Integrated Lateral Flow Bioassays and Biosensors

Abstract

Methods and systems for quantifying an analyte in a biological sample include a lateral flow assay (LFA). An integrated LFA analyte measurement device includes a first sensor that generates a signal relative to the concentration of the measured analyte. The LFA device may also include one or more additional sensors for auxiliary measures to assist in correcting, adjusting, or interpreting the output of the first sensor. The LFA device may cooperate with a remote database for data storage, analysis, and access.

Description

[Technical field]

[0001] INCORPORATION BY REFERENCE OF PRIORITY APPLICATION This application claims the benefit of U.S. Provisional Application No. 63 / 240,732, filed September 3, 2021, which is incorporated herein by reference in its entirety. All applications for which a foreign or domestic priority claim is identified in the Application Data Sheet filed with this application are incorporated herein by reference under 37 CFR 1.57.

[0002] This application describes biochemical systems and methods. More specifically, this application describes methods and systems for quantifying analytes in biological samples. The methods and quantification of analytes aid in the diagnosis, treatment and management of disease, and / or monitor health and well-being, human performance, or aid in law enforcement. [Background technology]

[0003] Technologies for the rapid identification and quantification of analytes in biological samples are essential for many applications, including the diagnosis, treatment and management of disease, identification of biological threats, maintenance of normal health and well-being, optimization of human performance, and the identification of controlled substances.

[0004] Immunoassay is one standard method of analyzing biological samples. The traditional immunoassay method is the enzyme-linked immunosorbent assay (ELISA). ELISA testing has several drawbacks. It is a complex, multi-step process that involves antibody-antigen complexation, multiple repeated washing steps, manual addition of reagents, and long analysis times using expensive laboratory equipment. Furthermore, significant training of laboratory technicians is required to perform laboratory ELISA testing.

[0005] An alternative to ELISA testing is biosensors, where a measurable signal is generated in the presence of a recognition molecule. Biosensors based on resonance, optical, thermal, and electrochemical measurements have been developed for point-of-care (POC) testing applications. When properly implemented, these approaches simplify the testing process and reduce the time of the assay, while maintaining sufficient precision, accuracy, and selectivity for a given application.

[0006] Biosensors have been widely reported in the academic literature on proof-of-concept, but have not been widely used for field or POC diagnostics (with few notable exceptions, such as pregnancy tests, as discussed below).

[0007] The lack of widespread adoption of biosensors may be due to the complexity of mass production of many reported designs. The reported processes may require many steps (antigen or antibody immobilization, blocking, washing), relatively expensive materials, and cleanroom infrastructure. There remains a need for portable, affordable, rapid, sensitive, and specific diagnostic biosensors, especially in low- and middle-income countries and other resource-limited regions.

[0008] Lateral flow assays (LFAs) have several advantages over other biosensor platforms, including relatively low cost, an established manufacturing process for lateral flow immunochromatographic assays, and excellent shelf life. Although qualitative paper-based LFA test strips, such as pregnancy test kits, are commonly available commercially, quantification of biomarker concentrations remains a challenge due to the ambiguity of the chromatographic output. This limits test results to applications where a binary presence / absence test result is acceptable. Integrating lateral flow strips with a quantitative element allows more informative data to be recorded, enabling novel applications. Summary of the Invention [Problem to be solved by the invention]

[0009] The integration of quantitative elements brings several additional challenges. First, biological samples such as blood, urine, or saliva may vary dramatically in pH and ionic content or contain contaminants that may affect the performance of the test and affect the reliability of the test results. One way to overcome these confounding factors is the treatment or pretreatment of the sample before analysis to normalize the ionic content and remove contaminants. However, this pretreatment introduces additional complexity and cost into the testing procedure. Ideally, the test system would not require sample treatment prior to the analysis of the sample.

[0010] Second, the quantitative element may require precise timing between the addition of the sample and the initiation of the stimulation or measurement of the secondary measurement process to allow for a defined incubation period. Ideally, the test system would integrate an automatic or intuitive timing element to reduce the possibility of user error, or errors introduced by variations in sample viscosity in the measurement process, improving the accuracy of the results.

[0011] Third, quantitative methods may require precise and well-directed addition of reagents after sample incubation. Ideally, the test system would integrate automated or user-friendly elements that direct and control the addition of such reagents.

[0012] The novel quantitative lateral flow biosensor described in this patent addresses the above challenges and enables an integrated, low-cost, portable and rapid quantitative testing tool that does not require administration by highly skilled personnel.

[0013] The assignee of the present application has filed prior patent applications describing systems, methods and devices for testing, measuring and analyzing saliva, measuring a subject's hydration level, and measuring other substances (e.g., sweat) and / or physiological parameters in a human or animal subject. These prior patent applications include U.S. Patent Application Publication No. 16 / 197,530, filed November 21, 2018, No. 16 / 598,000, filed October 11, 2018, No. 17 / 159770, filed January 27, 2021, and No. 17 / 149181, filed January 14, 2021. All of these patent applications referenced above are incorporated by reference into this application and may be referred to hereinafter as the "incorporated applications."

[0014] This application adds to the art in the incorporated applications by describing systems and methods for identifying or quantifying analytes in a biological sample.

[0015] These and other aspects and embodiments are described in more detail below in connection with the accompanying drawings. [Means for solving the problem]

[0016] In some embodiments, the devices and techniques described herein relate to a system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, the system comprising a first sensor generating a signal relative to the concentration of the one or more analytes, one or more additional sensors for one or more respective auxiliary measures to assist in interpreting the signal of the first sensor, and an analytical device to facilitate testing.

[0017] In some embodiments, one of the additional sensors is integrated into the lateral flow system. In some embodiments, one of the additional sensors uses the same analytical device but is not integrated into the lateral flow system. In some embodiments, one of the auxiliary measures is pH. In some embodiments, one of the auxiliary measures is the concentration of an ion. In some embodiments, one of the auxiliary measures is osmolality. In some embodiments, one of the auxiliary measures is temperature.

[0018] In some embodiments, one of the auxiliary measures is used to correct the first sensor's signal. In some embodiments, one of the auxiliary measures is used to flag an error. In some embodiments, one of the auxiliary measures is used to provide a diagnostic status of the first sensor's signal. In some embodiments, one of the auxiliary measures is data collected at a secondary analytical device. In some embodiments, one of the auxiliary measures is a set of answers provided by an individual providing a biological sample. In some embodiments, one of the auxiliary measures is a set of observations annotated by an individual operating the system. In some embodiments, one of the auxiliary measures is used to provide a diagnostic status of the first sensor's signal.

[0019] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes of a biological sample using a lateral flow system, the system comprising one or more sensors each generating a signal for the concentration of a respective one of the one or more analytes, integrated microfluidics and materials for collection and processing of the biological sample, and an analytical device to facilitate testing.

[0020] In some embodiments, the integrated microfluidics and materials control the physical variation of the biological sample. In some embodiments, the physical variation includes viscosity or bubble content. In some embodiments, the integrated microfluidics and materials control the chemical variation of the biological sample. In some embodiments, the chemical variation includes pH or ion content.

[0021] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes of a biological sample using a lateral flow system, the system comprising one or more sensors each generating a signal for the concentration of a respective one of the one or more analytes, one or more integrated automatic timing elements, and an analytical device to facilitate testing.

[0022] In some embodiments, the timing element includes multiple membranes with variable flow rates. In some embodiments, the timing element includes a fluid sensing electrode that starts a timer of the analyzer. In some embodiments, the timing element includes a fluid sensing electrode that initiates a measurement. In some embodiments, the timing element includes a fluid sensing electrode positioned to identify when a sufficient sample has been collected. In some embodiments, the output of the timing element is used to flag an error.

[0023] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes of a biological sample using a lateral flow system, the system comprising one or more sensors each generating a signal for the concentration of a respective one of the one or more analytes, integrated packaging of the liquids required to initiate a chemical reaction, and an analytical device to facilitate testing.

