Methods and devices for detecting analytes

Abstract

This invention relates to a method, system, and kit of parts suitable for detecting the presence of a target in a sample. If a target is present in the sample, a sandwich composite is formed comprising the target, magnetically movable particles, and a catalyst. The composite is separated from the other components of the sample by magnetic separation and then detected.

Description

[Technical Field]

[0001] This invention relates to a method, system, and kit of parts suitable for detecting the presence of an analyte in a sample. When a target analyte is present in the sample, a sandwich complex is formed comprising the analyte, magnetically movable particles, and a catalyst. The complex is separated from other components of the sample by magnetic separation and then detected. [Background technology]

[0002] High-sensitivity immunoassays are crucial tools in the point-of-care diagnostics industry. The need for high-sensitivity immunoassays is increasing as novel clinically important biomarkers are identified, many of which exist at very low concentrations. While laboratory-based high-sensitivity immunoassays exist, they typically require numerous assay steps and therefore must be performed by trained laboratory technicians. Many immunoassays incorporate labeled reagents conjugated to antibodies that specifically bind to the target antigen.

[0003] Enzyme-coupled immunosorbent assays (ELISAs) are used to detect the presence of an analyte, such as an antigen, in a sample through the use of a probe (e.g., an antibody) that binds to the analyte. The analyte is immobilized on a solid surface, and the probe is applied to the surface so that it binds to the analyte. The probe is typically coupled to a redox catalyst, such as an enzyme. Thus, if the analyte is present, a complex is formed containing the analyte, the probe, and the redox catalyst. Any unbound probe is removed from the assay by washing. Then, if the analyte is present, a substrate is added to the assay so that the redox catalyst of the formed complex acts on the substrate to catalyze its conversion to another product. This conversion reaction may be detectable, for example, by a change in color, photoemission, or a change in electrical signal.

[0004] Sandwich ELISA is a type of ELISA in which the analyte is not directly immobilized on the surface itself. Rather, additional probes, such as another antigen, are used (in addition to the one bound to the redox catalyst) to bind to different locations on the analyte, thereby immobilizing it on the surface. As a result, a sandwich complex is formed containing the immobilizable probe, the target analyte, and the probe bound to the redox catalyst. Again, before the addition of the substrate to the redox catalyst, any unbound probes are removed from the assay by washing, and if the analyte is present, the redox catalyst of the formed sandwich complex is capable of acting on the substrate and catalyzing its conversion to another product. For an overview of electrochemical sandwich assays for biomarker incorporation aptamers, antibodies, and nanomaterials for the detection of specific protein biomarkers, see D. Neupane and KJ Stine, Appl. Sci., 2021, 11, 7087.

[0005] These assays require a washing step that removes any unbound labeled antibodies so that, after the washing step, the only remaining redox catalysts are those bound to the probe that is bound to the captured analyte (i.e., those forming a sandwich complex with the target analyte). In this way, a signal proportional to the amount of analyte present in the sample can be generated. Many products are currently under development that attempt to automate the assay approach, often by performing an automated washing step. These automated washing steps require complex and expensive equipment and often require specialized washing solutions. These existing automated products are expensive and complex and have many potential modes of failure. The expensive equipment, in turn, means that these point-of-care solutions cannot be made available to users at a low cost, which limits product adoption.

[0006] An immunosensing probe containing horseradish peroxidase (HRP)-labeled anti-cancer fetal antigen antibody-functionalized magnetic beads is described by N. Gan, L. Jia, and L. Zheng in Int. J. Mol. Sci., 2011, 12(11), 7410-7423. As part of the synthesis of the immunosensing probe, it is described that non-adsorbed antibodies can be separated from the probe by magnetic separation, followed by washing of the final mixture. The immunosensing probe is used as part of a sandwich assay in which a sandwich complex is immobilized on an electrode surface and the analyte is detected electrochemically. To update the electrode surface, it is described that the magnetic nanoprobe can be released from the electrode surface by mixing a solution containing the electrode in the presence of a 0.3 mT magnetic field, where the magnetic field lines are oriented perpendicular to the electrode surface.

[0007] WO 2010 / 004241 (The Secretary of State for Innovation Universities & Skills of her Majesty's Britannic Government) describes an assay in which an antigen adheres to a carrier device, magnetic particles, and silver sol particles. It is described that the antigen can be made mobile, for example by applying a magnetic field, and then detected using labeled silver. WO 2020 / 032294 A1 (BBB Inc.) describes further examples of biosensors using magnetic nanoparticles.

