Systems, devices and methods for amplifying lateral flow assay signals

Abstract

The lateral flow assay devices, systems and methods described herein detect analytes present in a sample at low levels or concentrations by amplifying the signal generated in the detection zone. The signal generated can exceed the detection threshold of the measurement system and increase the sensitivity of the lateral flow assay device, system and method. On the one hand, the lateral flow device includes a signal amplification conjugate that is bound to a complex that is bound to an immobilized capture agent in the detection zone, causing a chain reaction of binding events in the detection zone, which results in amplification of the signal generated in the detection zone.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 686,606, filed June 18, 2018, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates generally to lateral flow devices, testing systems and methods. More specifically, the present disclosure relates to lateral flow assay devices capable of measuring analytes present in low amounts or concentrations in a sample by amplifying the detection signal. Background Art

[0004] Immunoassay systems including lateral flow assays provide reliable, inexpensive, portable, rapid, simple diagnostic tests. Lateral flow assays can quickly and accurately detect the presence of target analytes in a sample, and in some cases quantify them. Advantageously, lateral flow assays can be minimally invasive and used as point-of-care testing systems.

[0005] Lateral flow assays generate a signal that can be measured to indicate the presence, absence, or amount of a target analyte in a sample. However, when the analyte is present in a sample at low levels or concentrations, the signal generated by the labeled analyte captured in the test area of ​​the lateral flow assay may not exceed the detection threshold of the measurement system to read and process the signal. Therefore, the lateral flow assay may not provide a reliable or accurate indication of the presence of the target analyte in the sample. Summary of the invention

[0006] Therefore, one aspect of the present disclosure is to provide an improved lateral flow assay that can detect the presence, absence or content of an analyte in a sample. The embodiments of the methods and systems described herein can measure the presence of an analyte, and in some cases, measure the content or concentration of an analyte, even when the analyte is present at low levels or low concentrations and generally causes the signal generated to be at a level below the detection threshold of the measurement system. Therefore, the embodiments of the systems, devices and methods described herein are very sensitive to even low levels or low concentrations of target analytes. In addition, the embodiments of the methods and systems described herein can determine that there is no target analyte in a sample with a higher confidence than existing methods and systems. The absence of a detectable signal in an embodiment of the present disclosure can reliably indicate that there is no target analyte in a sample (contrary to the situation where the analyte actually exists but is at such a low concentration - the signal generated falls below the detection threshold of the system). Therefore, embodiments of the present disclosure can reduce false negative readings. As will be described in detail below, according to the lateral flow assay of the present disclosure, the signal generated in the test area of ​​the lateral flow assay such as the capture zone is amplified to allow a conventional measurement system to detect the level of the target analyte present in a sample at low levels or low concentrations.

[0007] Signals generated by assays according to the present disclosure are described herein in the context of optical signals generated by reflective markers such as, but not limited to, gold nanoparticle markers and latex particles of different colors. Although embodiments of the present disclosure are described herein by reference to "optical" signals, it should be understood that the assays described herein may use any suitable material for markers to generate signals, including, but not limited to, fluorescent latex bead markers that generate fluorescent signals and magnetic nanoparticle markers that generate signals indicative of changes in magnetic fields associated with the assay.

[0008] Embodiments of lateral flow assays described herein are particularly advantageous in diagnostic tests in which the target analyte is present at low levels or concentrations on the test strip. The target analyte may be present at low concentrations on the test strip for many reasons, including but not limited to the presence of a limited amount of the target analyte in the sample applied to the test strip and a limited volume of the sample being applied to the test strip. Although embodiments of the present disclosure are described with reference to detecting a target analyte at low concentrations, it should be understood that the present disclosure can also detect low levels of target analytes.

[0009] Some embodiments disclosed herein relate to a lateral flow assay for detecting a target analyte in a sample. The lateral flow assay includes a first conjugate comprising a first label, a reagent configured to specifically bind to the target analyte, and a first binding partner; a second conjugate upstream of the first conjugate along a fluid flow path of the lateral flow assay, wherein the second conjugate comprises a second label and a second binding partner configured to specifically bind to the first binding partner; and a detection zone downstream of the first conjugate and the second conjugate along the fluid flow path of the lateral flow assay, the detection zone comprising an immobilized capture agent that specifically binds to the target analyte.

[0010] The first conjugate can be present in a sample receiving zone of a lateral flow assay, or wherein the first conjugate is present in a first conjugate zone downstream of the sample receiving zone. The second conjugate can be present in a buffer receiving zone upstream of the sample receiving zone, or wherein the second conjugate is present in a second conjugate zone downstream of the buffer receiving zone and upstream of the sample receiving zone. The first conjugate can be configured to dissolve and move to the detection zone after the fluid sample is applied to the lateral flow assay. The second conjugate can be configured to dissolve and move to the detection zone after the first conjugate moves to the detection zone.

[0011] The reagent configured to specifically bind to the target analyte can be an antibody or antibody binding fragment that specifically binds to the target analyte. The first binding partner and the second binding partner can include a binding pair selected from the following: antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid chain / complementary nucleic acid chain, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / avidin, receptor / ligand and virus / cell receptor. The first binding partner and the second binding partner include a biotin / avidin binding pair, and the avidin can include streptavidin or neutravidin. The first binding partner and the second binding partner can include an antigen / antibody binding pair, and the antigen can include a peptide or a decapeptide. The target analyte can be a target biological substance or an environmental substance. The target analyte can be an influenza virus. The influenza virus can be influenza A virus, influenza B virus or influenza C virus. The immobilized capture agent can be an antibody or antibody binding fragment that specifically binds to the target analyte. The test strip can include a nitrocellulose membrane. The lateral flow assay can also include a control area, which contains an immobilized capture agent that specifically binds to the first conjugate.

[0012] The first and second markers can be selected from metal nanoparticles, blue latex beads, metal nanoparticles, colored latex particles, colored latex beads, magnetic particles, carbon nanoparticles, quantum dots, up-converting phosphors, organic fluorophores, textile dyes, enzymes or liposomes. The first marker and the second marker can include gold nanoparticles. The first marker and the second marker can be configured to generate an optical signal, a fluorescent signal or a magnetic signal.

[0013] The lateral flow assay may also include a housing comprising a sample well positioned laterally above or upstream of the first conjugate, a buffer well positioned laterally above or upstream of the second conjugate zone, and a reading window accessible to the detection zone. The buffer well, the second conjugate zone, the sample receiving well, and the first conjugate zone may be spatially separated along the fluid flow path of the lateral flow assay.

[0014] Further embodiments disclosed herein relate to methods for performing a lateral flow assay. The method comprises applying a first conjugate to a lateral flow test strip at or downstream of a sample receiving zone of the lateral flow test strip; and applying a second conjugate to the test strip at or downstream of a buffer receiving zone upstream of the sample receiving zone. The first conjugate and the second conjugate may be applied to the test strip simultaneously. The first conjugate and the second conjugate may be applied to the test strip by air jet deposition.

[0015] Other embodiments described herein relate to methods for detecting a target analyte in a sample. The method includes applying the sample to a lateral flow assay. The lateral flow assay includes a first conjugate comprising a first marker, a reagent configured to specifically bind to the target analyte, and a first binding partner; a second conjugate upstream of the first conjugate along the fluid flow path of the lateral flow assay, wherein the second conjugate comprises a second marker and a second binding partner configured to specifically bind to the first binding partner; and a detection zone downstream of the first conjugate and the second conjugate along the fluid flow path of the lateral flow assay, the detection zone comprising an immobilized capture agent that specifically binds to the target analyte. The method also includes binding a complex to an immobilized capture agent in the detection zone, wherein the complex comprises a target analyte bound to the first conjugate. After binding the complex, the method includes releasing the second conjugate to flow along the fluid flow path of the lateral flow assay. The method further includes binding the second conjugate to the complex bound in the detection zone.

[0016] The method can include detecting a signal generated by the complex and the second conjugate combined in the detection zone. The method can also include binding the first conjugate that is not bound to the target analyte to the second conjugate that is bound to the complex in the detection zone. The first label and the second label can be configured to generate an optical signal, a fluorescent signal or a magnetic signal. The second conjugate can be released in 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes after the sample is applied to the lateral flow device.

[0017] Binding the complex to the immobilized capture agent in the detection zone may include labeling the target analyte with a first conjugate to form a complex; and binding the complex to the immobilized capture agent in the detection zone. Releasing the second conjugate may include applying a buffer solution to the second conjugate or to a location on the lateral flow assay upstream of the second conjugate. Applying a buffer solution may include pouring a buffer solution into a buffer receiving well positioned laterally above or upstream of the second conjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A top view of an example lateral flow device according to the present disclosure is shown, including buffer wells, sample wells, and a reading window.

[0019] Figure 2 An example assay test strip of a lateral flow signal amplification assay according to the present disclosure is shown.

[0020] Figure 3 An example method of amplifying a signal produced by an assay test strip according to the present disclosure is schematically illustrated.

[0021] Figure 4 Shown are the results of a signal amplification reaction performed on a lateral flow device according to the present disclosure, indicating signal intensity as a function of read time for a control signal (without signal amplification conjugate - ◆) and a test signal (with signal amplification conjugate - ■).

[0022] Figure 5 The results of a signal amplification reaction performed on a lateral flow device according to the present disclosure are shown, indicating signal intensity as a function of analyte concentration, including control signals for assays performed without a signal amplification conjugate after 10 minutes (◆) and 20 minutes (▲), and test signals with a signal amplification conjugate after 10 minutes (■) and 20 minutes (●).

[0023] Figure 6 A flow chart illustrating an example method for detecting low concentrations of a target analyte in a sample using a lateral flow assay. DETAILED DESCRIPTION

[0024] Conventional lateral flow sandwich assays include a test strip having a conjugate pad (pad) (or labeling zone) arranged along a fluid flow path, a capture or test zone, and an absorbent pad, which is usually but not necessarily arranged along the longitudinal axis of the test strip. The test strip may include a sample receiving zone or hole upstream of the conjugate pad. The conjugate pad includes a labeled analyte-specific antibody, which is generally referred to as a conjugate. The label in the conjugate may include an analyte-specific antibody conjugated to a detector molecule, such as, but not limited to, gold nanoparticles. The capture zone includes an immobilized capture agent, such as an analyte-specific antibody immobilized on a carrier material such as a nitrocellulose membrane. The absorbent pad assists the flow of a liquid sample applied to the conjugate pad (or sample receiving zone, if present) through the carrier material to the capture zone. When the liquid sample is applied to the test strip (at the sample receiving hole or conjugate pad), the labeled analyte-specific antibody is rehydrated and becomes mobile. When there is an antibody-specific target analyte, the analyte will bind to the labeled analyte-specific antibody and form an analyte-antibody-detector complex that moves downstream through the carrier material to the test zone. In the test zone, the analyte in the analyte-antibody-detector complex interacts with the analyte-specific antibody immobilized in the capture zone to form an antibody-analyte-antibody-detector structure, which is often referred to as a sandwich structure. When a threshold number of these sandwich structures are formed in the capture zone, the signal generated by the detector molecules in the sandwich structure can be detected by the measurement system.