[0024] In some embodiments, the integrated liquid packaging is opened manually by a system operator to initiate a step in the measurement process, hi some embodiments, the integrated liquid packaging is opened automatically by the analytical device to initiate a step in the measurement process.

[0025] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, the system comprising one or more sensors each generating a signal for the concentration of a respective one of the one or more analytes, an integrated cutting element separating segments of the lateral flow system, and an analytical device to facilitate testing.

[0026] In some embodiments, the cutting element is combined with an integrated liquid pack.

[0027] In some embodiments, the technology described herein relates to a system for quantifying the concentration of one or more analytes of a biological sample using a lateral flow system, comprising one or more integrated ion-selective electrodes, ion-loaded liposomes, conjugate molecules that act to dissolve the liposomes, and an analytical device that facilitates testing. In some embodiments, the ion-loaded liposomes are immobilized near one or more ion-selective electrodes. In some embodiments, the ion-loaded liposomes are tagged with a capture ligand. In some embodiments, the presence of a target analyte in a biological sample prevents the capture of the conjugate molecule by the capture ligand, resulting in the dissolution of the liposomes and an increase in ion concentration.

[0028] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, the system comprising one or more integrated electrodes, one or more molecularly imprinted polymers targeting one or more analytes, one or more labeled conjugate molecules, and an analytical device to facilitate the testing.

[0029] In some embodiments, each of the one or more molecular imprinted polymers is disposed on one of the one or more integrated electrodes. In some embodiments, at least one of the labeled conjugate molecules binds to at least one of the molecular imprinted polymers in competition with at least one of the analytes. In some embodiments, one of the labeled conjugate molecules binds to one or more analytes, and the resulting complex binds to one of the molecular imprinted polymers.

[0030] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, the system comprising one or more integrated electrodes, one or more aptamers targeting the one or more analytes, one or more labeled conjugate molecules, and an analytical device to facilitate the testing.

[0031] In some embodiments, each aptamer is disposed on one of the integrated electrodes.In some embodiments, the labeled conjugate molecule competes with the analyte to bind to the aptamer.In some embodiments, the labeled conjugate molecule binds to one or more analytes, and the resulting complex binds to the aptamer.

[0032] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, the system comprising one or more integrated electrodes, one or more temperature altering elements, and an analytical device to facilitate testing.

[0033] In some embodiments, the temperature altering element includes a resistive heating element integrated into the lateral flow system. In some embodiments, the temperature altering element includes a liquid pack that, when opened, provides an endothermic reaction and reduces the temperature of the lateral flow system. In some embodiments, the temperature altering element is disposed within the analytical device and the temperature of the lateral flow system is altered using a thermally conductive material. In some embodiments, the analytical device prompts the user to apply the sample for analysis only after the desired temperature has been reached.

[0034] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, the system comprising one or more integrated electrodes, one or more integrated quality control elements, and an analytical device to facilitate testing.

[0035] In some embodiments, the quality control element is irreversibly altered by exposure to a temperature outside of a desired range. In some embodiments, the quality control element is irreversibly altered by exposure to a humidity outside of a desired range. In some embodiments, the quality control element is irreversibly altered when a lateral flow system is used. In some embodiments, the analytical device automatically measures at least one of the one or more quality control elements and does not perform an analysis when at least one quality control element indicates improper storage or use.

[0036] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, the system comprising one or more integrated electrodes for type and / or batch tracking, an analytical device to facilitate testing, and a mobile application.

[0037] In some embodiments, type and / or batch tracking is achieved using the resistance value of the integrated electrodes. In some embodiments, type and / or batch tracking is used to indicate if a lateral flow test is out of date.

[0038] In some aspects, the technology described herein relates to a system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, the system comprising one or more integrated electrodes, an analytical device to facilitate testing, and a mobile application.

[0039] In some aspects, the analytical device wirelessly communicates the measurement data with an internet-connected mobile device. In some aspects, the system further includes a centralized database for receiving the measurement data from the analytical device, the centralized database being accessible by multiple individuals via a mobile application or web portal. In some aspects, the centralized database performs further analysis of the measurement data using additional complementary data. In some aspects, the centralized database is responsive to the measurement data and automatically sends alerts to third parties based on predefined parameters.

[0040] In any of the embodiments, the analytical device presents results and / or interpretive advice directly to the operator. In some embodiments, the analytical device wirelessly communicates with a cell phone or tablet for presentation, interpretation, tracking and / or analysis of the data.

[0041] For purposes of summarizing the disclosure, certain aspects, advantages, and novel features are described herein. It is to be understood that not all such aspects, advantages, or features may be embodied in any particular embodiment of the disclosure, and that one of ordinary skill in the art will recognize from the disclosure herein myriad combinations of such aspects, advantages, or features. [Brief description of the drawings]

[0042] [Figure 1] Structure of a conventional lateral flow assay. [Figure 2A] Representative structures of conventional electrochemical sensors with various electrode configurations. [Figure 2B] Representative structures of conventional electrochemical sensors with various electrode configurations. [Figure 2C] Representative structures of conventional electrochemical sensors with various electrode configurations. [Diagram 3] An embodiment of a lateral flow assay using ion selective electrodes and liposomes for detection of a target analyte via an analyte concentration dependent signal. [Figure 4] An embodiment of a lateral flow assay using molecularly imprinted polymers for the detection of target analytes. [Diagram 5] An embodiment of an aptamer-based lateral flow assay for the detection of a target analyte. [Figure 6A] An embodiment of a lateral flow assay in which pH is a secondary measure, obtained using an integrated pH sensor. [Figure 6B] The measurement results were corrected using the lateral flow assay in Figure 6A. [Figure 6C] The measurements were excluded using the lateral flow assay in Figure 6A. [Figure 6D] The lateral flow assay of Figure 6A is used to improve clinical interpretation of the results. [Figure 7] FIG. 1A shows an embodiment of a lateral flow assay in which integrated microfluidics functions to collect saliva directly from the tongue and then filter particulate matter and air bubbles; FIG. 1B shows an embodiment of a lateral flow assay in which integrated microfluidics functions to collect saliva directly from the tongue and then automatically mix a running buffer with the collected sample. [Figure 8]FIG. 1A shows an embodiment of a lateral flow assay in which a flow detection electrode is placed under the membrane to monitor the progress of the sample flow and is used to initiate the measurement; FIG. 1B shows an embodiment of a lateral flow assay in which several membranes with variable flow rates are placed to regulate the addition of secondary compounds required to initiate a chemical reaction. [Figure 9] FIG. 1A shows an embodiment of a lateral flow assay in which a pre-measured liquid pack is manually opened by a system operator to initiate a step in the measurement process; FIG. 1B shows an embodiment of a lateral flow assay in which a pre-measured liquid pack is opened by an analytical device. [Figure 10] FIG. 1A shows an embodiment of a lateral flow assay in which depression of a cleaving element is used to separate regions of a membrane; FIG. 1B shows an embodiment of a lateral flow assay in which depression of a cleaving element separates regions of a membrane and causes the release of liquid. [Figure 11] FIG. 1 shows an embodiment of a lateral flow assay in which a heating or cooling element is integrated to regulate the temperature of the assay. [Figure 12] FIG. 1 shows an embodiment of a lateral flow assay in which a temperature sensing element is used as a quality control element. [Figure 13] FIG. 1 shows an embodiment of a lateral flow assay integrated electrode used for batch and / or type tracking. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] This application describes various embodiments and features of systems and methods for the identification or quantification of analytes in biological samples. These analytes can be, but are not limited to, antibodies, proteins, peptides, hormones, nucleic acids, drugs, and pathogens. Although the following disclosure focuses on the analysis of saliva or blood, the embodiments described below, or variations of those embodiments, can be used for the analysis of any other biological sample, such as urine, feces, or sweat.

[0044] The structure of a conventional lateral flow assay The structure of a conventional lateral flow assay (LFA) 100 is shown in FIG.