[0008] WO 2007 / 010368 (Inverness Medical Switzerland GmbH) describes an assay device comprising a first reagent containing magnetic particles and a second reagent containing detectable components. It is stated that the first and second reagents can each independently bind to the analyte in the sample, and that when a magnetic field is applied, the detectable components can be selectively concentrated in the detection zone.

[0009] The assay method and device are described in US 10,509,032 B (Alere Switzerland GmbH). The device is configured to create a sample liquid and a sample liquid-air interface, and when a second liquid comes into contact with the interface, a liquid-liquid interface is formed, magnetically sensitive particles can be located at the liquid-air interface (through an applied magnetic field). The magnetically sensitive particles are configured to transport the analyte across the interface into the second liquid.

[0010] US 2008 / 0160634 A1 (Intel Corp) describes a device for detecting an analyte in a sample. This device includes a fluid network and integrated circuit components. The fluid network includes multiple zones, such as a sample zone, a cleaning zone, and a detection zone. The fluid network contains magnetic particles and / or signal particles. A sample containing the analyte is introduced, and the analyte interacts with the magnetic particles and / or signal particles through an affinity agent. It is possible to functionally couple a microcoil array or mechanically movable permanent magnets to the fluid network and activate them to generate a magnetic field within a portion of the fluid network, thereby moving the magnetic particles from the sample zone to the detection zone. A detection element is present that detects an optical or electrical signal from the signal particles, thus indicating the presence of the analyte.

[0011] Many of the assays and devices described above have one or more drawbacks, including low sensitivity (e.g., due to high background signal), numerous fluid chambers, washing steps, complex and / or expensive test strip designs, and / or the need for relatively large sample volumes. There is still a need for low-cost, highly sensitive immunoassays that deliver high levels of target signal amplification made possible by enzymatic amplification, but do not require washing steps. Immunoassays are preferably easy to use and manufactured reliably at low cost and in high volume. The present invention provides alternative methods, systems, and kits that address these needs and one or more of the problems and / or limitations described above. [Overview of the Initiative]

[0012] This specification describes how a target, such as an analyte, in a sample can be detected by a sandwich ELISA-type process that does not require any washing steps. If present, the target is detected as part of a sandwich complex comprising the target, a target-binding portion anchored to a magnetically movable particle, and a target-capturing portion conjugated to a catalyst. The resulting sandwich complex is movable and fixable to a second position on or within the test strip by activating or generating (e.g., applying) a magnetic field. Upon magnetic movement to the second position, the complex separates from the unbound target-capturing portion conjugated to the catalyst, and the catalyst present at the second position consists only of the target-bound target-capturing portion conjugated to the target.

[0013] The ability to selectively move the complex to a second position can facilitate the provision of methods and systems for detecting targets in a sample without the need for a washing step. In particular, placing any additional reagents required for the spontaneous amplification of a catalyst-regulated reaction (e.g., excess catalyst substrate and optionally mediator species) only at the second position may mean that any signal produced by the catalyst is limited to the catalyst bound as part of the complex. The signal produced by the catalytic process may be detected, for example, by detecting the presence of the product produced by the catalyst, or in some cases, by detecting the presence of the converted mediator species.

[0014] To avoid the need for a complex strip design involving a washing step and / or multiple fluid chambers, the inventors have identified that a particular configuration of the test strip is particularly suitable in minimizing the risk of cross-contamination, where unbound / non-composited catalysts move (e.g., diffuse) toward the catalyst substrate at a second position (e.g., within the detection zone) when the test strip is used in the various methods described herein. By positioning the sample inlet between the first and second positions, after the introduction of the sample fluid, the flow of the sample across the first position means that any target capture portions conjugated to the catalyst resuspended in the sample fluid are less likely to move and / or diffuse toward the catalyst substrate located at the second position (because the fluid flow moves away from the second position). Thus, the risk of background signaling associated with unbound / non-composited catalysts can be reduced. This can help improve the overall sensitivity of the method.

[0015] In several examples, the inventors have found that certain catalysts can convert a catalytic substrate into another product that is detectable, for example, by sensing an electrical response upon application of electrical stimulation, or optically. When the catalytic substrate is located at a second position on or within a test strip, the catalyst in the complex catalyzes the conversion of the catalytic substrate into another detectable product. Thus, if the complex is magnetically moved and fixed at the second position, and the catalytic substrate is in excess of the concentration of the complex, the detectable product can accumulate rapidly. The detectable product is generated only when the complex moves to the second position (and optionally fixed at the second position) and the catalytic substrate is available to the complex. Detection of the product may be performed by sensing an electrical response upon application of electrical stimulation, or optically, for example, by detecting a color change. The detected signal may be directly proportional to the concentration of the complex at the electrode, and then directly proportional to the concentration of the target.