[0025] The measurement system can process the detected signal to determine the presence of the target analyte in the sample and in some cases determine its content. If the target analyte is not present in the sample, then the analyte-antibody-detector complex will not be formed, and therefore, the sandwich structure will not be formed in the capture zone. In this example, there is no detector molecule in the capture zone, and the measurement system associates the absence of a signal from the capture zone with the absence of the target analyte in the sample. In the case where the target analyte is actually present in the sample but at a very low content or concentration, the number of sandwich structures formed in the capture zone can be very small. In some examples, the number of sandwich structures is so low that the signal generated by the detector molecule is below the detection threshold of the measurement system. In this case, the measurement system associates the absence of a signal from the capture zone (or the absence of a signal above the detection threshold of the system) with the absence of the target analyte in the sample, even if the target analyte is actually present in the sample but at a very low content or concentration. Therefore, conventional lateral flow sandwich assays have limited sensitivity for target analytes present at low levels or low concentrations.

[0026] The devices, systems, and methods described herein address these and other shortcomings by amplifying the signal generated in the presence of a target analyte in a sample applied to a lateral flow assay. Advantageously, even when the target analyte is present at low concentrations, embodiments of the devices, systems, and methods according to the present disclosure can amplify the signal generated at a test area of ​​a lateral flow assay, such as a capture zone, to a level above the detection threshold of a conventional reader. The lateral flow devices, test systems, and methods according to the present disclosure include a signal amplification conjugate that is bound to both (1) a first analyte-specific conjugate that is bound to an immobilized capture agent in the capture zone, and (2) a second, residual analyte-specific conjugate that is present in the capture zone but not bound to the immobilized capture agent. In a multi-stage chain reaction of binding events in the capture zone, the second, previously unbound residual analyte-specific conjugate (which has been bound to the first signal amplification conjugate in the capture zone) includes additional binding sites that interact with and bind to the second signal amplification conjugate, which itself interacts with and binds to the third, residual analyte-specific conjugate present in the capture zone, and so on.

[0027] The presence of additional analyte-specific conjugates in the capture zone, which may have been bound through binding interactions with the signal amplification conjugate, can increase the intensity of the signal generated in the capture zone to a level that exceeds the detection threshold of a conventional measurement system. The presence of a bound signal amplification conjugate in the capture zone can also increase the intensity of the signal generated in the capture zone to a level that exceeds the detection threshold of the measurement system. Embodiments of the present disclosure improve the sensitivity of lateral flow assays by increasing the signal generated by the lateral flow assay to a conventionally detectable range rather than altering the characteristics of the measurement system, which advantageously allows lateral flow assays according to the present disclosure to be backwards compatible with conventional lateral flow assay test systems.

[0028] Without being bound by any theory, it is believed that the signal amplification conjugate forms a scaffold on which additional analyte-specific conjugates that would normally pass through the capture zone can be captured and retained in the capture zone, resulting in an amplified signal generated in the capture zone. In addition to the detector molecules in the analyte-specific conjugate, the signal amplification conjugate may also include a detector molecule so that the signal amplification conjugate that has been bound in the capture zone also generates a signal in the capture zone, thereby helping to increase the intensity of the signal generated in the capture zone to a level above the detection threshold. Advantageously, embodiments of the present disclosure do not rely on or require that all or substantially all target analytes present in the sample are bound to the analyte-specific conjugate and to the immobilized capture agent in the capture zone so that the signal in the capture zone is amplified. An advantage of the present disclosure is that even very low levels of target analyte (e.g., in the case of a sample with a target analyte present at low concentrations) binding to the analyte-specific conjugate and the immobilized capture agent in the capture zone allows a chain reaction of binding events to occur in the capture zone, regardless of the presence or absence of the target analyte within the scaffold - which is formed after some amount (in some cases very small amounts) of the target analyte binds to the immobilized capture agent in the capture zone. Thus, embodiments of the systems, devices, and methods described herein can greatly improve the sensitivity of lateral flow assays for target analytes present in samples even in small amounts.

[0029] In addition to increasing the sensitivity of lateral flow assays for measuring the presence, absence or amount of a target analyte at low concentrations in a sample, the devices, systems and methods described herein can advantageously be performed in a point-of-care setting without the use of highly specialized, and in some cases time-consuming, sample preparation techniques and detection systems, such as polymerase chain reaction (PCR), that are typically used to analyze analytes at low concentrations in a sample. Embodiments of the lateral flow devices described herein provide users with a convenient, easy-to-use format, quickly generate a signal that can be detected by a conventional reader, and can be performed in a point-of-care setting rather than an off-site laboratory facility.

[0030] This description is intended to illustrate a test strip for measuring a target analyte in a test sample, even when present in low concentrations. Those skilled in the art will recognize that this example is intended to be exemplary, and that various changes and variations can be made to the lateral flow assay described herein. For example, a sample can include one or more target analytes present in low concentrations, and the assay test strip can measure one or more target analytes. In each of various iterations, the lateral flow assay includes amplifying the signal at the detection zone according to the features of the present disclosure to measure the analyte in the test sample.

[0031] When more than one target analyte is to be detected, such as multiple target analytes in a sample, the detection zone may include a separate capture zone specific for each target analyte. For example, a sample may include three target analytes: a first target analyte, a second target analyte, and a third target analyte. The detection zone of the lateral flow assay will thus include three capture zones: a first capture zone specific for the first target analyte, a second capture zone specific for the second target analyte, and a third capture zone specific for the third target analyte.

[0032] Thus, the present disclosure provides assay test strips for measuring a target analyte in a test sample, even when present at low concentrations; lateral flow assays comprising the test strips; methods of measuring a target analyte using the test strips described herein; and methods of making the assay test strips described herein.

[0033] Embodiments of lateral flow assays described herein are particularly advantageous in diagnostic tests where low levels of a target analyte are present on a test strip. Although embodiments of the present disclosure are described herein with reference to low levels of an analyte being present on a test strip because the analyte is present in a sample at a low concentration, it should be understood that embodiments of the present disclosure are also applicable when the sample contains an amount of analyte capable of generating a signal above the detection threshold but only a limited amount of the sample is applied to the test strip.

[0034] The various aspects of the device, test system and method are described more comprehensively below with reference to the accompanying drawings. However, the present disclosure can be implemented in many different forms. Based on the teachings herein, it will be appreciated by those skilled in the art that the scope of the present disclosure is intended to cover any aspect of the device, test system and method disclosed herein, whether implemented independently of any other aspect of the present disclosure or implemented in combination with any other aspect of the present disclosure. For example, using any number of aspects set forth herein, a device can be implemented or a method can be put into practice.

[0035] Although specific aspects are described herein, many variations and permutations of these aspects fall within the scope of the present disclosure. Although some benefits and advantages are mentioned, the scope of the present disclosure is not intended to be limited to specific benefits, uses or purposes. On the contrary, aspects of the present disclosure are intended to be more broadly applicable to different detection techniques and device configurations, some of which are shown by way of example in the accompanying drawings and in the following description.

[0036] Conventional lateral flow device

[0037] Lateral flow device described herein is the analytical device used in lateral flow chromatography. Lateral flow assay is the assay that can be performed on lateral flow device described herein. Lateral flow device can be implemented on assay test strip, but other forms can be suitable, such as dipstick, flow device or microfluidic device. In the test strip format, the fluid sample containing or suspected of containing analyte is placed in the sample receiving area. After the target analyte contacts the test strip, it becomes labeled. Then the target analyte (for example, by capillary action) marked now is flowed through the strip. The strip can be made of medium such as paper, nitrocellulose, cellulose, fiber or nylon or other materials that allow sample flow through the medium.

[0038] Such assays are referred to as sandwich assays. Sandwich assays according to the present disclosure are described in the context of reflective markers that produce optical signals (such as gold nanoparticle markers and latex particles of different colors), but it should be understood that assays according to the present disclosure may include latex bead markers configured to produce fluorescent signals, magnetic nanoparticle markers configured to produce magnetic signals, or any other markers configured to produce detectable signals. Sandwich lateral flow assays include a labeled conjugate deposited at a sample receiving area on a solid substrate. After the sample is applied to the sample receiving area, the labeled conjugate is dissolved or solubilized in the sample, and then the labeled conjugate recognizes and specifically binds to the first epitope on the analyte in the sample to form a marker-conjugate-analyte complex. The complex flows along the liquid front from the sample receiving area through the solid substrate to the detection area (sometimes referred to as a "test line" or "capture zone") where an immobilized capture agent (e.g., an immobilized analyte-specific antibody) is located. In some cases where the analyte is a polymer or contains multiple identical epitopes on the same monomer, the labeled conjugate deposited at the sample receiving area can be the same as the capture agent immobilized in the detection area. The immobilized capture agent recognizes and specifically binds to the epitope on the analyte, thereby capturing the marker-conjugate-analyte complex at the detection area. If the analyte is present in sufficient amounts, the presence of the labeled conjugate in the detection area provides a detectable signal at the detection area. In a non-limiting example, gold nanoparticles are used to label the conjugate because they are relatively cheap, stable, and provide an easily observable color indication due to the surface plasmon resonance properties of the gold nanoparticles.

[0039] The detection of the signal generated in the detection zone can indicate that the target analyte is present in the sample. For example, if the signal exceeds the detection threshold of the measurement system, the measurement system can detect the presence of the analyte in the sample and the content of the sample in some cases. However, the absence of any detectable signal in the detection zone can indicate that the target analyte is not present in the sample or that it may be present below the detection limit. For example, if the sample does not contain any target analyte, the sample will still dissolve the labeled conjugate and the labeled conjugate will still flow to the detection zone. However, the labeled conjugate will not bind to the immobilized conjugate in the detection zone. Instead, it will flow through the detection zone, through the control line (if present), and, in some cases, flow to the optional absorption zone. Some labeled conjugates will bind to the control reagent deposited on the control line and generate a detectable signal on the control line, indicating that the device is working properly. In the case where the analyte is present but its amount is below the detection limit, the marker-conjugate-analyte complex is combined in the detection zone, but will not be detected. In these cases, the absence of a detectable signal emitted from the detection zone means that the user cannot confirm with certainty whether the analyte is absent in the sample or the analyte is present in the sample below the detection limit of the measurement system.

[0040] Example lateral flow signal amplification device according to the present disclosure

[0041] The lateral flow assays, test systems and methods described herein address these and other shortcomings of lateral flow assays that are unable to determine the difference between an analyte being absent from a sample and an analyte being present in an amount below the detection limit. In addition to improving the qualitative measurement of an analyte, embodiments of the assays, test systems and methods of the present disclosure can also greatly improve the sensitivity of measurements by conventional readers, allowing for quantitative measurement of analytes in some cases.

