading the signal from background regions. The kinetic readout improves the quantitative dynamic range. The same reader can be used regardless of the number of test lines.

[0047] Figure 2 shows a diagram illustrating an embodiment of a combined enzymatic electrochemical sensor and lateral flow assay.

[0048] In one embodiment, the top of the graphical user interface includes information related to the project, the contract, the version name, the related work breakdown structure (WBS), and the related activity. Users may click on buttons to traverse to a different page,such as Task Details, Activity Relations, KC Insights, and Activity Log. The Start Time, End Time, number of comments, Delay Probability, and KC Risk data are also display for view.

[0049] Figure 3 shows an example of a configuration of an optical reader. An example of some configurations for a 300 micron beam width projected onto the test line is shown in the table below. The photodiode test line spacing is 3mm.

[0050] Figure 4 shows a diagram illustrating a PCB slit with a mirrored surfaces configuration. This enables a longer path length for the LED than the photodiode, while keeping the COGS low by mounting both or the same PCB.

[0051] Figure5 shows an example of an LFIA kinetic readout with an exemplary hook effect. It can be seen that measuring the signal of the sample line on the lateral flow assay over time as the sample flows over the test strip allows accurate quantification of the sample concentration even into the hook effect regime of the test strip. This is not possible with an endpoint readout alone as illustrated in the plot of Figure 5. The hook effect leads the highest concentration to have a lower endpoint signal than the mdium and high concentrations in this example.Time Negative Low Medium High Highest (into hook effect) 0 1 1 1 1 11 1 2 6 31 402 1 3 11 61 803 1 4 16 81 784 1 5 21 99 735 1 6 26 115.5 686 1 7 31 130.5 637 1 8 36 140.5 588 1 9 41 145.5 539 1 10 46 147.5 4810 1 11 51 148.5 43

[0052] Figure 6 illustrates a low cost assay reader that uses a DC motor to rotate the lead screw to translate the optical stage along the lateral flow assay strip. The DC motor 601 also rotates a four-toothed code wheel 602, which interrupts the light beam of an optical interrupter module 603 as it rotates. This allows the sped of the motor to b determined as well as the incremental linear motion of the optical stage relative to the assay strip. The code wheel and interrupter module can be eliminated by monitoring current surges that occur in a DC motor as it rotates.

[0053] Figure 7 shows a DC motor with three motor windings and a three-segment commutator. The arrows show the direction of currnt flow from the voltage source through the windings. As shown the left diagram, the brushes usually connect to two adjacent commutator segments with the third commutator segment floating. The voltage applied to the brushs causs current to flow through one winding directly and through the other two windings arranged in series.

[0054] As shown in the right diagram, as the motor rotates clockwise, the commutator rotates until one of the brushes straddles two adjacent commutator segments. At this point, one of the coils is shorted, and the applied voltage causes current to flow through the remaining two coils arranged in parallel. The resistance between the brushes is reduced and increased current flows from the voltage source.

[0055] The increased current is generated each time that a brush translates over a gap between commutator segments. With three commutator segments and two brushes, six current pulses are generated per revolution of the motors. By monitoring the current pulses, the speed and rotation of the motor can be determined.

[0056] Figure 8 shows an example of current pulses as a pulse width modulated (PWM) voltage is applied to a DC motor for approximately 50 ms. The motor accelerates, rotates approximately two turns and then the voltage source is turned off.

[0057] One embodiment of the present invention pertaining to LED spatial filtering addresses a unique challenge stemming from its innovative use of a low-cost LED and motor scanning assembly, which forms the core of its invention. In this design, unexpected stray light from the LED presents a distinct obstacle in the precision optical system. This stray light, which may originate from the LED itself or other components of the assembly, can lead to an increased illumination angle on the optical mask, causing undesirable optical artifacts such as halos or other distortions. These effects are particularly problematic given the BDA-01's requirement for precise image formation, which is critically required to achieve excellent quantitation. To overcome this unique challenge, the project implements a specialized dualmask near-field / far field optical spatial filter. This approach employs two carefully designed optical masks: one positioned in the near-field of the LED emitter and another in the far-field. By utilizing this configuration, the system achieves superior control and shaping of the optical output, effectively managing the unexpected stray light from the low-cost LED and associated components. This solution allows the project to maintain its cost-effective design while significantly reducing stray light-induced artifacts and achieving the desired illumination pattern, crucial for the accuracy and performance of the motor scanning assembly. It also reduces alignment accuracy when the near and far field masks are combined in a monolithic assembly.

[0058] In one embodiment, the kinematic mount is used for test cartridge mounting, ensuring precise and repeatable positioning. The lack of this feature induces deleterious variation in the quantitation. This mounting technique employs carefully designed contact points between the cartridge and the reader, guaranteeing consistent placement of each test strip for scanning. The current and best embodiment uses a three dovetails, with one spring loaded, and a reference surface against a circular curve, to completely constrain the cartridge. By incorporating kinematic mounting features into both the cartridge body holder (integrated into the reader) and the cartridge body, the mounting system achieves high repeatability. This kinematic mount design minimizes positioning errors that could affect calibration and quantitative accuracy, particularly valuable when scanning multiple test strips of the sametype or for use in lot calibration. This approach ensures that the test cartridge is held securely in a fixed position, allowing the optical sub-assembly to perform precise and consistent scans. By completely constraining the cartridge and eliminating any degrees of freedom, the kinematic mount ensures reliable and consistent measurements across different cartridges and reader units, optimizing the system's overall performance and accuracy.