[0045] In a conventional LFA 100, a biological sample is first added to the sample pad 110. In some embodiments, the sample pad 110 comprises a cellulose fiber filter or a woven mesh. The sample pad 110 acts primarily to regulate the flow of the sample to downstream materials. The sample pad 110 may also act to modify sample parameters such as pH (e.g., by adding reagents to the sample pad 110), act as a filter to remove particulate matter from the sample, or contain a blocking solution to improve binding specificity.

[0046] In some embodiments, sample parameters such as pH or viscosity can be normalized to facilitate more sensitive measurements. Alternatively or additionally, the biological sample can be first mixed with a running buffer before being placed on the sample pad 110. A similar approach involves adding a running buffer to the sample pad 110 after the sample.

[0047] The sample then flows through the conjugate pad 112. In some embodiments, the conjugate pad 112 can include a glass fiber filter, a cellulose filter, a surface-treated polyester or polypropylene filter, or other filter. The conjugate pad 112 can include one or several labeled molecules (conjugates) that can bind to the target analyte (e.g., in the case of a "sandwich" LFA), or one or several labeled molecules similar to the target analyte (e.g., in the case of a "competitive" LFA). The test sample rehydrates the conjugates in or on the conjugate pad 112 such that the conjugates are present in the sample at a constant concentration.

[0048] Conjugate molecules include, but are not limited to, antigens, monoclonal or polyclonal antibodies, complementary nucleotide sequences or aptamers. Labels include, but are not limited to, enzymes (e.g., horseradish peroxidase-HRP, alkaline phosphate-AP), electroactive moieties / redox mediators (e.g., ferrocene, thionine), metal nanoparticles (e.g., gold), polymer microspheres (e.g., latex), redox polymers with metal complexes, and liposomes. In some embodiments, signaling tracers such as fluorescers, dyes, enzymes, redox mediators, and ions can be incorporated into liposomes. Thus, LFAs can be implemented as integrated sensors, such as integrated immunosensors, integrated hydration sensors, integrated pH sensors, etc.

[0049] The sample / conjugate mixture then flows along a membrane 114 to which several capture reagents are attached. In some embodiments, the membrane 114 can comprise one or more of nitrocellulose, cellulose acetate, glass fiber membrane, nylon, polyvinylidene, fluoride, etc. The capture reagent can be an antigen, a monoclonal or polyclonal antibody, a complementary nucleotide sequence, an aptamer, or a synthetic antibody such as a molecularly imprinted polymer.

[0050] At locations referred to as "test lines" 120, a capture ligand is immobilized to capture an analyte or a competitive conjugate molecule. A single LFA 100 can include several test lines 120. In some embodiments, an LFA 100 can include two, three, four, five, six, or more test lines 120, each having an immobilized capture ligand. In some embodiments, two or more immobilized capture ligands are the same capture ligand. In some embodiments, two or more immobilized capture ligands are different capture ligands.

[0051] At a location designated as control line 122, a ligand is specifically immobilized to the conjugate molecule to act as a check for conjugate solubilization and validity of the test. In some embodiments, additional control lines 122 may contain specific immobilized ligands selected to check additional sample characteristics. One or more additional calibration lines 124 may target one or more unrelated targets and may be included to control for any inherent background interference within the sample.

[0052] Finally, an absorbent or wick pad 116 is placed after the test and control lines to facilitate additional sample flow across the membrane. In some embodiments, the wick pad 116 can be a cellulose fiber filter, cotton fiber, a cotton / glass mixture, or other suitable material. In some embodiments, the wick pad 116 can reduce background signal and increase the sensitivity of the test.

[0053] Lateral Flow Assay Formats Two common assay formats used in lateral flow strips are the sandwich assay and the competitive assay.

[0054] Sandwich assays are used to detect relatively large analytes such as proteins, whereas competitive assays are used for small analytes such as hormones, where the analyte is too small for two capture molecules to bind simultaneously.

[0055] In sandwich LFA, a capture molecule for one binding site of the analyte is conjugated to a label and a second capture molecule for the other binding site is immobilized at a test line, the presence / concentration of the label at the test line indicates the presence / concentration of the target analyte.

[0056] In some embodiments of competitive LFA, a capture molecule that targets a specific binding site is immobilized at the test line. The molecule that contains this binding site is conjugated to a label and then competes with any analyte present in the sample for binding at the test line. The presence / concentration of the label at the test line indicates the inverse of the absence / concentration of the target analyte.

[0057] In another embodiment of competitive LFA, the target analyte is immobilized at a test line. A labeled capture molecule binds to any analyte present in the sample, and any remaining capture molecules bind to the test line. Again, the presence / concentration of the label at the test line indicates the inverse of the absence / concentration of the target analyte.

[0058] Lateral Flow Assay Detection Format In conventional LFAs, the presence of the conjugate at the test or control line is determined using optical or electrochemical methods.

[0059] For optical measurements, the color intensity of the test line is a function of the conjugate concentration. For some labels, additional reagents may need to be added to the LFA after the sample has passed through the membrane to initiate a color-changing chemical reaction or increase color intensity.

[0060] Measurement results can be judged by eye (typical for qualitative LFA testing), or an external or integrated optical reader can be used to more objectively or quantitatively assess the strength of the optical signals at the test and calibration lines.

[0061] For electrochemical methods, one or several sensors are integrated into the lateral flow strip. The positions of the sensor and conjugate are selected such that a signal is generated when the conjugate is in proximity to the test or control line. For some conjugates, additional reagents may need to be added to the membrane to initiate the electrochemical measurement.

[0062] As shown in FIGS. 2A-2C, an embodiment of an electrochemical sensor 218 for electrochemical measurements can include one or more working electrodes 260, counter electrodes 262, and reference electrodes 264. For example, the electrochemical sensor 218 shown in FIG. 2A can include one working electrode 260, one counter electrode 262, and one reference electrode 264. The electrochemical sensor 218' shown in FIG. 2B can include two working electrodes 260, one counter electrode 262, and one reference electrode 264. In some embodiments, each working electrode 260 is paired with a respective counter electrode 262, and the strip includes a single reference electrode 264. In some embodiments, the counter electrode 262 is not required. For example, the electrochemical sensor 218'' shown in FIG. 2C includes one working electrode 260 and one reference electrode 264, but in some embodiments, multiple working electrodes 260 can be used with one reference electrode 264. This type of electrochemical sensor can be interfaced with an integrated or external analytical system that can use one of a variety of measurement methods including amperometry, impedance, potentiometry, or voltammetry.

[0063] The bound conjugate produces a signal that depends on the relative concentration of conjugate at the test or control line, which is proportional or inversely proportional to the concentration of the analyte.

[0064] In some embodiments, such as the sensor 300 shown in FIG. 3, the sensor 300 can include an ion-selective electrode in which the working electrode has an ion-selective membrane. In some embodiments, the sensor 300 can correspond to the sensor 100 in some or all respects, including the conjugate pad 312, the test line 320, and the control line 322, each of which generally corresponds to the respective conjugate pad 112, the test line 120, and the control line 122 as described above. In some embodiments, the sensor 300 can include an ion or ion solution loaded liposome 342 immobilized above the ion selective electrode 318. The lysis agent can be conjugated to the target analyte as an analyte lysis conjugate 330 that is loaded into the conjugate pad 312. In some embodiments, the ion-loaded liposome can be tagged with a capture ligand.

[0065] If a conjugate molecule 330 is not captured by the capture ligand 340 (e.g., due to competition with analyte present in the sample), it continues to flow into the region containing liposomes 342, disrupting the liposome bilayer and causing an analyte-dependent increase in the relative ion concentration. The ion selective electrode 318 can be used to measure the ion concentration as an indication of the analyte concentration of the sample.

[0066] Molecularly imprinted polymers are an alternative to traditional antibody-based capture of analytes in lateral flow assays. In contrast to antibodies, these polymers are chemically inert, can withstand extremes of pH and temperature, have long-term stability, and are insoluble in water and most organic solvents.