[0042] Figure 1 An example lateral flow device 100 according to the present disclosure is shown. An assay test strip is received within a housing of the device 100. In this non-limiting example of the lateral flow device 100, the housing defines a buffer well 110, a sample well 120, and a reading window 130. The size and shape of the lateral flow device 100 can be adapted for ease of use, rapid delivery of test results, portability, proper operation and placement within an automatic reader, economy of material use and cost, or other considerations. The size and shape are therefore not limited to any particular size or shape, and can be easily modified to suit the requirements of a particular need or a particular use case.

[0043] Figure 2 An example assay test strip 101 according to the present disclosure is shown. The assay test strip 101 may be received or housed in Figure 1The lateral flow device 100 of the present invention is provided. The example assay test strip 101 in this non-limiting embodiment includes a matrix, an assay membrane having a sample receiving area and a buffer receiving area, a detection area, and an absorbent pad. It should be understood that the present disclosure is not limited to this example assay test strip, and other assay test strips with different features can be implemented according to the present disclosure.

[0044] exist Figure 2 In an embodiment, the assay test strip 101 includes a backing card 102, a conjugate pad 103 having a sample receiving area 121 and a buffer receiving area 111, a membrane 104 having a detection area 131, and an absorbent pad 105. The membrane 104 can be a nitrocellulose material. The assay test strip 101 is contained within a lateral flow device 100 so that the buffer receiving area 111 can be entered through the buffer well 110, the sample receiving area 121 can be entered through the sample well 120, and the detection area 131 can be entered through the reading window 130. The result of the assay can be measured at the reading window 130 by measuring the signal (if any) generated in the detection area 131.

[0045] The backing card 102 may be any suitable material sufficient to support the assay test strip, for example, a water-impermeable layer such as a solid plastic, a laminate, a composite material, etc. The absorbent pad 105 helps promote capillary action and fluid flow through the membrane, and may include any suitable material for absorbing fluids, including, for example, nitrocellulose, a cellulosic material, a porous polyethylene pad, a glass fiber filter paper, etc. The absorbent material may be treated with a surfactant to aid in the wicking process.

[0046] The fluid is configured to flow from the conjugate pad 103 to the absorbent pad 105 along the longitudinal axis of the assay test strip 101. The components of the assay test strip 101 will be described with reference to this fluid flow direction. For example, the membrane 104 is located downstream of the conjugate pad 103 and upstream of the absorbent pad 105. For another example, the sample receiving area 121 is located downstream of the buffer receiving area 111 and upstream of the detection area 131.

[0047] The detection zone 131 of film 104 includes an immobilized capture agent configured to specifically bind the target analyte when the target analyte is present in the sample. Film 104 may further include an additional detection zone for detecting more than one target analyte, and may include one or more control zones. Film 104 may be transparent in the visible region to minimize undesirable read interference in order to accurately determine the presence and / or content of the target analyte. Capture agent can be fixed on film 104 or in film 104 using any suitable method, including, for example, deposition, spraying, soaking, immersion, pouring or injecting capture agent on film 104 or in film 104. For example, capture agent can be deposited and immobilized on film 104 by preparing a solution including capture agent and spraying the solution onto film 104 with air jet technology. In another example, capture agent is deposited by preparing a solution with capture agent and pouring the solution, spraying the solution, preparing the solution as a powder or gel placed or rubbed on a test strip, or any other suitable method.

[0048] The capture agent can be immobilized in the detection zone 131 of the assay test strip 101 in any suitable amount. Advantageously, embodiments of the present disclosure can use capture agents of a type and amount typical for conventional lateral flow sandwich assays to detect the presence, absence, and amount of target analytes at low concentrations. In some embodiments, the immobilized capture agent is present in an amount in the range of about 0.1-20 μL / test strip.

[0049] In this example implementation, the conjugate pad 103 is placed over a portion of the backing card 102. When the assay test strip 101 is contained within the lateral flow device 100, the buffer receiving area 111 is accessible through the buffer well 110 and, in this case, is located laterally below the buffer well 110. The sample receiving area 121 is accessible through the sample well 120 and, in this case, is located laterally below the sample well 120. In some cases, the conjugate pad 103 can be secured to the backing card 102. The conjugate pad 103 can be any suitable material that allows fluid to flow through the material, such as fiber (including glass fiber), polyester, or other material that provides fluid flow through the conjugate pad 103. In this example implementation, the conjugate pad 103 includes an analyte-specific conjugate 200 deposited at the sample receiving area 121. In accordance with the present disclosure, embodiments of the analyte-specific conjugate 200 may also be referred to as a "first conjugate." The analyte-specific conjugate 200 is configured to dissolve when a fluid is received in the sample receiving area 121. As will be described below with reference to Figure 3As described, the analyte-specific conjugate 200 includes a first label 210 (e.g., a detector molecule), a first binding partner 230, and a reagent 220 that specifically binds to a target analyte 221 (when present in a sample). Thus, the analyte-specific conjugate 200 is configured to specifically bind to the target analyte (if present) in a sample received in a sample receiving zone.

[0050] In one example, the analyte-specific conjugate 200 is placed on or in the sample receiving area 121. In another example, the analyte-specific conjugate 200 is placed on or in a first conjugate zone located downstream of the sample receiving area 121, so that the sample (and any target analyte, if present) added to the sample receiving area 121 will flow through the first conjugate zone and interact with the analyte-specific conjugate 200. The analyte-specific conjugate 200 can be placed on or in the assay test device 101 using any suitable method, including, for example, depositing, spraying, soaking, immersing, pouring or injecting the analyte-specific conjugate on or in the sample receiving area 121. For example, the analyte-specific conjugate can be deposited by preparing a solution with the analyte-specific conjugate and spraying the solution with an air jet technique. In another example, the analyte-specific conjugate can be prepared in a solution and deposited by pouring the solution, spraying the solution, formulating the solution as a powder or gel placed or rubbed on a test strip, or any other suitable method.

[0051] The analyte-specific conjugate 200 may be provided in any suitable amount at the sample receiving area 121 (or other suitable location) of the assay test strip 101. Advantageously, embodiments of the present disclosure may detect the presence, absence, and amount of a target analyte at low concentrations using a type and amount of analyte-specific conjugate 200 typical for conventional lateral flow sandwich assays. In some embodiments, the analyte-specific conjugate is deposited in an amount in the range of approximately 0.1-20 μL / test strip.

[0052] Embodiments of systems, devices, and methods according to the present disclosure include a signal amplification conjugate 250. Embodiments of the signal amplification conjugate 250 may also be referred to as a "second conjugate" according to the present disclosure. Figure 3 As described, the signal amplification conjugate 250 includes a second label 211 and a second binding partner 231. The second binding partner 231 is configured to specifically bind to the first binding partner 230 of the analyte-specific conjugate 200. The signal amplification conjugate 250 is present in or added to a position upstream of the analyte-specific conjugate 200. Figure 2In one example described, the signal amplification conjugate 250 is added to the assay test strip 101 during the manufacturing process. In another example, the signal amplification conjugate 250 is added to the assay test strip 101 by an operator during a testing event. It should be understood that embodiments of the present disclosure are not limited by the manner in which the signal amplification conjugate 250 is introduced into the lateral flow assay, and other examples beyond those described herein are also suitable.

[0053] exist Figure 2 In one example shown in , the signal amplification conjugate is placed on or in the buffer receiving zone 111. In another example, the signal amplification conjugate 250 is placed on or in a second conjugate zone located downstream of the buffer receiving zone 111 and upstream of the sample receiving zone 121, such that the sample (and any target analyte, if present) added to the sample receiving zone 121 will not interact with the signal amplification conjugate 250 in the second conjugate zone. This spatial orientation also ensures that the buffer added to the buffer receiving zone 111 will flow downstream through the second conjugate zone and dissolve the signal amplification conjugate 250. Although Figure 2 The non-limiting embodiment of the present disclosure does not use a sample solution to rehydrate the signal amplification conjugate 250, but it should be understood that other embodiments are suitable. For example, another embodiment of the present disclosure can use a single solution (such as a sample solution) to rehydrate both the analyte-specific conjugate 200 and the signal amplification conjugate 250. In this case, the sample solution can be applied at different times to ensure that the signal amplification conjugate 250 moves down the fluid path to the detection zone of the test strip after the analyte-specific conjugate 200 has moved down the fluid path to the detection zone.

[0054] The signal amplification conjugate 250 can be placed on or in the assay test device using any suitable method, including, for example, depositing, spraying, soaking, immersing, pouring or injecting the signal amplification conjugate 250 on or in the buffer receiving area 111 (or other suitable location). For example, the signal amplification conjugate 250 can be deposited by preparing a solution with the signal amplification conjugate 250 and spraying the solution using an air jet technique. In another example, the signal amplification conjugate 250 can be prepared in a solution and deposited by pouring the solution, spraying the solution, formulating the solution as a powder or gel that is placed or rubbed on a test strip, or any other suitable method.

[0055] It should be understood that the signal amplification conjugate 250 can be added to the assay test strip in areas other than the buffer receiving zone 111. For example, in one non-limiting embodiment, the lateral flow device 100 does not include a separate or dedicated location, such as a buffer well or buffer receiving zone, to receive the signal amplification conjugate 250 onto the assay test strip 101. After the analyte-specific conjugate 200 has dissolved in the sample receiving zone 121 and moved to the detection zone 131, the signal amplification conjugate 250 can be added to the assay test strip 101 at any suitable location.

[0056] Embodiments of the signal amplification conjugate 250 of the present disclosure can be provided in any suitable amount in the buffer receiving area 111 (or other suitable location) of the assay test strip 101. In some embodiments, the signal amplification conjugate 250 is deposited in an amount in the range of about 0.1-20 μL / test strip. In some embodiments, the signal amplification conjugate 250 is deposited on the assay test strip 101 in an amount of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μL / test strip.

[0057] The signal amplification conjugate 250 is placed or added to the assay test strip 101 upstream of the location where the analyte-specific conjugate 200 is placed or added to the assay test strip 101. During the process of manufacturing the assay test strip 101 or by an operator using the assay test strip 101 to perform a test event, the signal amplification conjugate 250 and the analyte-specific conjugate 200 may be placed on the test strip at the same time or at different times. In one non-limiting example, the signal amplification conjugate 250 and the analyte-specific conjugate 200 are both added to the assay test strip 101 at the same time or substantially at the same time but at different locations of the assay test strip 101, for example, by introducing or depositing the signal amplification and analyte-specific conjugates on the test strip at the same time or nearly at the same time. In this example, the analyte-specific conjugate 200 is added to or positioned on the assay test strip 101 downstream of the location where the signal amplification conjugate 250 is added or positioned. This spatial orientation minimizes interactions between the conjugates before the analyte-specific conjugate moves to the detection zone 131 and binds at the detection zone 131. In another non-limiting example, the signal amplification conjugate 250 is added to the assay test strip 101 at any suitable location and using any suitable method or technique, including but not limited to those described herein, before or after the addition of the analyte-specific conjugate 200. For example, if the signal amplification conjugate 250 is added after the analyte-specific conjugate 200 has moved to and bound at the detection zone 131, the signal amplification conjugate 250 can be added to the same or substantially the same location as the analyte-specific conjugate 200 was initially added.