[0016] If detection is performed by applying an electrical stimulus, the second position may be close to the working electrode. The electrical stimulus applied to the working electrode may be selected such that electrons are transferred between the working electrode and the detectable product, and therefore the reaction occurs only if the detectable product is present. Thus, the reaction occurs only if the complex moves to the second position (and optionally becomes fixed there) and the catalytic substrate is available to the complex. In other words, and without being constrained by theory, if the catalytic substrate present at or near the working electrode does not produce a signal (e.g., at the potential applied to the working electrode), and the catalyst (e.g., enzyme) is not present, then when the electrical stimulus (e.g., measurement potential) is applied, no signal (e.g., current) will be produced. However, if the catalyst (e.g., enzyme) is present at or near the working electrode as a result of binding to the target and forming a complex, the catalytic substrate may be spontaneously converted into a detectable product by the catalyst in proportion to the concentration of the target.

[0017] In yet another example, if the catalyst is a redox catalyst, the redox mediator and redox catalyst substrate are positioned at a second location on or within a test strip, and if the redox mediator is in an "active" state (i.e., an oxidized state that allows for the transfer of electrons to or from the redox catalyst), the redox catalyst in the complex catalyzes the conversion of the redox catalyst substrate to another product, while simultaneously catalytically converting the active redox mediator to a converted redox mediator or an inactive redox mediator (i.e., an oxidized redox mediator that does not allow for the transfer of electrons to or from the redox catalyst). Therefore, if the complex is magnetically moved to a second location (and optionally fixed there), and the active redox mediator is in excess relative to the concentration of the complex, the converted (or inactive) redox mediator can rapidly accumulate. The converted (or inactive) redox mediator is generated only when the complex moves to a second position (and optionally becomes fixed there) and the redox mediator and redox catalyst substrate are available to the complex. The converted (or inactive) redox mediator can then be detected. The converted or inactive redox mediator can be detected by sensing an electrical response upon application of electrical stimulation, or optically, for example, by detecting a color change. The detected signal may be directly proportional to the concentration of the complex at the electrode, and then directly proportional to the concentration of the target.

[0018] If detection is by application of electrical stimulation, the second position may be close to the working electrode. The electrical stimulation applied to the working electrode may be selected such that electrons are transferred between the working electrode and the converted or inactive redox mediator (but not between the converted or active redox mediator), and therefore the reaction occurs only when the converted or inactive redox mediator is present. Thus, the reaction occurs only when the complex moves to the second position (and optionally becomes fixed there) and the redox mediator and redox catalyst substrate are available to the complex. In other words, and without being constrained by theory, if the active or unconverted mediator present at or near the working electrode is initially in an oxidized state that does not produce a signal (e.g., at the potential applied to the working electrode), and then, in the absence of a redox catalyst (e.g., an enzyme), then when the electrical stimulation (e.g., the measurement potential) is applied, no signal (e.g., a current) will be produced. However, if a redox catalyst (e.g., an enzyme) is present at or near the working electrode, the active or unconverted mediator may be spontaneously converted to another oxidation state (e.g., converted or inactive) by the catalyst, in proportion to the concentration of the target, as it binds to the target and forms a complex.

[0019] Unexpectedly, when detection is by applying an electrical stimulus, the amplified signal can be detected by moving the complex to a second position, optionally fixing it, and then sensing the electrical reaction immediately after applying the electrical stimulus. That is, it is not necessary to delay sensing the reaction for a long time (e.g., several tens of minutes to several hours) after first applying the electrical stimulus to allow the signal to increase. This is because, when the redox mediator is in a "converted" or "inactive" state, i.e., in an oxidized state formed after the mediator transfers electrons to or from the redox catalyst, the electrical reaction occurs by the transfer of electrons between the electrode and the redox mediator, or by the transfer of electrons between the electrode and the detectable product. As described above, if the complex is magnetically moved to a second position (and optionally fixed in the second position), and the active (or unconverted) redox mediator and / or catalytic substrate are in excess relative to the concentration of the complex, the converted or inactive redox mediator, or the detectable product, can rapidly accumulate and thereby provide an amplified signal. In other words, and without being constrained by theory, the inventors can create conditions that allow spontaneous catalytic (e.g., enzymatic) signal amplification to occur by placing a suitable amplification reagent on or near the working electrode (and not elsewhere on the test strip, for example). In other words, once the redox catalyst reaches the working electrode region, it can begin to convert the substrate, and if the catalyst is a redox catalyst, the redox catalyst can then be regenerated by an active or unconverted mediator so that the redox catalyst (e.g., an enzyme) can continue to convert the substrate. As this occurs, the stock of active or unconverted mediator can be successively converted into inactive or converted mediators. This stock of converted mediators can be detected (e.g., electrochemically measured) at the end of the assay. In such examples, the signal produced from this converted mediator would be proportional to the concentration of the analyte in the sample.In cases where the substrate and the active, unconverted mediator are present in excess, this reaction can be continuous, thus amplifying the signal (and the longer the amplification, the greater the amplification). Therefore, the methods, kits, and systems described can provide exceptional signal amplification, which can further be spontaneous and continuous.