[0067] In some embodiments, such as the sensor 400 shown in FIG. 4, the capture molecule is a molecularly imprinted polymer 440 on the surface of the working electrode 418 as part of a competitive LFA. In some embodiments, the sensor 400 can correspond to the sensor 100 in some or all respects, including the conjugate pad 412, the membrane 414, and the wick pad 416, each of which generally corresponds to the respective conjugate pad 112, the membrane 114, and the wick pad 116 as described above. In some embodiments of the sensor 400, an analyte-labeled conjugate 430 is present on the conjugate pad 412 and competes with the analyte present in the sample for binding to the polymer. When the labeled conjugate molecule 430 is captured by the molecularly imprinted polymer 440, a signal is generated that can act as an indicator of the analyte concentration of the sample. The analyte concentration-dependent signal can be measured at the electrode 418.

[0068] In some embodiments, the label is conjugated to an antibody that targets the analyte as part of a sandwich LFA. If the analyte is present, it is bound by the labeled antibody and captured by the polymer of the working electrode, generating a signal that can act as an indicator of the analyte concentration of the sample.

[0069] Aptamers, single-stranded nucleic acid molecules that bind with high affinity to target molecules, can be used in place of or in combination with traditional antibody-based capture of analytes in lateral flow assays. In contrast to antibodies, aptamers have higher thermal stability and can be mass-produced via traditional polynucleotide manufacturing routes.

[0070] In some embodiments, such as the sensor 500 shown in FIG. 5, the capture molecule may be an aptamer 540 on the surface of the working electrode 418 as part of a competitive LFA. In some embodiments, the sensor 500 may correspond to the sensor 100 in some or all respects, including the conjugate pad 512, the membrane 514, and the wick pad 516, each of which generally corresponds to the respective conjugate pad 112, the membrane 114, and the wick pad 116 as described above. In some embodiments of the sensor 500, an analyte-labeled conjugate 530 is present on the conjugate pad 512 and competes with the analyte present in the sample for binding to the aptamer 540. When the labeled conjugate molecule 530 is captured by the aptamer 540, a signal is generated that may act as an indicator of the analyte concentration of the sample. The aptamer 540 may be immobilized on the working electrode 518, and / or on a test line (not shown), and / or on other suitable locations. An analyte concentration-dependent signal may be measured at the electrode 518.

[0071] In some embodiments, the label is conjugated to an antibody of the analyte label conjugate 530 that targets the analyte as part of the sandwich LFA. If the analyte is present, it is bound by the labeled antibody and captured by the aptamer 540 of the working electrode 518, generating a signal that can act as an indicator of the analyte concentration of the sample.

[0072] Use of ancillary measures to correct, eliminate, or enhance LFA results Many compounds and reactions typically used in LFAs are sensitive to reaction conditions such as temperature, pH, or ion concentration. These sensitivities can be taken into account in LFAs to create more robust integrated sensors, e.g., robust integrated immunosensors.

[0073] One such compound is an enzyme, a biological molecule that increases the rate of a chemical reaction. Enzymes can be used in LFAs as part of a conjugate molecule, whereby the enzymatic reaction produces an optical or electrochemical signal. Certain enzymes have an optimal pH at which they function, outside of which the enzymatic activity is reduced or lost.

[0074] Another such compound is an aptamer, a single-stranded nucleic acid molecule, or a short peptide that binds to a target molecule with high affinity. The selectivity of an aptamer is sensitive to solution conditions (pH and ion concentration) that can affect the conformation of the target binding site and subsequent performance if not properly compensated for.

[0075] As mentioned above, these effects can be reduced using running buffers or chemical additives. In some situations, this may not be possible or desirable due to the additional complexity introduced by these components or the additional diagnostic value of the regulation parameters.

[0076] In some embodiments, the LFA is paired with one or more additional biosensors that perform ancillary measures to correct, eliminate, enhance, and / or aid in the interpretation of the LFA measurements.

[0077] In some embodiments, the secondary measure is pH, which can be determined using a pH sensor 602 integrated below the sample membrane of an analyte biosensor 604 of an LFA 600, as shown in Figure 6A. This pH measurement can be used in one or more of several ways, as shown in Figures 6B-6D.

[0078] In some embodiments, pH may be known to affect the binding of conjugates to a test line or the efficiency of an enzymatic reaction of a sample 610, for example as shown in FIG. 6B. The LFA 600 and an analyzer 620 may be used to collect and measure parameters of the sample 610. An analyte sensor output 614 from an analyte sensor 604 is generated along with an auxiliary sensor output 612 from an auxiliary sensor (e.g., pH sensor) 602. A reference database 630 may be used along with the measured pH 612 and the measured analyte 612 for correction of the test strip output to generate a corrected result 650 to improve the accuracy of the assay.

[0079] In some embodiments, the pH of the biological sample 610' is within a range, and samples outside this range cannot be accurately measured, as shown, for example, in FIG. 6C. The pH can be used to identify samples that are outside of acceptable pH parameters to better identify erroneous results. The LFA 600 and the analyzer 620 can be used to collect and measure parameters of the sample 610'. An analyte sensor output 614 from the analyte sensor 604 is generated along with an auxiliary sensor output 612 from an auxiliary sensor (e.g., pH sensor) 602. A reference database 630' can be used along with the measured pH 612 and the measured analyte 612 to exclude test strip results that are known to be outside the acceptable range or to identify our outlier data that is otherwise erroneous. The remaining results can be used to generate excluded results 650' to improve the accuracy of the analysis.

[0080] In some embodiments, as shown for example in FIG. 6D, the pH of the biological sample 610″ is indicative of saliva flow rate, which impacts interpretation of analyte concentrations due to biological processes. Enhanced pH assessment can provide additional diagnostic information that can be used to better interpret the results of the LFA 600, and the LFA 600 and analyzer 620 can be used to collect and measure parameters of the sample 610″. An analyte sensor output 614 from the analyte sensor 604 is generated along with an auxiliary sensor output 612 from an auxiliary sensor (e.g., pH sensor) 602. A reference database 630″ can be used along with the measured pH 612 and the measured analyte 612 to adjust or provide context for the test strip results and can be used to generate improved clinical interpretation results 650″ and improve the accuracy of the assay.

[0081] In some embodiments, the pH can be measured and one or more of the functions described above can be used. For example, the LFA 600 and analyzer 620 can be used to collect the analyte sensor output 614 and the auxiliary sensor output 612, and both outputs 612, 614 can be used along with multiple databases 630, 630' and 630'' to provide a final result that is pH corrected, filters out erroneous or invalid data, and includes improved clinical interpretation.

[0082] In some embodiments, the auxiliary measure is osmolality or osmolality, determined using an impedance biosensor integrated below the sample membrane, which measurement can similarly be used to correct, eliminate, or enhance the output of the LFA.

[0083] In some embodiments, the auxiliary measure is temperature, determined using a temperature sensor integrated beneath the sample membrane and / or handheld measurement device, which measurement can similarly be used to correct, eliminate, or enhance the output of the LFA.

[0084] In some embodiments, the auxiliary measurements are not integrated into the LFA, but instead are performed on separate biosensors, in which the analytical device can interface with two or more biosensors and perform multicomponent analyses that integrate each measurement.

[0085] In some embodiments, the auxiliary measurement is kinematic data and is collected by a second analytical device, which can commingle data with the first analytical device for multicomponent analysis.

[0086] In some embodiments, the auxiliary measurements are a set of answers and / or a set of observations provided and / or annotated by the individual providing the sample or the individual operating the analytical device, in which the answers and / or observations are used to contextualize the output of the LFA.