[0058] In some embodiments, the lateral flow device 100 does not include a separate or dedicated location, such as a buffer well or buffer receiving area, which includes a signal amplification conjugate 250 before a test event using the assay test strip 101. Instead, during the test event, the operator introduces the signal amplification conjugate 250 to the assay test strip 101. For example, the signal amplification conjugate 250 can be introduced to the assay test strip after the sample is introduced to the assay test strip. Specifically, the sample is first placed in the sample receiving area, and the sample is allowed to migrate through the test assay strip in a suitable time period. The time period can be a sufficient amount of time for the analyte-specific conjugate 200 to dissolve, form a binding interaction with the target analyte in the sample (if present), move to the detection zone, and form a binding interaction with the immobilized capture agent in the detection zone. After incubation for a suitable period of time, a solution including the signal amplification conjugate 250 is introduced into the lateral flow device 100. The signal amplification conjugate 250 then migrates through the assay test strip 101 to the detection zone 131.

[0059] The delay period between adding the sample and the signal amplification conjugate 250 to the assay test strip 101 can be adjusted based on various parameters, including but not limited to the identity of the target analyte, the fluid containing or suspected of containing the target analyte, the analyte-specific conjugate 200, the signal amplification conjugate 250, and the material properties of the assay test strip 101. Examples of delay periods include but are not limited to 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, and 15 minutes. Thus, in this example, at the beginning of the test event, the signal amplification conjugate 250 is not present on the assay test strip 101. It should therefore be understood that embodiments of the present disclosure for amplifying the signal of an analyte present at low concentrations, such as at concentrations below the detection limit, can be accomplished using n signal amplification conjugates 250 in a variety of ways, including preparing a test strip in which the signal amplification conjugate 250 is present or adding the signal amplification conjugate 250 in a later step.

[0060] Embodiments of the systems, devices and methods according to the present disclosure can detect target analytes very quickly. The total time from the start of the test event (defined as the time when the sample is added to the test strip) to the end of the test event (defined as the time when the measurement system detects the signal generated in the detection zone (if present)) can be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes and 15 minutes.

[0061] Figure 3 Schematically illustrates example embodiments of systems, devices, and methods according to the present disclosure. Without being bound by any particular theory, reference will now be made to Figure 3 The process of combining the analyte-specific conjugate 200 with the target analyte 221, the capture agent 240 immobilized in the capture zone 242, and the signal amplification conjugate 250 is described, but it should be understood that the present disclosure is not limited to Figure 3 Schematic diagram of .

[0062] The analyte-specific conjugate 200 includes a first label 210, a first binding partner 230, and a reagent 220 that specifically binds to a target analyte 221 (when present in a sample). The reagent 220 is configured to specifically bind to the target analyte 221 and may include an antibody or a binding fragment of an antibody that specifically binds to the target analyte 221. Figure 2The described method deposits or adds the analyte-specific conjugate 200 to the sample receiving area 121 (or any other suitable location) of the assay test strip 101. The sample is added to the assay test strip 101, for example, at the sample receiving area 121. The analyte-specific conjugate 200 dissolves into the sample, and the reagent 220 specifically binds to the target analyte 221 when the target analyte 221 is present in the sample, forming an analyte-reagent-first marker complex 225. The complex 225 flows through the assay test strip 101 to the detection zone 131, which includes an immobilized capture agent 240 configured to specifically bind to the target analyte 221. The complex 225 is captured at the detection zone 131, as shown. Figure 3 shown.

[0063] After a suitable period of time has passed to allow the complex 225 to travel to the detection zone 131 and be captured therein, the signal amplification conjugate 250 according to the present disclosure is allowed to travel downstream along the assay test strip 101 to the detection zone 131. The signal amplification conjugate 250 includes a second label 211 and a second binding partner 231. In one non-limiting implementation, the signal amplification conjugate 250 is present on the assay test strip 101 (in a manner including the above referenced) prior to adding the sample. Figure 2 The signal amplification conjugate 250 is deposited in any suitable manner (such as those described above) and is caused to travel downstream to the detection zone 131 by applying a buffer solution to the signal amplification conjugate 250. In this implementation, the previously deposited signal amplification conjugate 250 is deposited upstream of the sample receiving zone 121, for example, in a buffer receiving zone 111 located upstream of the sample receiving zone 121. This spatial orientation of the signal amplification conjugate 250 in the buffer receiving zone 111 upstream of the analyte-specific conjugate 200 in the sample receiving zone 121 ensures that adding the sample to the sample receiving zone 121 will cause the analyte-specific conjugate 200, rather than the signal amplification conjugate 250, to dissolve and mobilize. In another non-limiting implementation, the signal amplification conjugate 250 is added to the assay test strip 101 after a suitable period of time has passed since the sample was added to the sample receiving zone. In this implementation, the buffer solution including the signal amplification conjugate 250 may be added to the assay test strip 101 at the buffer receiving zone 111 (if present), at the sample receiving zone 121, or at any other suitable location of the assay test strip 101.

[0064] In these implementations, embodiments of the present disclosure allow the analyte-specific conjugate 200 to migrate and bind to the capture agent 240 in the detection zone 131 before being exposed to and forming a binding interaction with the signal amplification conjugate 250. Embodiments of the present disclosure advantageously allow the timing of release of the signal amplification conjugate 250 to be adjusted to suit the specific characteristics of the lateral flow assay and to be triggered only when a buffer solution is added to the system (either to the buffer receiving zone 111 containing the signal amplification conjugate 250, or to any suitable location when the buffer solution itself contains the signal amplification conjugate 250). These embodiments of the present disclosure also advantageously prevent the interaction between the analyte-specific conjugate 200 and the signal amplification conjugate 250 from occurring in locations of the assay test strip 101 where they are not desired, i.e., locations upstream of the detection zone 131. Thus, embodiments of the present disclosure that allow the timing of release of the signal amplification conjugate 250 to be optimal (after a sufficient amount of the analyte-specific conjugate 200 has moved to and bound to the detection zone 131) advantageously maximize the occurrence of binding interactions between the analyte-specific conjugate 200 present in the detection zone (whether bound or not bound to the capture agent 240) and the optimal location - the signal amplification conjugate 250 in the detection zone 131. This results in the aggregation and amplification of the signals generated by the conjugates 200, 250 in the detection zone to a level above the detection threshold.

[0065] After adding the buffer solution to the assay test strip 101 and either introducing the signal amplification conjugate 250 directly into the system or dissolving the signal amplification conjugate 250 already present in the system, a second series of binding events will occur due to the properties of the signal amplification conjugate 250 of the present disclosure. As described above, the second binding partner 231 specifically binds to the first binding partner 230 of the analyte-specific conjugate 200. Without being bound by any particular theory, it is believed that when the signal amplification conjugate 250 flows through the assay test strip 101 to the detection zone 131, the second binding partner 231 of the signal amplification conjugate 250 specifically binds to the first binding partner 230 of the complex 225 bound to the capture agent 240, whereby the signal amplification conjugate 250 is captured in the detection zone 131. The second label 211 of the now bound signal amplification conjugate 250 generates a signal in the detection zone. In this way, the signal from a single analyte-specific conjugate 200 bound to the detection zone is amplified. In some embodiments, a single analyte-specific conjugate 200 in a complex 225 is bound to a plurality of signal amplification conjugates 250, which themselves contribute to the signal generated by the single analyte-specific conjugate 200 in the complex 225. In some embodiments of the present disclosure, even this first order binding interaction between the complex 225 and one or more signal amplification conjugates 250 may be sufficient to increase the signal generated in the detection zone above a threshold detection level.

[0066] Advantageously, embodiments of the present disclosure can produce even higher levels of signal amplification than is possible through first order binding interactions alone. In embodiments of the systems, devices, and methods described herein, the amplification of the signal produced in the detection zone is further enhanced by the ability of the now bound signal amplification conjugate 250 to bind to residual analyte-specific conjugate 200 present in the detection zone but not bound to the capture agent 240. In conventional lateral flow assays, such residual analyte-specific conjugate 200 can typically pass through the detection zone 131 or remain dispersed in the detection zone. In the presence of a signal amplification conjugate 250 according to the present disclosure, the residual analyte-specific conjugate 200 can be bound to the scaffold of the conjugate anchored to the detection zone 131 through the complex 225, and further contribute to the aggregation signal produced in the detection zone 131. The signal produced by embodiments of the present disclosure can increase in intensity exponentially. Thus, in some embodiments, the signal intensity generated at the detection zone 131 can be increased by 2-100 times due to signal amplification, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 times. It should be understood that the present disclosure is not limited to these example amplification levels, and that embodiments of the present disclosure enable amplification to other amplification levels.

[0067] Advantageously, the above-described chain reaction of binding events can occur in the presence of even very low levels of target analyte in the sample. Without being bound by any particular theory, it is believed that the residual analyte-specific conjugate 200 can form a binding interaction with the signal amplification conjugate 250 regardless of whether the residual analyte-specific conjugate 200 is bound to an analyte in the sample. In other words, the binding interactions that occur after the first interaction between the complex 225 and the signal amplification conjugate 250 rely on the first interaction to ensure that the chain reaction of binding events occurs in a local area (detection zone), but they do not rely on additional target analytes to form a scaffold of the binding conjugate 200, 250 pair that amplifies the signal generated by the complex 225.

[0068] The present disclosure is also advantageous in that the absence of a signal at the detection zone 131 can be associated with the absence of the target analyte in the sample with high confidence. In the absence of the target analyte in the sample, the complex 225 will not form at the detection zone 131 and bind to the capture agent 240. In the absence of this binding event, the signal amplification conjugate 250 will not be bound in the detection zone 131 and will pass through the detection zone 131 to the absorbent pad 151.

[0069] As used herein, buffer refers to a solution used in a lateral flow assay that does not interfere with binding interactions, does not denature the target analyte or other components of the device, and is neutral in order to allow the components to flow through the test strip. Various buffers are well known, and any of the various buffers can be used for specific analytes, compounds, and components used in the lateral flow assays described herein. In some embodiments, the buffer is phosphate buffered saline (PBS).

[0070] Embodiments of the present disclosure have been described with reference to adding a tracking buffer to a buffer receiving zone located upstream of a sample receiving zone, and with reference to adding a buffer solution including a signal amplification conjugate to any suitable location of an assay test strip after the sample has been added. However, it will be understood that other methods of adding or releasing a buffer solution to the system will be suitable and may be used in embodiments of the present disclosure. For example, the tracking buffer solution may be prepackaged in a pouch or other suitable structure and incorporated upstream of a signal amplification conjugate deposited on an assay test strip and a sample receiving hole of the assay test strip. At a suitable time after the sample has been added to the assay test strip, the tracking buffer solution may be released by rupturing the pouch using any suitable mechanism.