[0059] In one embodiment in the design of the photodiode optical mask / slit, stray light affected the photodiode's performance, specifically decreasing the signal to background ratio. In the low-cost design, stray light can potentially enter the photodiode, negatively impacting both the upper and lower limits of quantitation. To overcome this issue, an optical mask was developed, such as a pinhole or slit (preferred form), to narrow the photodiode's field of view. This modification was implemented in the context of the project's test strip design, which typically involves highly scattering materials like nitrocellulose for the strip background and white plastic for the cartridge body. By implementing this optical mask, the reader system significantly reduced the amount of scattered light reaching the photodiode from these sources. This enhancement leads to an improved signal-to-background ratio, thereby enhancing the overall quantitative accuracy of the system. This solution renders the optical reader more resistant to variations in test cartridge configurations, maintaining consistent performance across different cartridge designs.

[0060] In one embodiment, a significant advancement was made by integrating the LED two- part (near and far field) mask and the photodiode mask into a single, monolithic part. This innovative design choice offered several key advantages that met the project's goals of costeffectiveness and performance optimization. By combining these two critical components into one unit, the system greatly simplifies the manufacturing process, as the integrated part can be easily molded for mass production. This approach not only streamlines production but also substantially reduces alignment complexity during assembly. The unified design ensures that the LED mask and photodiode mask are inherently aligned, minimizing the potential for errors that could occur with separate components. Consequently, this integration significantly eases system assembly, leading to reduced assembly costs and improved consistency across units. The simplified integration process contributes to the overall robustness of the BDA-01 system, increasing its reliability in various operating conditions.

[0061] Although the subject matter has been described in language specific to structural features and / or methodological acts, it is to be understood that the subject matter defined in this disclosure is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing this disclosure.

[0062] Embodiments according to the invention are thus described. While the present invention has been described in particular embodiments, the invention should not be construed as limited by such embodiments, but rather construed according to the following claims.

Claims

CLAIMSWhat is claimed is:

1. A lateral flow test device for testing a sample, comprising: a reader that reads a test strip, wherein the test strip comprises: a test pad for accepting the sample; a conjugate pad adjacent to the test pad that includes conjugates for marking target particles in the sample as the sample flows from the test pad onto the conjugate pad; at least one test line that includes antibodies that bind to marked target particles; a mechanism that controls the reader to periodically read the test line to measure marked target particles, wherein a plurality of measurement data is accumulated over a period of time for a same test line of the test strip, until an endpoint time duration is reached.

2. The lateral flow test device of Claim 1 further comprising:.a light emitting diode for illuminating the test line; a photo detector for detecting light reflected from the test line; a processor that receives a signal from the photo detector; a recorder that records the measurement data taken over the period of time.

3. The lateral flow test device of Claim 2, wherein the measurement data comprises at least a first measurement read at a first time, a second measurement read at a later second time, and an endpoint measurement read at an endpoint in time.

4. The lateral flow test device of Claim 3, wherein results from the first measurement and the second measurement determine a slope which is used to calibrate a result of the endpoint measurement.

5. The lateral flow test device of Claim 4, wherein the slope is used to detect a hook effect.

6. The lateral flow test device of Claim 4, wherein multiple measurements of concentration data is used to extrapolate a concentration curve which can be used to verify a concentration measured at the endpoint.

7. The lateral flow test device of Claim 1, wherein a plurality of test lines are periodically read.

8. The lateral flow test device of Claim 2 further comprising a slit through which the light from the light emitting diode passes through.

9. The lateral flow test device of Claim 7 further comprising a motor to physically move the test strip relative to the reader.

10. A method for determining an endpoint measurement of a biological test sample comprising: reading the test sample for markers at a first time; reading the test sample for markers at a second time; reading the test sample for markers at an endpoint time; using results from reading the test sample at the first time and reading the test sample at the second time to verify a result from the reading of the test sample at the endpoint time.

11. The method of claim 10 further comprising: shining a light onto a test area; periodically reading light reflected from the test area to determine a plurality of concentrations of markers at different times; determining a rate at which the concentrations of markers changes over time; using the rate to verify a concentration of markers at the endpoint time.

12. The method of Claim 11, wherein the test area is part of a lateral flow test strip.

13. The method of Claim 11 further comprising: determining a slope corresponding to the plurality of concentration of markers; using the slope to help quantify a result from reading the concentration of markers at the endpoint time.

14. The method of Claim 13 further comprising: determining a hook effect based on the slope corresponding to the plurality of concentration of markers.

15. The method of Claim 14, wherein a negative slope indicates a hook effect.

16. The method of Claim 10 further comprising: shining the light through a slit onto the test area.

17. The method of Claim 10 further comprising: moving the test area relative to a light source generating the light.

18. A reader for reading a test line of a lateral flow test strip, comprising: a light source for shining a light onto a test line of the test strip; a detector for generating a signal corresponding to light that is reflected from the test line; a processor that converts the signal into measurement data, wherein the processor measures the reflected light at different times during a testing process to corroborate an endpoint measurement.

19. The reader of Claim 18, wherein the measurement data comprising a kinetic readout can be used to detect a hook effect.

20. The reader of Claim 18 further comprising: a slit through which the light passes through before the light shines on the test line; a motor that moves the test strip relative to the reader.

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