[0087] In some embodiments, a system of one or more analytical devices and one or more sensors cooperate to provide one or more assays corrected or interpreted with multiple auxiliary measurements. For example, in some embodiments, one analytical device can use one sensor that measures a body fluid analyte that is adjusted with auxiliary measurements of pH and temperature to provide a final measurement of the analyte. In other exemplary embodiments, a system can include one analytical device with one sensor that measures a body fluid analyte that is adjusted with auxiliary measurements of pH and sample temperature, and a second analytical device with a first sensor for kinetic data and a second sensor for ambient temperature, and the system provides a final measurement of the analyte adjusted for sample pH, sample temperature, kinetic data, and ambient temperature.

[0088] Integration of sample collection and processing microfluidics within the LFA, eliminating the need for external processing Some conventional LFAs require the sample to be collected by an external device and / or some form of sample processing, such as the addition of a running buffer, prior to application to the LFA sample pad.

[0089] These processes serve to normalize sample parameters such as pH, osmolality, or viscosity prior to measurement, and / or to remove potential contaminants from the sample, such as blood cells, air bubbles, or food debris that may interfere with downstream measurement processes. In some situations, these processes are undesirable due to the additional complications they introduce.

[0090] In some embodiments, the LFA is paired with integrated sampling microfluidics to enable direct sampling and / or automated processing of biological samples within the LFA.

[0091] In some embodiments, such as the sensor 700 shown in FIG. 7A, the integrated sample collection and processing microfluidics 710 functions to collect saliva 704 directly from the tongue 702 and then filter particulate matter and air bubbles from the sample 704 before metering it to the sample pad. In some embodiments, such as the sensor 700' shown in FIG. 7B, the integrated sample collection and processing microfluidics 710' includes the features of the sample collection and processing microfluidics 710 and the sample mixing microfluidics 712 described above. The sample collection and processing microfluidics 710' functions to collect saliva 704 directly from the tongue 702 and then automatically mix the running buffer with the collected sample in the sample mixing microfluidics 712 before metering it to the sample pad. In some embodiments, the buffer may be stored in the buffer reservoir 706 before mixing. In some embodiments, the mixing microfluidics 712 is separate from the sample collection and processing microfluidics 710.

[0092] In some embodiments, the sample is collected in an external receptacle and then sampled with the LFA, hi some embodiments, the integrated microfluidics functions to automatically mix the running buffer with the collected sample before metering it onto the sample pad.

[0093] In some embodiments, the sample is collected in an external receptacle that then physically interfaces with the end of the LFA. The integrated microfluidics functions to automatically draw the sample from the attached receptacle and mix the running buffer with the collected sample prior to metering onto the sample pad.

[0094] Integration of an automatic timing element inside the LFA to eliminate the need for manual timing Some conventional LFAs require additional manual steps to be performed after sample addition and before measurement results can be obtained.

[0095] Examples of this include adding a buffer to initiate a chemical reaction, inserting an LFA into the measurement device, or folding a secondary structure to redirect the flow of the sample. Typically, these steps are prompted after a set time or after the sample has advanced past a certain point on the membrane.

[0096] In some embodiments, the LFA is paired with one or more integrated timing elements that can be used to automate or expedite time-sensitive processes.

[0097] Some implementations, such as the electrochemical sensor 818 shown in FIG. 8A, may include this timing element. The electrochemical sensor 818 may correspond to the sensor 218 in some or all respects, including the working electrode 860, the counter electrode 862, and the reference electrode 864, each of which generally corresponds to the respective working electrode 260, the counter electrode 262, and the reference electrode 264 as described above. The electrochemical sensor 818 may also include a fluid sensing electrode 872 disposed under the membrane to confirm the progress of the sample flow. For example, as the fluid 870 flows through the sensing point or line on the membrane, the fluid sensing electrode 872 may generate a signal that is used by the analytical device to start a timer 874. The timer 874 may be started in the analytical device to count down for a predetermined period of time. The duration of the timer 874 can be between 0.2 seconds and 180 seconds, e.g., 0.2 seconds, 0.5 seconds, 1 second, 2 seconds, 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 45 seconds, 60 seconds, 90 seconds, 120 seconds, 150 seconds, 180 seconds, or other durations within that range. In some embodiments, the duration of the timer 874 can be between 0.5 and 10 minutes, e.g., 0.5, 1, 1.5, 2, 3, 5, 8, or 10 minutes, and other durations within that range. For example, a 2 minute timer 874 can be used to count down to the start of a measurement. This avoids the need for the user to start a measurement at a point when the reaction has progressed sufficiently. Other durations of the timer 874 can be used as pauses or wait times for other functions, such as delays to allow for proper sample collection, delays to collect additional parameters as described above, delays to verify communication or other connectivity, etc.

[0098] In some implementations, a fluid detection electrode 872 is placed near the wick pad 110 to indicate when the sample has passed the test line 120, the control line 122, or the calibration line 124. Detection of fluid at this point can be used to trigger a measurement. This avoids the need for a user to monitor the progress of the flow and begin a measurement once the sample has progressed sufficiently along the membrane 114.

[0099] In some embodiments, several membranes with variable flow rates can be arranged to regulate the addition of secondary compounds necessary to initiate a chemical reaction. For example, the LFA 800 shown in FIG. 8B can include a test strip base that can correspond to the LFA 100 in some or all respects, including a sample pad 810, a test line 820, a wick pad 816, and an electrode 818, each of which generally corresponds to a respective sample pad 110, test line 120, wick pad 116, and electrode 218 as described above. The sensor 800 can also include a labeled conjugate 812 that corresponds to the conjugate pads 112, 312, 412, and 512 described above. The sensor 800 can also include a primary membrane 815 with a fast flow rate. A second membrane 878 can be selected with a slower flow rate such that delivery of the substrate solution 876 to the test line 820 is delayed relative to the primary membrane 815. This allows the first compound to effectively bind to the test line before delivery of the second compound. An insulating material 874 may be used to separate the faster flowing primary membrane 815 from the slower flowing secondary membrane 878 .

[0100] In some embodiments, several fluid detection electrodes are integrated into the LFA, and an unexpected output from the timing element (e.g., more than 5 minutes elapsed between fluid detection at electrode 1 and fluid detection at electrode 2) is used to flag a measurement error.

[0101] Integrated packaging of liquid chemicals within the LFA to reduce user errors when adding substrate solutions In some conventional LFAs, it may not be possible to automate the addition of chemicals before obtaining a measurement result, such as when the chemicals must be stored in the liquid phase and cannot be dried on the conjugate pad. In such cases, these chemicals may need to be stored separately from the LFA materials and manual addition of these chemicals by an operator may be required.

[0102] In some embodiments, the LFA is paired with an integrated fluid pack that can be used to store reagents. Opening a pre-measured fluid pack adds a set volume of reagent to a set location. The use of a fluid pack reduces the risk of user error by regulating the volume and location where the fluid is deposited.

[0103] In some embodiments, such as the LFA 900 shown in Figure 9A, the integrated liquid pack 980 is manually opened by a system operator 982 to initiate a step in the measurement process. The liquid pack 980 may be part of the enzyme substrate that is deposited on the surface of the test line 920 after binding of the enzyme conjugate molecule.

[0104] In some embodiments, such as the LFA 900' shown in FIG. 9B, the integrated liquid pack 980' is opened by the analyzer 986. For example, the analyzer 986 can include an automated pressurizing arm 984 to open the liquid pack 980' when the LFA 900' is inserted into the analyzer or when the sample reaches the test line 920. In some embodiments, the analyzer includes a piercing, cutting, or clamping mechanism to open the liquid pack 980' when the LFA 900' is inserted into the analyzer. The liquid pack 980' and / or the LFA 900' can include perforations, frangibles, pins, rails, or other components that cooperate with the analyzer and / or mechanisms to facilitate aligning and / or opening the liquid pack 980'. These features can further reduce the possibility of user error by regulating the timing and location at which the liquid pack 980' is opened.

[0105] Integration of a cutting element to regulate deposition of substrate solution In some LFAs, the addition of substrate can be disturbed due to the continuous flow of solution onto the absorbency pad or backflow onto the sample and conjugate pads. To reduce the effect of this factor, it may be necessary to isolate the area of ​​the membrane containing the test and control lines before adding the substrate solution.