[0071] In some embodiments, the signal amplification conjugate 250 does not bind to other conjugates (such as to the analyte-specific conjugate 200) before reaching the detection zone 131. In some embodiments, the binding of the signal amplification conjugate 250 to other conjugates can cause precipitation of the bound conjugate, causing the precipitated label to be smeared through the test strip, thereby interfering with or confusing the results. As described above, embodiments of the present disclosure include features that prevent or minimize such precipitation of bound conjugates upstream of the detection zone 131. These features ensure that the signal amplification conjugate 250 remains dissolved in the solution and does not bind to the analyte-specific conjugate 200 before reaching the analyte-specific conjugate 200 captured in the complex 225 of the detection zone 131. In one example, an embodiment of the present disclosure includes depositing the signal amplification conjugate 250 upstream of the analyte-specific conjugate 200 in the sample receiving zone 121 on the assay test strip 101, and dissolving the signal amplification conjugate 250 only after the analyte-specific conjugate has first had a chance to flow through the lateral flow device to the detection zone 131. In another example, embodiments of the present disclosure include adding the signal amplification conjugate 250 to the buffer solution applied to the assay test strip 101 only after the analyte-specific conjugate 20 has first had a chance to flow through the lateral flow device to the detection zone 131 .

[0072] The first label 210 of the analyte-specific conjugate 200 can be a label identical to the second label 211 of the signal amplification conjugate 250. In a non-limiting example, the first label 210 and the second label 211 can be colloidal gold labels. The colloidal gold label can include gold nanoparticles. The first label 210 and the second label 211 can include any suitable label that can provide a detectable signal. Thus, in some embodiments, the first label 210 and the second label 211 are enzymes, colloidal metal particles (also referred to as metal nanoparticles, such as gold or silver nanoparticles), colored latex particles, radioactive isotopes, cofactors, ligands, chemiluminescent or fluorescent agents, protein-adsorbed silver particles, protein-adsorbed iron particles, protein-adsorbed copper particles, protein-adsorbed selenium particles, protein-adsorbed sulfur particles, protein-adsorbed tellurium particles, protein-adsorbed carbon particles, or protein-coupled dye capsules, or any other suitable label.

[0073] As described above, the reagent 220 is a reagent that specifically binds to the target analyte 221, and may include an antibody or antibody binding fragment that specifically binds to the target analyte 221. Thus, the reagent 220 may be an anti-analyte antibody. The first binding partner 230 of the analyte-specific conjugate 200 specifically binds to the second binding partner 231 of the signal amplification conjugate 250, and thus the first and second binding partners 230, 231 constitute a binding pair. The binding pair may include, for example, a binding pair selected from antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid chain / complementary nucleic acid chain, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / avidin, receptor / ligand or virus / cell receptor. In a non-limiting example, the first binding partner 230 is avidin (or a derivative or analog thereof) and the second binding partner 231 is biotin (or a derivative or analog thereof). For example, the analyte-specific conjugate 200 may be biotinylated and the signal amplification conjugate 250 may include an anti-biotin antibody or a streptavidin binding partner. In another non-limiting example, the binding pair is an antigen / antibody, such as a peptide and an antibody. In some embodiments, the peptide is a decapeptide. In a further non-limiting example, the binding pair is doxorubicin and anti-doxorubicin. In a still further non-limiting example, the binding pair is methotrexate and anti-methotrexate. In yet another non-limiting example, the binding pair is FITC and an anti-FITC antibody. It should be understood that embodiments of the present disclosure can use any suitable first binding partner 230 that specifically binds to a second binding partner 231 and is not limited to the above examples.

[0074] Embodiments of the present disclosure are advantageous in that the second binding partner 231 of the signal amplification conjugate 240 can be selected to specifically bind to the first binding partner 230 of the analyte-specific conjugate 200 without interfering with the binding of the target analyte 221 to the analyte-specific antibody of the analyte-specific conjugate 200. In one example, a different protein / peptide can be co-conjugated with the analyte-specific antibody on the first label 210 to form the analyte-specific conjugate 200. In another example, another protein / peptide can be first biotinylated and then co-conjugated with the analyte-specific antibody on the first label 210 to form the analyte-specific conjugate 200. In a further example, a small molecule, such as biotin, is added to the analyte-specific antibody, which is then conjugated to the first label 210 of the analyte-specific conjugate 200, and the signal amplification conjugate 250 includes the small molecule-specific binding protein / antibody. In this example, the small molecules added to the analyte-specific antibody are selected so that they do not interfere with the binding of the analyte to the analyte-specific antibody in the analyte-specific conjugate 200. In still another example, in addition to biotin, the protein / peptide can be conjugated to another molecule - there is another antibody or binding protein against it. Thus, the added protein / peptide, biotin, or other molecule conjugated to the first marker 210 can provide a binding site for an antibody against the protein / peptide, biotin, or other molecule on the signal amplification conjugate 250 without interfering with the analyte-specific binding interaction between the target analyte 221 and the analyte-specific antibody conjugated to the first marker 210.

[0075] In some embodiments, the lateral flow device includes one or more control areas. The control area can be in the detection area or separated from the detection area. In some embodiments, the control area can be a positive control area, which can include small molecules conjugated to proteins, such as bovine serum albumin (BSA). The antibody of the positive control labeling of the specific binding small molecule can be deposited on the conjugate pad. When the antibody of the positive control labeling is rehydrated with a liquid sample, it flows toward the positive control area and is bound to the small molecule to form a semi-sandwich structure. In some embodiments, the positive control area includes an immobilized capture agent that is specifically bound to an analyte-specific conjugate, so that the analyte-specific conjugate that is not bound to the target analyte passes through the detection area (test line) and is specifically bound to the immobilized capture agent at the control line, generating a signal indicating the normal operation of the lateral flow device. It should be understood that any suitable control area can be implemented in the embodiments of the present disclosure.

[0076] Embodiments of the readers and data analyzers disclosed herein can process signal measurements obtained from a control zone to correct the signal measured at the detection zone or to alert an operator that a test is invalid.

[0077] As used herein, "analyte" generally refers to a substance to be detected. In some embodiments, an analyte is those of low concentrations sometimes found in a sample, such as being at a concentration below the detection limit of a measurement system. Some analytes are usually present in a sample at high concentrations, but may be present in a specific sample to be tested at low concentrations due to a specific disease, due to the stage of the disease, or due to sample processing. For example, in the initial stages of a disease or disease or after the peak stage of a disease or disease, an analyte may be present in a sample at low concentrations. It should be understood that embodiments of the present disclosure are applicable to measuring any analyte present in or suspected of being present in a sample at low levels or a lateral flow assay wherein the sensitivity of measuring such an analyte is low.

[0078] Analytes can include antigenic substances, haptens, antibodies and combinations thereof. Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including those for therapeutic purposes and those for illegal purposes), drug intermediates or by-products, bacteria, viral particles and metabolites or antibodies of any of the above substances. In some embodiments, the target analyte is an influenza virus, such as influenza A virus, influenza B virus or influenza C virus. For the purpose of biological or environmental target substances, additional analytes may be included.

[0079] In some embodiments, the sample may include one or more target analytes, and thus the lateral flow device may be configured to detect one or more target analytes. In order to detect one or more target analytes, the lateral flow device according to the present disclosure may include one or more analyte-specific conjugates 200, wherein each analyte-specific conjugate 250 specifically binds to the target analyte. Thus, for example, for a desired number of target analytes present or suspected to be present in the sample, the first analyte-specific conjugate 200A specifically binds to the first target analyte 221A, the second analyte-specific conjugate 200B specifically binds to the second target analyte 221B, the third analyte-specific conjugate 200C specifically binds to the third target analyte 221C, and so on. In some embodiments, each analyte-specific conjugate that specifically binds to the target analyte does not specifically bind to any other target analyte. In addition, in some embodiments, each analyte-specific conjugate may have the same label or a label different from each other analyte-specific conjugate. Thus, for example, each analyte-specific conjugate may include a unique label different from any other label of the analyte-specific conjugate. In addition, in the presence of one or more target analytes, the lateral flow device can include one or more capture lines, each capture line including an immobilized capture agent that specifically binds to a particular target analyte. For example, for a desired number of target analytes being analyzed in a test sample, a first capture agent 240A immobilized in a capture zone 242A specifically binds to a first target analyte 221A, a second capture agent 240B immobilized in a capture zone 242B specifically binds to a second target analyte 221B, a third capture agent 242C immobilized in a capture zone 242C specifically binds to a third target analyte 221C, and so on.

[0080] In some embodiments in which the lateral flow device is configured to measure one or more target analytes, the device further includes a signal amplification conjugate 250. The signal amplification conjugate 250 may include one binding partner of a binding pair that specifically binds to the other binding partner of the binding pair. In some embodiments, for a desired number of target analytes, the signal amplification conjugate is bound to a first analyte-specific conjugate, a second analyte-specific conjugate, a third analyte-specific conjugate, and so on, thereby amplifying the signal of each target analyte. In some embodiments, the signal amplification conjugate is bound to one, two, three or more analyte-specific conjugates, thereby amplifying the signal only for a desired number of analytes, for example, amplifying the signal only for target analytes that are known or suspected to be present in a sample at low concentrations.

[0081] It will be appreciated that target analytes are typically present in a sample at various concentrations, and thus, it may not be necessary to perform signal amplification for each target analyte, but only for those target analytes that are known or suspected to be present at low concentrations, such as at concentrations below the detection limit of the measurement system. Thus, in some embodiments, a first analyte-specific conjugate that specifically binds to a first analyte present at a low concentration includes a binding partner, while a second analyte-specific conjugate that specifically binds to a second analyte present at a concentration above the detection limit does not include a binding partner, whereby the signal amplification conjugate will bind to the first analyte-specific conjugate and not to the second analyte-specific conjugate, thereby amplifying the signal of the first target analyte. For example, various iterations of the present disclosure may be implemented by amplifying any desired signal, including signals below the detection limit of the measurement system.

[0082] The following non-limiting examples illustrate features of the lateral flow devices, testing systems, and methods described herein and are in no way intended to limit the scope of the present disclosure.

[0083] Example 1

[0084] Preparation of lateral flow assays according to the present disclosure

[0085] The following example describes the preparation of a lateral flow assay according to the present disclosure to measure an analyte present in a sample at low concentrations. In this non-limiting example, the target analyte is influenza A virus ("Flu A").

[0086] A test strip having a conjugate pad with a sample receiving area and a buffer receiving area was prepared. The analyte-specific conjugate was prepared in solution and the solution was sprayed on the sample receiving area by air jet deposition. In short, anti-Flu A antibody and biotin were incubated with gold nanoparticles to form the analyte-specific conjugate. The analyte-specific conjugate was deposited on the sample receiving area on the conjugate pad in an amount of 7 μL / test strip by spraying a solution including the analyte-specific conjugate with an air jet. The conjugate pad was heated to dry the analyte-specific conjugate to the conjugate pad. The amount of anti-Flu A antibody used to prepare the analyte-specific conjugate deposited on the conjugate pad was approximately 260 ng per test strip.

[0087] The signal amplification conjugate is prepared in solution and the solution is sprayed on the buffer receiving area by air jet deposition. In short, anti-biotin is incubated with gold nanoparticles to form the signal amplification conjugate. The signal amplification conjugate is deposited on the buffer receiving area on the conjugate pad in an amount of 2 μL / test strip by spraying a solution including the signal amplification conjugate with an air jet. The conjugate pad is heated to dry the signal amplification conjugate to the conjugate pad. The amount of anti-biotin antibody used to prepare the signal amplification conjugate deposited on the conjugate pad is approximately 74 ng per test strip.

[0088] Additionally, a test strip having a detection zone was prepared. The detection zone included an immobilized capture agent that specifically binds to Flu A. In this example, an anti-Flu A antibody was deposited in the detection zone at 2.4 mg / mL at 0.75 μL / cm.

[0089] In this embodiment, the detection zone also includes a positive control capture zone. The positive control capture zone is prepared to ensure that the assay is operating normally. In this embodiment, the positive control capture zone includes immobilized bovine serum albumin (BSA-anti-biotin) derived with anti-biotin. The immobilized BSA-biotin captures the signal amplification conjugate present on the test strip, which is rehydrated with buffer and flows to the positive control zone, which indicates the normal operation of the assay. The signal amplification conjugate is captured at the positive control line, and the positive control signal indicates the normal operation of the assay.

[0090] The test strip is placed in a lateral flow device having a housing to hold the test strip. The lateral flow device includes a buffer well placed above the buffer receiving area, a sample well placed above the sample receiving area, and a reading window placed above the detection area, such as Figure 1 as displayed.

[0091] Example 2

[0092] Amplification of low concentration analyte signals

[0093] The following example illustrates the use of the lateral flow assay described in Example 1 to detect low concentrations of FluA in a sample.

[0094] The lateral flow assay prepared in Example 1 was contacted with a sample containing a low concentration of Flu A. In this non-limiting example, the Flu A antigen was a dilution of recombinant Flu A nucleoprotein expressed in E. coli and used as a crude cell lysate. As a control, a lateral flow assay prepared as in Example 1 but lacking the signal amplification conjugate was used. The sample with Flu A was added to the sample wells of the lateral flow device and the control lateral flow device. The resulting signal was measured at various time points, such as Figure 4 and as shown in Table 1.

[0095] Table 1: Signal changes over time

[0096]

[0097]

[0098] Samples without target analytes are also applied to lateral flow assays prepared with various amounts of signal amplification conjugates according to the present disclosure. Specifically, the signal amplification conjugates of the amounts of 0.5, 1, 1.5 and 3 μL are deposited on the test strips and compared with the control strips, where readings are obtained at 10 minutes and 20 minutes. The results are shown in Table 2, where each time point is measured twice. Samples without target analytes are deposited on the assay test strips to verify that no signal is generated at the test line when there is no target analyte in the sample. As shown in Table 2 below, for all samples without target analytes, the signal at the test line remains negative (the signal intensity measured at the test line is 0AU or substantially 0AU). Any signal below 1AU can be regarded as system noise, therefore being equivalent to the measured value of 0AU. Further, as shown in Table 2 below, the intensity of system noise measurement does not increase with the increase of the amount of signal amplification conjugate. Without being bound by any particular theory, it is believed that in the absence of the target analyte, the signal amplification conjugate will not amplify the signal because there is no initial signal to amplify (in other words, since the analyte is not present in the sample, a sandwich structure is not formed on the test line, and therefore there is no sandwich structure to generate a signal to be amplified by the signal amplification conjugate).

[0099] Table 2: Signal generated when sample is applied to the test strip in the absence of target analyte

[0100]

[0101] *Each reading shows two values ​​because each was performed twice.

[0102] Finally, the amount of Flu A in the sample was varied before being placed on the lateral flow device. Specifically, samples with 0%, 0.04%, 0.1%, 0.2% and 0.5% of Flu A (expressed as a percentage of the antigen concentration in the sample) were prepared and deposited on a lateral flow device and a control lateral flow device without any signal amplification conjugate. The signal intensity was read at 10 minutes and 20 minutes. The results are shown in Figure 2. Figure 5 and shown in Table 3.

[0103] Table 3: Signal intensity with different amounts of target analytes

[0104]

[0105]

[0106] Advantageously, the lateral flow assay according to the present disclosure amplifies the signal of the analyte present in the sample at a low concentration, such as a concentration below the detection limit, thereby allowing the analyte present in the sample at a low concentration to be reliably measured. In addition to determining that the target analyte is present in the sample, the embodiments of the present disclosure can also be used to quantitatively measure the amount of the target analyte present in the sample. For example, a solution of the target analyte with a known content can be applied to a determination test strip prepared according to an embodiment of the present disclosure. The measurement system can measure the intensity of the signal generated by the strip, and the data can be used to develop a dose response curve. After a test event using the test strip prepared according to an embodiment of the present disclosure, the measurement system can measure the signal generated by the test strip, and the test signal is compared with the signal drawn in the dose response curve to determine the content of the target analyte in the test sample. It should be understood that other methods of using the embodiments of the present disclosure to quantitatively measure the amount of the target analyte are possible.

[0107] Methods for detecting target analytes in a sample using lateral flow assays according to the present disclosure

[0108] Figure 6 An example method 600 of detecting low concentrations of a target analyte in a sample using a lateral flow assay according to the present disclosure is shown. The method 600 begins at step 605, where a lateral flow assay as described herein is provided. At step 610, a sample is applied to a sample well of a lateral flow device.

[0109] In some embodiments, applying the sample at the sample well includes contacting the sample with a lateral flow assay. The sample can be contacted with the lateral flow assay by external application, such as using a dropper or other applicator, to introduce the sample into the sample well. In some embodiments, the sample reservoir can be directly immersed in the sample, such as when the test strip is immersed in a container containing the sample. In some embodiments, the sample can be poured, dripped, sprayed, placed or otherwise contacted with the sample reservoir.

[0110] The method then moves to step 615, where a sample is applied at the sample well to dissolve an analyte-specific conjugate ("first conjugate") present in the sample receiving area or a first conjugate area downstream of the sample receiving area. The analyte-specific conjugate may include a first label, a first binding partner, and a reagent that specifically binds to a target analyte. As described above, depending on the number of analytes to be analyzed, the device may include a second analyte-specific conjugate comprising a label, a binding partner, and a reagent that specifically binds to a second target analyte, and the device may also include a third analyte-specific conjugate comprising a label, a binding partner, and a reagent that specifically binds to a third target analyte, or additional analyte-specific conjugates. In some embodiments, the analyte-specific conjugate may not include a binding partner because a specific target analyte may not need to be amplified due to the expected or typical concentration of the specific target analyte in the sample.

[0111] The analyte-specific conjugate (or more than one analyte-specific conjugate, if this is the case) can be integrated on the sample receiving area by physical binding or chemical bonding. After the sample is added to the sample well, the sample dissolves the analyte-specific conjugate, releasing the bond (binding) that holds the analyte-specific conjugate to the conjugate pad.

[0112] Next, moving to step 620, the target analyte (if present in the sample) is labeled with the analyte-specific conjugate. The analyte-specific conjugate binds to the target analyte (if present in the sample) to form a complex. It should be understood that step 615 and step 620 can occur substantially simultaneously.

[0113] The method then moves to step 625, where the complex moves along the fluid flow path of the assay test device and binds to the immobilized capture agent at the detection zone of the lateral flow assay, forming a sandwich structure. It should also be understood that the implementation of the present disclosure can form a sandwich structure in the detection zone in many different ways. For example, steps 615, 620 and 625 can be replaced by step 615A, where the target analyte and the analyte-specific conjugate flow separately to the detection zone, the target analyte binds to the immobilized capture agent in the detection zone, and then the analyte-specific conjugate binds to the now bound target analyte, forming an antibody-analyte-antibody-first marker structure, commonly referred to as a sandwich structure.

[0114] After the complex is bound to the immobilized capture agent (or otherwise formed into a sandwich structure), the method then moves to step 630, where a buffer is applied to the buffer well (or other suitable location) of the device. At step 635, the buffer dissolves the signal amplification conjugate ("second conjugate") present in or positioned downstream of the buffer receiving area. The signal amplification conjugate includes a second label and a second binding partner that specifically binds to the first binding partner of the analyte-specific conjugate. The signal amplification conjugate can be integrated in the buffer receiving area (or other suitable location) by physical binding or chemical bonding. After the buffer is added to the buffer well, the buffer dissolves the signal amplification conjugate, releasing the bond (binding) that holds the signal amplification conjugate to the conjugate pad (or other suitable location) of the buffer receiving area. It should be understood that steps 630 and 635 can be replaced with alternative methods to add the signal amplification conjugate to the lateral flow assay.

[0115] The method then moves to step 640, where the signal amplification conjugate flows with the buffer to the detection zone. In the detection zone, the second binding partner forms a binding pair with the first binding partner of the analyte-specific conjugate in the complex bound to the capture agent in the detection zone. As described above, this first binding interaction between the signal amplification conjugate and the complex bound to the capture agent in the detection zone can amplify the signal generated in the detection zone to a level exceeding the threshold detection level. Therefore, in some example implementations, the method then moves to step 650, where the amplified signal is detected in the detection zone.

[0116] Embodiments of the methods according to the present disclosure may also include subsequent binding interactions as described above. According to the present disclosure, a chain reaction of binding events occurs, and a series of first binding pair and second binding pair interactions result in the formation of an aggregation of analyte-specific conjugates and signal amplification conjugates in the detection zone. The aggregation of conjugates in the detection zone results in amplification of the signal generated in the detection zone, in some cases causing exponential amplification of the signal. This chain reaction of binding events occurs in Figure 6 This is shown in step 645, where residual, unbound first conjugate present in the detection zone binds to the signal amplification conjugate of the complex that has been bound to the detection zone.

[0117] Then move to step 650, the method includes detecting the signal generated in the detection zone. The detection zone may include a capture zone for capturing each complex (where more than one target analyte will be detected and / or quantified). For example, the detection zone may include a first capture zone for capturing a first complex, a second capture zone for capturing a second complex, and a third capture zone for capturing a third complex. The first immobilized capture agent in the first capture zone binds the first analyte (if present) and the first complex. When the first complex is bound to the first immobilized capture agent in the first capture zone and the signal amplification conjugate is bound thereto, a first amplified signal is detected. The first amplified signal may include an optical signal as described herein. Any suitable measurement system may be used to detect the signal generated in the detection zone at step 650, including but not limited to visual inspection of the device and optical detection using an optical reader. The signal detected at step 650 may be associated with the presence, absence or content of the target analyte in the sample.

[0118] In some embodiments, the sample is obtained from a source including an environment or a biological source. In some embodiments, it is suspected that the sample has one or more target analytes, including one or more target analytes present at a low concentration, such as a concentration below the detection limit of the measurement system. In some embodiments, it is suspected that the sample does not have any target analytes. In some embodiments, a sample is obtained and analyzed to verify the absence or presence of multiple analytes. In some embodiments, the fluid sample is blood or plasma. In some embodiments, the fluid sample is applied in an amount of 50 to 100 μL.