[0106] In some embodiments, the LFA is paired with an integrated cutting element that is used to isolate segments of the lateral flow system.

[0107] In some implementations, such as the LFA 1000 shown in FIG. 10A, depressing the cutting element 1090 causes one or more blades 1094 to cut the membrane. The blades 1094 may be positioned at or near the test line 1020 such that they do not cut the electrodes required for the measurement. In some implementations, the cutting element 1090 may include a cover 1096 or button for ease of operation. The cover 1096 may also help distribute pressure evenly across the multiple blades 1094. In some implementations, the cutting element 1090 may also include a spring 1092 configured to hold the cutting element open and return the blades to a raised position when pressure is released.

[0108] In some embodiments, such as the LFA 1000' shown in FIG. 10B, an integrated cutting element 1090' can be combined with an integrated fluid pack 1070. The cutting element 1090' corresponds in many ways to the cutting element 1090, including the cover 1096, spring 1092, and blade 1094, as described above. When the cutting element 1090' is pressed, the fluid pack 1070 is also pressed, expelling fluid. The same pressing motion causes the cutting element 1090' to lower the blade 1094, isolating an area of ​​the membrane where the substrate solution is being expelled.

[0109] Integration of heating or cooling elements to improve the performance of the LFA For some LFAs, performance such as sample flow, analyte binding, signal generation, etc. may be affected by the temperature of the LFA and / or the sample. In some situations, it may be difficult or impossible to compensate for these effects using a temperature sensor, and rather, it is necessary or more convenient to modify the temperature of the LFA.

[0110] In some embodiments, the LFA incorporates a temperature modifier, such as a heating and / or cooling element, that is used to modify the temperature of the LFA to a desired temperature.

[0111] In some embodiments, such as the LFA 1100 shown in FIG. 11, the temperature modifier 1135 can be a heating element implemented, for example, as an integrated resistive heating coil. The LFA 1100 can be similar to other LFA devices described above, including a conjugate pad 1112, a membrane 1114, and a wick pad 1116, which correspond to the conjugate pad 112, the membrane 114, and the wick pad 116 described above, respectively. When the LFA 1100 is inserted into an analytical device, the temperature of the LFA 1100 is determined using an integrated temperature sensing element. If the temperature is too low, as determined by the temperature sensing element, the analytical system provides power to the temperature modifier 1135 (heating element) to heat the device until the desired temperature is achieved, and then prompts the user to apply a sample for analysis.

[0112] In some embodiments, the temperature modifier 1135 may be a cooling element implemented, for example, as an integrated fluid pack containing two separate chemicals (e.g., urea and water). When an LFA, for example, the LFA 1100, is inserted into an apparatus for analysis, the temperature of the LFA 1100 is determined using an integrated temperature sensing element. If the temperature is too high, the analysis system activates the temperature modifier 1135 to cool the apparatus. In some embodiments, the temperature modifier 1135 mixes two fluids resulting in an endothermic reaction that cools the LFA 1100. When the desired temperature is achieved as determined by the temperature sensing element, the user is then prompted to apply the sample for analysis. In some embodiments, the temperature modifier 1135 heats or cools the LFA as described above. In some embodiments, the temperature modifier 1135 both heats and cools as determined by a temperature sensor. In addition to those described above, other temperature modification mechanisms such as air or liquid cooling may be used as appropriate, along with pumps, fans, and the like. In some embodiments, time is used to adjust the temperature, for example, by waiting a predetermined period of time for the sample to warm or cool.

[0113] In some embodiments, the heating or cooling element 1135 is integrated into the analytical system and the LFA, e.g., the LFA1100, includes an integrated material with high thermal conductivity. When the LFA1100 is inserted into an analytical device, the temperature of the LFA1100 is determined using an integrated temperature sensing element (not shown). If the temperature is outside of the desired range, the temperature modifier 1135 activates the heating or cooling element to change the temperature within the analytical system, which in turn changes the temperature of the LFA1100 via the conductive element. When the desired temperature is achieved as determined by the temperature sensing element, then the analytical system prompts the user to apply a sample for analysis.

[0114] Integrating quality control elements to prevent inappropriate LFA use For some LFAs, there may be chemical or material components that are sensitive to suitable storage conditions, such as temperature or humidity, and / or have a limited shelf life. Additionally, for some LFAs (such as those that use electrochemical rather than optical methods), it may not be clear whether the LFA has been used previously.

[0115] In some embodiments, the LFA incorporates a quality control element that can be used to identify whether a test strip has been properly stored, is expired, or has previously been used.

[0116] In some embodiments, such as the LFA 1200 shown in FIG. 12, the quality control element 1237 is a temperature sensing element integrated into the LFA 1200. The LFA 1200 may be similar to other LFA devices described above, including a conjugate pad 1212, a membrane 1214, and a wick pad 1216, which correspond to the conjugate pad 112, the membrane 114, and the wick pad 116 described above, respectively. If the LFA 1200 is exposed to a temperature above a threshold, a characteristic (e.g., color, resistance, phase) of the quality control element 1237 (e.g., the temperature sensing element) is irreversibly altered. When the LFA 1200 is inserted into a device for analysis, a characteristic of the temperature sensing element 1237 is measured by the device. If the characteristic indicates exposure to a temperature above a threshold, the LFA 1200 is identified as expired and cannot be used to perform an analysis.

[0117] In some embodiments, the quality control element 1237 is a moisture detection element integrated into the LFA, e.g., the LFA 1200 in the wick pad 1216. If the LFA 1200 has previously been used to analyze a sample, the quality control element 1237 (e.g., the moisture detection element) is wet and the properties of the quality control element 1237 are irreversibly altered. When the LFA 1200 is inserted into an instrument for analysis, the properties of the moisture detection element 1237 are measured by the instrument. If the properties indicate exposure to moisture, the LFA 1200 is marked as used and cannot be used to perform an analysis.

[0118] In some embodiments, LFAs 1200 having quality control elements 1237 are identified as expired, as described above. In some embodiments, expired LFAs may be marked as such. For example, expired LFAs 1200 may be marked visually with a stamp, hole, brand, or other visible indicator, or may be marked electrically by breaking traces, shorting connections, etc. Integration of batch tracking elements to aid in the identification of test strips.

[0119] For an analytical system to be compatible with multiple types of LFAs, it is necessary to include mechanisms that allow the system to identify the type of LFA, initiate the correct measurement process, and properly interpret the output of the LFA.

[0120] Additionally, due to manufacturer and batch-to-batch variations in the production of LFAs, it may also be necessary to interpret the output of the LFA using manufacturer- or batch-specific reference or calibration data. To accomplish this, a mechanism capable of identifying the LFA manufacturer and / or batch is required. In the present invention, the LFA incorporates electrodes or other structures that can be used to identify the type, manufacturer, and / or batch of the LFA.

[0121] In some implementations, such as the circuit 1318 shown in FIG. 13, the electrode arrangement 1318 for a particular LFA manufacturing batch is arranged and sized to have a particular resistance. In this sense, the particular resistance acts as an identifier for the LFA manufacturer, batch, and / or type. After an LFA carrying the electrode arrangement or circuit 1318 is inserted into an analytical device, this resistance is measured by the analytical device and used to identify the type, manufacturer, and / or batch of the LFA. The analytical device references a reference database to identify the correct measurement process and / or calibration data associated with this LFA type, manufacturer, and batch.

[0122] In some implementations, the encoded batch and type tracking data can be further used to identify whether a test strip is old, counterfeit, region locked, proprietary, etc. In some implementations, the resistance-based identification of the circuit 1318 can be used to trigger a firmware update of the analysis unit to enable proper processing of an LFA carrying the circuit 1318. For example, the batch encoding 1318 in the electrode arrangement 1318 can prompt the handheld device to download additional software or firmware to enable measurement of additional analytes or to update a reference database (e.g., reference database 630 with compensation or calibration parameters described above) for proper processing.