[0119] In some embodiments, the detected signal is an optical signal, a fluorescent signal, or a magnetic signal.

[0120] Example test systems comprising lateral flow assays according to the present disclosure

[0121] Lateral flow assay test system described herein can include lateral flow assay test device (such as but not limited to test strip), system housing including a port configured to receive all or part of the test device, reader including a light source and a photodetector, data analyzer and combination thereof. System housing can be made of any of a variety of materials, including plastic, metal or composite material. System housing forms a protective enclosure (enclosure) of the components for diagnosing the test system. System housing also defines a receiver (receptacle), which mechanically positions (register) the test strip relative to the reader. Receiver can be designed to receive any of various different types of test strips. In some embodiments, system housing is a portable device allowing lateral flow assay in various environments, and the environment is included on a workbench, on the scene, at home or in a facility for family, business or environmental applications.

[0122] The reader may include one or more optical components for optically inspecting the exposed area of ​​the detection zone of the test strip and being able to detect multiple capture zones within the detection zone. In some implementations, the reader includes at least one light source and at least one photodetector. In some embodiments, the light source may include a semiconductor light emitting diode and the photodetector may include a semiconductor photodiode. Depending on the nature of the marker used in the test strip, the light source may be designed to emit light within a specific wavelength range or light with a specific polarization. For example, if the marker is a fluorescent marker, such as a quantum dot, the light source will be designed to illuminate the exposed area of ​​the capture zone of the test strip with light within a wavelength range that induces fluorescence emission from the marker. Similarly, the photodetector may be designed to selectively capture light from the exposed area of ​​the capture zone. For example, if the marker is a fluorescent marker, the photodetector will be designed to selectively capture light within the wavelength range of the fluorescence emitted by the marker or light with a specific polarization. On the other hand, if the marker is a reflective marker, the photodetector will be designed to selectively capture light within the wavelength range of the light emitted by the light source. To this end, the photodetector may include one or more optical filters that limit the wavelength range or polarization axis of the captured light. The signal from the label can be analyzed using visual observation or a spectrophotometer to detect the color of a chromogenic substrate; a radiation counter to detect radiation, such as a 125 1 gamma counter; or a fluorometer that detects fluorescence in the presence of light of a certain wavelength. In the case of using an enzyme-linked assay, a spectrophotometer can be used to perform quantitative analysis of the amount of the target analyte. If desired, the lateral flow assay described herein can be automated or automatically performed, and signals from multiple samples can be detected simultaneously. In addition, multiple signals can be detected for multiple target analytes, including when the markers used for each target analyte are the same or different.

[0123] The data analyzer processes the signal measurements obtained by the reader. Typically, the data analyzer can be implemented in any computing or processing environment, including in a digital electronic circuit or in computer hardware, firmware or software. In some embodiments, the data analyzer includes a processor (e.g., a microcontroller, a microprocessor or an ASIC) and an analog-to-digital converter. The data analyzer can be incorporated into the housing of the diagnostic test system. In other embodiments, the data analyzer is positioned in a separate device, such as a computer, which can communicate with the diagnostic test system by wired or wireless connection. The data analyzer can also include a circuit for transmitting the result to an external source via a wireless connection to perform data analysis or review results.

[0124] Generally, the result indicator can include any of a variety of different mechanisms for indicating one or more results of the assay test. In some implementations, the result indicator includes one or more lights (e.g., light emitting diodes) that are activated to indicate, for example, that the assay test is complete. In other implementations, the result indicator includes an alphanumeric display (e.g., a two or three character light emitting diode array) for presenting the assay test result.

[0125] The test system described herein can include a power source for powering active components of the diagnostic test system, including a reader, a data analyzer, and a result indicator. The power source can be implemented by, for example, a replaceable battery or a rechargeable battery. In other embodiments, the diagnostic test system can be powered by an external host device (e.g., a computer connected via a USB cable).

[0126] Features of Example Lateral Flow Devices

[0127] The lateral flow devices described herein include a device housing. The housing of any lateral flow device described herein, including the top housing or the bottom housing, can be made of any suitable material, including, for example, vinyl resin, nylon, polyvinyl chloride, polypropylene, polystyrene, polyethylene, polycarbonate, polysulfone, polyester, urethane or epoxy resin. The housing can be prepared by any suitable method, including, for example, by injection molding, compression molding, transfer molding, blow molding, extrusion molding, foam molding, thermoforming, casting, layer deposition or printing.

[0128] Lateral flow devices as described herein may include sample wells in which a fluid sample is introduced into a test strip, such as, but not limited to, immunochromatographic test strips present in a lateral flow device. In one example, the sample may be introduced into the sample well by external application, such as using a dropper or other applicator. The sample may be poured or squeezed onto the sample well. In another example, the sample well may be directly immersed in the sample, such as when the test strip is immersed in a container containing the sample.

[0129] The lateral flow device described herein may include a buffer well in which a buffer is introduced into a test strip, such as, but not limited to, an immunochromatographic test strip present in a lateral flow device. In one example, the buffer may be introduced into the buffer well by external application, such as with a dropper or other applicator. The buffer may be poured or squeezed onto the buffer well. In another example, the buffer well may be directly immersed in the buffer, such as when the test strip is immersed in a container containing the buffer.

[0130] Lateral flow devices as described herein may include solid supports or substrates. Suitable solid supports include, but are not limited to, nitrocellulose, the pore walls of reaction disks, porous plates, test tubes, polystyrene beads, magnetic beads, membranes and microparticles (such as latex particles). Any suitable porous material may be used in lateral flow devices as described herein, having sufficient porosity to allow analyte-specific conjugates and signal amplification conjugates to pass through and suitable surface affinity to immobilize capture agents. For example, the porous structure of nitrocellulose has excellent adsorption and adsorption quality for a variety of reagents (e.g., immobilized capture agents). Nylon has similar characteristics and is also suitable. Microporous structures are available, and materials with gel structures in a hydrated state are also useful.

[0131] Further examples of useful solid carriers include: natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, cellulose esters - especially those with nitric and carboxylic acids, mixed cellulose esters and cellulose ethers; nitrogen-containing natural polymers, such as proteins and derivatives, including cross-linked or modified gelatin; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers that can be prepared with a suitable porous structure, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and partially hydrolyzed derivatives thereof, polyvinyl acetate and polyvinyl acetate; ... Acrylamide, polymethacrylate, copolymers and terpolymers of the above condensation polymers, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali metals and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clay, alumina, talc, kaolin, zeolite, silica gel or glass (these materials can be used as filters for the above polymer materials); and mixtures or copolymers of the above categories, such as graft copolymers obtained by initiating polymerization of synthetic polymers on pre-existing natural polymers.

[0132] Lateral flow devices as described herein may include a porous solid carrier in the form of a sheet or strip, such as nitrocellulose. The thickness of such a sheet or strip may vary over a wide range, for example, about 0.01 to 0.5 mm, about 0.02 to 0.45 mm, about 0.05 to 0.3 mm, about 0.075 to 0.25 mm, about 0.1 to 0.2 mm, or about 0.11 to 0.15 mm. The pore size of such a sheet or strip may similarly vary over a wide range, for example, about 0.025 to 15 microns, or more specifically about 0.1 to 3 microns; however, pore size is not intended to be a limiting factor in selecting a solid carrier. The flow rate of the solid carrier, if applicable, can also vary over a wide range, such as about 12.5 to 90 sec / cm (i.e., 50 to 300 sec / 4cm), about 22.5 to 62.5 sec / cm (i.e., 90 to 250 sec / 4cm), about 25 to 62.5 sec / cm (i.e., 100 to 250 sec / 4cm), about 37.5 to 62.5 sec / cm (i.e., 150 to 250 sec / 4cm), or about 50 to 62.5 sec / cm (i.e., 200 to 250 sec / 4cm). In a specific embodiment of the device described herein, the flow rate is about 35 sec / cm (i.e., 140 sec / 4cm). In other specific embodiments of the device described herein, the flow rate is about 37.5 sec / cm (i.e., 150 sec / 4cm).

[0133] The surface of the solid support can be activated by a chemical process that causes the reagent (e.g., immobilized conjugate) to be covalently attached to the support. As described below, the solid support can include a conjugate pad. Many other suitable methods can be used to immobilize the reagent (e.g., immobilized capture agent) to the solid support, including, but not limited to, ionic interactions, hydrophobic interactions, covalent interactions, etc.

[0134] Unless otherwise physically constrained, the solid support may be used in any suitable shape, such as a film, sheet, strip or plate, or may be coated on or bonded to or laminated to a suitable inert support, such as paper, glass, plastic film or fabric.

[0135] The lateral flow devices described herein may include a conjugate pad, such as a membrane or other type of material including a capture agent. The conjugate pad may be cellulose acetate, cellulose nitrate, polyamide, polycarbonate, fiberglass, membrane, polyethersulfone, regenerated cellulose (RC), polytetrafluoroethylene (PTFE), polyester (e.g., polyethylene terephthalate), polycarbonate (e.g., 4,4-hydroxy-diphenyl-2,2'-propane), alumina, mixed cellulose esters (e.g., a mixture of cellulose acetate and cellulose nitrate), nylon (e.g., polyamide, hexamethylenediamine, and nylon 66), polypropylene, PVDF, high-density polyethylene (HDPE) + nucleating agent "aluminum dibenzoate" (DBS) (e.g., 80u 0.024 HDPE DBS (Porex)) and HDPE.

[0136] Lateral flow devices as described herein are used for low sensitivity samples, such as samples having low concentrations of analytes or having typical concentrations of analytes but low sample volumes." Sensitivity" refers to the ratio of actual positives correctly identified as such (e.g., the percentage of subjects correctly identified as having an infection, latent or symptomatic condition). Sensitivity can be calculated as the sum of the number of true positives divided by the number of true positives and the number of false negatives.

[0137] Lateral flow devices as described herein can accurately measure multiple target analytes in many different types of samples. Samples can include samples or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans), and include fluids, solids, tissues and gases. Before being applied to the lateral flow device of the present disclosure, the sample can be processed. In a first non-limiting example, a whole blood sample can be processed to obtain plasma or serum, and plasma or serum can be applied to a lateral flow device according to the present disclosure. In a second non-limiting example, one or more sample preparation steps, such as but not limited to a cell lysis step, are used to process a sample containing cells to release intracellular proteins for detection. The processed sample can be applied to a lateral flow device according to the present disclosure. Biological samples include urine, saliva and blood products, such as plasma, serum, etc. However, this embodiment should not be interpreted as limiting the sample types applicable to the present disclosure.