[0123] Communicating data with a central database to facilitate analysis and use of the data In some embodiments, the analytical device presents results and / or interpretation advice directly to the operator. For example, results including raw, corrected, calibrated, adjusted, filtered, and interpreted results may be presented to a user of the system, such as the individual providing the biofluid sample, the person operating the analytical device, or a remote user. The data may be presented in a user interface, for example, to select, mark, adjust, zoom, collate, or otherwise view, modify, manipulate, and / or annotate the data. To improve the usefulness of the measurement data, in some embodiments, it may be beneficial for the results of the LFA to be transmitted to a central database. These results may then be relayed to other individuals for action or as part of a more complex analysis.

[0124] In some embodiments, the analysis system communicates with a mobile device to relay the LFA results to a centralized database. In some embodiments, the analysis device alternatively or additionally communicates wirelessly with a cell phone or tablet for presentation, interpretation, tracking and / or analysis of the data.

[0125] In some embodiments, the analytical system wirelessly communicates the LFA results and associated information to an internet-connected mobile device, which then relays the results to a centralized database. This database can be accessed via the individual providing the sample, the individual operating the analytical system, or other individuals authorized to view the measurement results. In some embodiments, the centralized database can be accessed via a mobile application and / or a web portal.

[0126] In some embodiments, additional analysis of the LFA results is performed in a centralized database, optionally integrating the results and other data, to aid in interpreting the LFA results.

[0127] In some embodiments, the centralized database automatically alerts third parties based on the measurement results if one or more specified criteria are met to help improve the immediacy of response to LFA results.

[0128] Other variations Although some embodiments describe LFA devices, the systems, devices, and / or methods disclosed herein can be applied to other types of therapeutics that can be used standalone or in addition to LFA diagnostics. The systems, devices, and / or methods disclosed herein can be extended to any medical device, particularly any diagnostic device that tests bodily fluids, although other fluid testing may also be appropriate. For example, the systems, devices, and / or methods disclosed herein can be used with devices that provide one or more additional diagnoses and / or treatments based on the diagnosis. The systems and methods disclosed herein are not limited to medical devices and can be utilized by any liquid testing device.

[0129] Any implementation of the transmission of data described herein may be performed securely, for example utilizing one or more of encryption, https protocol, secure VPN connections, error checking, delivery confirmation, etc.

[0130] Any values ​​of thresholds, limits, durations, etc. presented herein are not intended to be absolute and therefore may be approximated. Additionally, any thresholds, limits, durations, etc. presented herein may be fixed or changed automatically or by a user. Additionally, as used herein, relative terms such as exceed, over, less than, etc. with respect to a reference value are intended to encompass equal to the reference value. For example, exceeding a reference value that is positive can encompass being equal to or greater than the reference value. Additionally, as used herein, relative terms such as exceed, over, less than, etc. with respect to a reference value are intended to encompass the inverse of the disclosed relationships such as less than or equal to, less than, greater than, etc. with respect to the reference value.

[0131] A feature, material, characteristic, or group described in connection with a particular aspect, embodiment, or example should be understood to be applicable to any other aspect, embodiment, or example described herein, unless to the contrary. All of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Protection is not limited to the details of any of the foregoing embodiments. Protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), or any novel one, or any novel combination of the steps of any method or process so disclosed.

[0132] Although certain embodiments have been described, these embodiments are presented as examples only and are not intended to limit the scope of protection. Indeed, the novel methods and systems described herein may be embodied in a variety of other forms. Furthermore, various omissions, substitutions, and modifications of the forms of the methods and systems described herein may be made. Those skilled in the art will appreciate that in some implementations, the actual steps performed in the illustrated and / or disclosed processes may differ from those shown in the figures. Depending on the implementation, some of the steps described above may be deleted and others may be added. For example, the actual steps and / or order of steps performed in the disclosed processes may differ from those shown in the figures. Depending on the implementation, certain steps may be implemented in different locations or structures. For example, various components shown in the figures or described herein may be implemented as software and / or firmware of a processor, controller, ASIC, FPGA, and / or dedicated hardware. The software or firmware may include instructions stored in a non-transitory computer-readable memory. The instructions may be executed by a processor, controller, ASIC, FPGA, or dedicated hardware. Hardware components such as controllers, processors, ASICs, FPGAs, and the like may include logic circuits. Furthermore, the features and attributes of the specific embodiments disclosed above can be combined in different ways to form additional embodiments, all of which are within the scope of the present disclosure.

[0133] The user displays and interface screens illustrated and described herein may include additional and / or alternative components. These components may include menus, lists, buttons, text boxes, labels, radio buttons, scroll bars, sliders, check boxes, combo boxes, status bars, dialog boxes, windows, and the like. The user interface screens may include additional and / or alternative information. The components may be arranged, grouped, and / or displayed in any suitable order.

[0134] Provided herein are lateral flow assay devices and methods of using such devices to detect biomarkers, analytes, ion concentrations, etc. in a fluid sample obtained from a subject. One of skill in the art will understand that such lateral flow assay devices can be used to detect any of the biomarkers described herein or additional biomarkers.

[0135] The terms "immobilized" or "embedded" refer interchangeably to reversibly or irreversibly immobilized molecules (e.g., analytes or binding agents). In some instances, reversibly immobilized molecules are immobilized in a manner that allows the molecules or portions thereof (e.g., at least about 25%, 50%, 60%, 75%, 80% or more of the molecules) to be removed from their immobilized locations without substantial denaturation or aggregation. For example, molecules can be reversibly immobilized to an absorbent material (e.g., an absorbent pad) by contacting the absorbent material with a solution containing the molecules, thereby soaking the solution and reversibly immobilizing the molecules. Reversibly immobilized molecules can then be removed by wicking the solution from the absorbent material or from one area of ​​the absorbent material to another. In some instances, molecules can be reversibly immobilized to an absorbent material by contacting the absorbent material with a solution containing the molecules, soaking the solution therewith, and then drying the absorbent material containing the solution. The reversibly immobilized molecules can then be removed by contacting the absorbent material with another solution of the same or different composition, thereby solubilizing the reversibly immobilized molecules, and then wicking the solution from the absorbent material or from one region of the absorbent material to another.

[0136] Irreversibly immobilized molecules (e.g., binding agents or analytes) are immobilized such that they are not, or are not substantially, removed from their location under mild conditions (e.g., a pH of about 4-9, a temperature of about 4-65° C.). Exemplary irreversibly immobilized molecules include those mentioned above, in addition to protein analytes or binding agents bound to nitrocellulose, polyvinylidene fluoride, nylon or polysulfide membranes by standard blotting techniques (e.g., electroblotting). Other exemplary irreversibly immobilized molecules include protein analytes or binding agents bound to glass or plastic (e.g., microarrays, microfluidic chips, glass histology slides or plastic microtiter plates having wells with bound protein analytes therein).

[0137] The term "binding agent" refers to an agent that specifically binds to a molecule such as an analyte. Binding agents, aptamers, ions, binding molecules, and analytes are described in many contexts herein, but those skilled in the art will understand that other binding agents can be used instead as preferred by the user. A wide variety of binding agents are known in the art, including antibodies, aptamers, affixers, lipocalins (e.g., anticalins), thioredoxin A, bilin-binding proteins, or proteins containing ankyrin repeats, the Z domain of Staphylococcus aureus protein A, or fibronectin type III domain. Other binding agents include, but are not limited to, biotin / streptavidin, chelators, chromatography resins, affinity tags, or functionalized beads, nanoparticles, and magnetic particles.

[0138] The terms "bind" and "effectively bind" refer to a molecule (e.g., a binding agent such as an aptamer) that binds to a target with an affinity that is at least 2-fold greater than a non-target compound, e.g., at least about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1000-fold, or more than 1000-fold.