[0138] Lateral flow devices as described herein may include markers. The marker may take many different forms, including detectable binding or being able to bind to analyte, analyte analog, detector reagent or binding partner molecules or compositions by spectral, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The example of marker includes enzyme, colloidal gold particles (also referred to as gold nanoparticles), colored latex particles, radioisotopes, cofactors, ligands, chemiluminescence or fluorescent agents, protein-adsorbed silver particles, protein-adsorbed iron particles, protein-adsorbed copper particles, protein-adsorbed selenium particles, protein-adsorbed sulfur particles, protein-adsorbed tellurium particles, protein-adsorbed carbon particles and protein-coupled dye capsules. The attachment of a compound (e.g., a detector reagent) to a marker may be by covalent bonds, adsorption processes, hydrophobic bonds and / or electrostatic bonds, such as in chelates, etc., or these bonds and combinations of interactions and / or may involve linking groups.

[0139] The term "specific binding partner" or "binding partner" refers to members of a pair of molecules that interact by means of specific, non-covalent interactions that depend on the three-dimensional structure of the molecules involved. Typical specific binding partner pairs include antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid strand / complementary nucleic acid strand, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / (streptavidin), receptor / ligand, and virus / cell receptor, or various combinations thereof.

[0140] As used herein, the term "immunoglobulin" or "antibody" refers to a protein that binds to a specific antigen. Immunoglobulins include, but are not limited to, polyclonal, monoclonal, chimeric and humanized antibodies, Fab fragments, F(ab')2 fragments, and include the following classifications of immunoglobulins: IgG, IgA, IgM, IgD, IgE and secretory immunoglobulins (sIg). Immunoglobulins generally include two identical heavy chains and two light chains. However, the terms "antibody" and "immunoglobulin" also include single-chain antibodies and double-chain antibodies. The term antibody used herein refers to an antibody as a whole or any fragment thereof, including its binding fragment. Thus, it can be imagined that when referring to a labeled antibody that specifically binds to a target analyte, the term refers to a labeled antibody or a fragment thereof that specifically binds to a target analyte. Similarly, when referring to a capture agent or antibody, the term refers to a capture antibody or a fragment thereof that specifically binds to a target analyte.

[0141] The antibodies in the lateral flow devices, test systems and methods according to the present disclosure may include polyclonal antibodies. The polyclonal antibodies for measuring any target analyte disclosed herein include, but are not limited to, antibodies produced from serum by active immunization of one or more of: rabbits, goats, sheep, chickens, ducks, guinea pigs, mice, donkeys, camels, rats and horses. The antibodies in the lateral flow devices, test systems and methods according to the present disclosure may include monoclonal antibodies. Antibodies for binding to target analytes are known in the art or can be easily developed by methods known in the art.

[0142] The lateral flow device according to the present disclosure includes an immobilized capture agent. The immobilized capture agent includes a reagent capable of binding to an analyte, the analyte including a free (unlabeled) analyte and / or a labeled analyte (e.g., an analyte bound to an analyte-specific conjugate, as described herein). The immobilized capture agent includes an unlabeled specific binding partner that is specific for (i) a target analyte bound by an analyte-specific conjugate, (ii) a free analyte, or (iii) an auxiliary specific binding partner, which itself has specificity for the analyte, such as in an indirect assay. As used herein, an "auxiliary specific binding partner" is a specific binding partner that is bound to a specific binding partner of an analyte. For example, an auxiliary specific binding partner may include an antibody that is specific for another antibody, e.g., a goat anti-human antibody.

[0143] The lateral flow devices described herein may include a "detection zone" or "detection zone", which is an area including one or more capture zones or capture zones and is an area in which a detectable signal can be detected. The lateral flow devices described herein may include a "capture zone" or "capture zone", which is an area of ​​the lateral flow device in which a capture agent is immobilized. The lateral flow devices described herein may include more than one capture zone. In some cases, different capture agents are immobilized in different capture zones (such as a first immobilized capture agent in a first capture zone and a second immobilized capture agent in a second capture zone). On a lateral flow substrate, multiple capture zones can have any orientation relative to each other; for example, a first capture zone can be at the distal or proximal end of a second (or other) capture zone along the path of fluid flow, or vice versa. Optionally, the first capture zone and the second (or other) capture zone can be aligned along an axis perpendicular to the path of fluid flow so that the fluid contacts the capture zones simultaneously or approximately simultaneously.

[0144] The lateral flow device according to the present disclosure includes an immobilized capture agent, which is immobilized so that the movement of the immobilized capture agent is restricted during the normal operation of the lateral flow device. For example, before and after the fluid sample is applied to the lateral flow device, the movement of the immobilized capture agent is restricted. The immobilization of the immobilized capture agent can be achieved by physical means such as barriers, electrostatic interactions, hydrogen bonds, bioaffinity, covalent interactions or combinations thereof.

[0145] The lateral flow device according to the present disclosure can measure biological products. Biological products include chemical or biochemical compounds produced by living organisms, including prokaryotic cell lines, eukaryotic cell lines, mammalian cell lines, microbial cell lines, insect cell lines, plant cell lines, mixed cell lines, naturally occurring cell lines or synthetic engineered cell lines. Biological products can include large macromolecules, such as proteins, polysaccharides, lipids and nucleic acids, as well as small molecules, such as primary metabolites, secondary metabolites and natural products.

[0146] It should be understood that the description, specific examples and data, although indicating exemplary embodiments, are given by way of example and are not intended to limit the various embodiments of the present disclosure. Various changes and modifications in the present disclosure will become apparent to those skilled in the art from the description and data contained herein, and are therefore considered to be part of the various embodiments of the present disclosure.

Claims

1. A lateral flow assay device for detecting a target analyte in a sample, comprising: a first conjugate comprising a first label, a reagent configured to specifically bind to the target analyte, and a first binding partner; a second conjugate upstream of the first conjugate along the fluid flow path of the lateral flow assay device, wherein the second conjugate comprises a second label and a second binding partner configured to specifically bind to the first binding partner, wherein the second binding partner is an antibody to the first binding partner, and wherein the first label and the second label are the same; and a detection zone downstream of the first conjugate and the second conjugate along the fluid flow path of the lateral flow assay device, the detection zone comprising an immobilized capture agent that specifically binds the target analyte, wherein the first conjugate is present in a sample receiving zone of a lateral flow assay device, or wherein the first conjugate is present in a first conjugate zone downstream of the sample receiving zone, and wherein the second conjugate is present in a buffer receiving zone upstream of the sample receiving zone, or wherein the second conjugate is present in a second conjugate zone downstream of the buffer receiving zone and upstream of the sample receiving zone. 2 . The assay device of claim 1 , wherein the first conjugate is configured to dissolve and migrate to the detection zone after applying a fluid sample to the lateral flow assay device. 3 . The assay device of claim 2 , wherein the second conjugate is configured to dissolve and move to the detection zone after the first conjugate moves to the detection zone. 4 . The assay device of claim 1 , wherein the reagent configured to specifically bind to a target analyte is an antibody or an antibody binding fragment that specifically binds to the target analyte.

5. The assay device of claim 1, wherein the first binding partner comprises biotin, and the second binding partner comprises an anti-biotin antibody.

6. The assay device of claim 1, wherein the target analyte is a target biological or environmental substance.

7. The assay device of claim 1, wherein the target analyte is influenza virus.

8. The assay device of claim 7, wherein the influenza virus is influenza A virus, influenza B virus, or influenza C virus.

9. The assay device of claim 1, wherein the immobilized capture agent is an antibody or antibody binding fragment that specifically binds to the target analyte.

10. The assay device of claim 1, wherein the test strip comprises a nitrocellulose membrane.

11. The assay device of claim 1, further comprising a control zone comprising an immobilized capture agent that specifically binds to the first conjugate.

12. The assay device of claim 1, wherein the first and second markers are selected from metal nanoparticles, colored latex beads, magnetic particles, carbon nanoparticles, quantum dots, upconversion phosphors, organic fluorophores, textile dyes, enzymes, or liposomes.

13. The assay device of claim 1, wherein the first and second markers are blue latex beads.

14. The assay device of claim 1, wherein the first marker and the second marker are colored latex particles.

15. The assay device of claim 1, wherein the first label and the second label comprise gold nanoparticles.

16. The assay device of claim 1, wherein the first label and the second label are configured to generate an optical signal, a fluorescent signal, or a magnetic signal.

17. The assay device according to claim 1, further comprising: A housing comprising a sample well positioned laterally above or upstream of the first conjugate zone, a buffer well positioned laterally above or upstream of the second conjugate zone, and a reading window accessible to the detection zone.

18. The assay device of claim 17, wherein the buffer well, the second conjugate zone, the sample receiving well, and the first conjugate zone are spatially separated along the fluid flow path of the lateral flow assay device.

19. A method for performing a lateral flow assay using the lateral flow assay device according to claim 1, comprising: applying the first conjugate to a lateral flow test strip at or downstream of a sample receiving area of ​​the lateral flow test strip; and The second conjugate is applied to the test strip at a buffer receiving zone upstream of the sample receiving zone or downstream thereof.

20. The method of claim 19, wherein the first conjugate and the second conjugate are applied to the test strip simultaneously.

21. The method of claim 19, wherein the first conjugate and the second conjugate are applied to the test strip by air jet deposition.

22. A method for detecting a target analyte in a sample, comprising: The sample is applied to a lateral flow assay device, which includes a first conjugate comprising a first label, a reagent configured to specifically bind to the target analyte, and a first binding partner; a second conjugate upstream of the first conjugate along the fluid flow path of the lateral flow assay device, wherein the second conjugate comprises a second label and a second binding partner configured to specifically bind to the first binding partner, wherein the second binding partner is an antibody to the first binding partner, and wherein the first label and the second label are the same; and a detection zone downstream of the first conjugate and the second conjugate along a fluid flow path of the lateral flow assay device, the detection zone comprising an immobilized capture agent that specifically binds the target analyte; binding a complex to the immobilized capture agent in the detection zone, wherein the complex comprises the target analyte bound to the first conjugate; After binding to the complex, releasing the second conjugate to flow along a fluid flow path of the lateral flow assay device; The second conjugate is bound to the complex bound in the detection zone.

23. The method of claim 22, further comprising detecting a signal generated by the complex and the second conjugate bound in the detection zone.

24. The method of claim 23, further comprising binding the first conjugate that is not bound to the target analyte to the second conjugate that is bound to the complex in the detection zone.

25. The method of claim 22, wherein the first marker and the second marker are configured to generate an optical signal, a fluorescent signal, or a magnetic signal.

26. The method of claim 22, wherein the second conjugate is released 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes after applying the sample to the lateral flow device.

27. The method of claim 22, wherein binding a complex to the immobilized capture agent in the detection zone comprises: labeling the target analyte with the first conjugate to form a complex; and The complex is bound to the immobilized capture agent in the detection zone.

28. The method of claim 22, wherein releasing the second conjugate comprises applying a buffer solution to the second conjugate or to a location on the lateral flow assay device upstream of the second conjugate.

29. The method of claim 28, wherein applying the buffer solution comprises pouring the buffer solution into a buffer receiving well positioned laterally above or upstream of the second conjugate.

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