[0139] As used herein, hypothetical language, particularly "can," "could," "might," "may," "eg," and the like, unless otherwise indicated or understood otherwise in the context in which they are used, is generally intended to convey that certain embodiments include certain features, elements, and / or conditions, but not other embodiments. Thus, such hypothetical language is not generally intended to imply that the features, elements, and / or conditions are in any way required by one or more embodiments, or that one or more embodiments necessarily include logic for determining whether or not those features, elements, and / or conditions should be included or implemented in any particular embodiment, with or without authorial input or prompting. Terms such as "comprising," "including," "having," and the like are synonymous and are used in an inclusive, open-ended manner and do not exclude additional elements, features, acts, operations, and the like. Also, the term "or" is used in an inclusive sense (not an exclusive sense), e.g., when used to join a list of elements, the term "or" means one, some, or all of the listed elements. Furthermore, the term "each" as used herein, in addition to having its ordinary meaning, can refer to any subset of the set of elements to which the term "each" applies. Furthermore, the words "herein," "above," "below," and words of similar meaning, when used in this application, refer to this application as a whole and not to any particular portions of this application.

[0140] Unless otherwise indicated, conjunctive language such as the phrase "at least one of X, Y, and Z" should be understood as generally used in context to convey that an item, term, etc. can be either X, Y, or Z, or any combination thereof. Thus, such conjunctive language is not generally intended to imply that at least one of X, at least one of Y, and at least one of Z are required for a particular embodiment to each be present.

[0141] As used herein, degree phrases such as "approximately," "about," "generally," and "substantially" refer to values, amounts, or characteristics that are close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result. For example, the terms "approximately," "about," "generally," and "substantially" can refer to amounts that are less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of the stated amount. As another example, in certain embodiments, the terms "nearly parallel" and "substantially parallel" refer to values, amounts, or characteristics that deviate from strictly parallel by 15 degrees, 10 degrees, 5 degrees, 3 degrees, 1 degree, or 0.1 degrees or less.

[0142] Unless otherwise noted, articles such as "a" or "an" should generally be construed to include one or more of the described items. Thus, phrases such as "an apparatus configured to" are intended to include the recited apparatus or apparatuses. Such one or more recited apparatuses can also be collectively configured to perform the recited recitation.

[0143] Although the present disclosure includes specific embodiments, examples, and applications, it will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, including embodiments that do not provide all of the features and advantages described herein, as well as obvious modifications and equivalents thereof. Thus, the scope of the present disclosure is not intended to be limited by the specific disclosures of preferred embodiments or embodiments herein, but may be defined by the claims presented herein or presented in the future. [Explanation of symbols]

[0144] 1 Electrode, 2 Electrodes, 100 Lateral Flow Assay (LFA), Sensor, 110 Sample Pad, Wick Pad, 112 Conjugate Pad, 114 Membrane, 116 Wick Pad, 120 Test Line, 122 Control Line, 124 Calibration Line, 218 Electrochemical Sensor, Electrode, 218' Electrochemical Sensor, 218'' Electrochemical Sensor, 260 Working Electrode, 262 Counter Electrode, 264 Reference Electrode, 300 Sensor, 312 Conjugate Pad, 318 Ion Selective Electrode, 320 Test Line, 322 Control Line, 330 Conjugate Molecule, Analyte Dissolving Conjugate, 340 Capture Ligand, 342 Ionic Solution Loaded Liposome, 400 Sensor, 412 Conjugate Pad, 414 Membrane, 416 Wick Pad, 418 Working Electrode, 430 Conjugate molecule, analyte labeled conjugate, 440 molecularly imprinted polymer, 500 sensor, 512 conjugate pad, 514 membrane, 516 wick pad, 518 working electrode, 530 analyte labeled conjugate, conjugate molecule, 540 aptamer, 602 pH sensor, 604 analyte sensor, analyte biosensor, 610 sample, 610' biological sample, 610'' biological sample, 612 auxiliary sensor output, analyte, 614 analyte sensor output, 620 analyzer, 630 reference database, 630' reference database, 630'' reference database, 650 corrected results, 650' excluded results, 650'' clinical interpretation results, 700 sensor, 700' sensor, 702 tongue, 704 sample, saliva, 706 buffer reservoir, 710 processing microfluidics, 710' Processing microfluidics, 712 Sample mixing microfluidics, 800 LFA, sensor, 810 Sample pad, 812 Labeled conjugate, 815 Primary membrane, 816 Wick pad, 818 Electrochemical sensor, electrode, 820 Test line, 860 Working electrode, 862 Counter electrode, 864 Reference electrode, 870 Fluid, 872 Fluid sensing electrode, 874 Insulating material, timer, 876 Substrate solution, 878 Second membrane, secondary membrane, 900 LFA, 900' LFA, 920 Test line, 980 Fluid pack, 980' Fluid pack, 982 System operator, 984 Automated pressurizing arm, 986 Analyser, 1000 LFA, 1000'LFA, 1020 test line, 1070 liquid pack, 1090 cutting element, 1090' cutting element, 1092 spring, 1094 blade, 1096 cover, 1100 LFA, 1112 conjugate pad, 1114 membrane, 1116 wick pad, 1135 temperature modifier, cooling element, 1200 LFA, 1212 conjugate pad, 1214 membrane, 1216 wick pad, 1237 quality control element, temperature sensing element, moisture sensing element, 1318 circuit, electrode arrangement, batch coding

Claims

1. 1. A system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, comprising: a first sensor that generates a signal in response to the concentration of the one or more analytes; one or more additional sensors for one or more respective auxiliary measures to assist in interpretation of the signal of the first sensor; [0023] an analytical device to facilitate testing; The system wherein the one or more auxiliary measures include temperature.

2. The system of claim 1 , wherein at least one of the one or more additional sensors is integrated into the lateral flow system.

3. 3. The system of claim 1, wherein at least one of the one or more additional sensors uses the same analytical device but is not integrated into the lateral flow system.

4. The system of any one of claims 1 to 3, wherein the one or more auxiliary measures further comprise at least one of pH, osmolality, and concentration of an ion.

5. The system of claim 1 , wherein at least one of the one or more auxiliary measures is used to correct the signal of the first sensor.

6. The system of claim 1 , wherein at least one of the one or more auxiliary measures is used to flag errors.

7. The system of claim 1 , wherein at least one of the one or more auxiliary measures is used to provide a diagnostic status of the signal of the first sensor.

8. The system of claim 1 , wherein at least one of the one or more auxiliary measures is data collected by a secondary analysis device.

9. 10. The system of claim 1, wherein at least one of the one or more auxiliary measures is a set of responses provided by the individual providing the biological sample.

10. The system of claim 1 , wherein at least one of the one or more auxiliary measures is a set of observations annotated by an individual operating the system.

11. The system of claim 1 , wherein at least one of the one or more auxiliary measures is used to provide a diagnostic status of the signal of the first sensor.

12. The system of claim 1 , wherein the analytical device presents results and / or interpretive advice directly to an operator.

13. The system of claim 1 , wherein the analytical device wirelessly communicates with a cell phone or tablet for data presentation, interpretation, tracking and / or analysis.

14. 1. A system for quantifying the concentration of one or more analytes in a biological sample using a lateral flow system, comprising: one or more sensors each generating a signal in response to the concentration of a respective one of the one or more analytes; integrated microfluidics and materials for collection and processing of said biological sample; and an analytical device that facilitates the testing.

15. The system of claim 14 , wherein the integrated microfluidics and materials controls physical variations in the biological sample.

16. The system of claim 15 , wherein the physical variation comprises viscosity or air bubble content.

17. 15. The system of claim 14, wherein the integrated microfluidics and materials controls chemical variations in the biological sample.

18. 18. The system of claim 17, wherein the chemical variation comprises pH or ionic content.

19. 15. The system of claim 14, wherein the analytical device presents results and / or interpretive advice directly to an operator.

20. 15. The system of claim 14, wherein the analytical device wirelessly communicates with a mobile phone or tablet for data presentation, interpretation, tracking and / or analysis.

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