[0020] As described above, the inventors have identified that certain configurations of the test strips are particularly suitable in minimizing the risk of background signaling associated with the migration of unbound / non-complexed catalysts toward the catalytic substrate at the second position.

[0021] Therefore, in view of a first aspect, the present invention relates to a method for detecting a target in a sample using a system including a test strip, wherein the test strip is: Assay chamber and The assay chamber and the sample inlet with fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, the catalyst substrate and are placed on or inside the assay chamber. Includes, The above sample inlet is positioned between the first and second positions. The system further: It includes a target binding portion anchored to a magnetically movable particle, Method: (a) Introducing the sample into the assay chamber through the sample inlet so that the sample moves or flows across to the first position, away from the second position, (b) At the first position, incubate for a certain period of time so that a complex is formed that includes a sample, a target capture moiety conjugated to a catalyst, and a target binding moiety tethered to a magnetically movable particle, which, when the target is present, each independently binds to the target, the target capture moiety conjugated to the catalyst, and the target binding moiety tethered to the magnetically movable particle. (c) Activate or generate a magnetic field so that the complex is separated from one or more other components of the mixture and the catalyst catalyzes the conversion of the catalyst substrate to another product, and move the complex to the second position. (d) Detect the catalysis Provided is the above method comprising.

[0022] Detection may be performed using any suitable detection method. In some embodiments, the detection may include electrochemical detection or optical detection.

[0023] From the perspective of a second aspect, the present invention is a test strip comprising: an assay chamber, a sample inlet in fluid communication with the assay chamber, at the first position, a target capture moiety conjugated to a catalyst disposed on or within the assay chamber, at the second position, a catalyst substrate disposed on or within the assay chamber and the sample inlet is disposed between the first and second positions Provided is the above test strip.

[0024] The test strip may be configured such that introduction of a sample into the assay chamber through the sample inlet moves or flows the sample in a direction away from the second position across the first position.

[0025] From the perspective of a third aspect, the present invention is a system: (a) Test strip: Assay chamber and The assay chamber and the sample inlet with fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, the catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is positioned between the first and second positions such that the introduction of the sample into the assay chamber through the sample inlet causes the sample to move or flow across the first position, away from the second position. The above test strip and, (b) A target binding portion anchored to a magnetically movable particle, (c) A magnetic field generator configured to activate or generate a magnetic field Includes, The system is configured such that when a magnetic field is activated or generated, magnetically mobile particles move to a second position. We provide the above system.

[0026] From a fourth aspect, the present invention relates to a kit of parts, comprising separate components: (a) Test strip: Assay chamber and The assay chamber and the sample inlet with fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, the catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is located between the first and second positions. The above test strip and, (b) A target binding portion anchored to a magnetically movable particle, (c) an electromagnet, wherein the activation of the electromagnet can be arranged to generate a magnetic field capable of moving a magnetically movable particle to a second position, or a permanent magnet, wherein the permanent magnet can be arranged to generate a magnetic field capable of moving a magnetically movable particle to a second position. It provides a kit of parts, including the following:

[0027] From the perspective of a fifth aspect, the present invention provides the use of a test strip according to the second aspect, a system according to the third aspect, or a kit of parts according to the fourth aspect for detecting the presence or absence of a target in a sample.

[0028] In some embodiments described above and of any aspect herein, the first location may be a reagent zone. In some embodiments, the first location may be the first end of the assay chamber. In some embodiments, a target binding portion anchored to a magnetically movable particle may be located at or near the first location.

[0029] In some embodiments, the second location may be a detection zone. In some embodiments, the second location may be the second end of the assay chamber or a location near it.

[0030] In some embodiments, the method for detecting a target in a sample may be performed in a single liquid (e.g., a single liquid mixture containing the sample and / or a fluid or a single liquid). In some embodiments, the method for detecting a target in a sample does not involve the step of bringing the sample into contact with one or more fluid interfaces (e.g., on a test strip). In some embodiments, the method for detecting a target in a sample does not require the use of one or more washing steps (e.g., using a buffer). In some embodiments, the method for detecting a target in a sample does not involve the use of any washing steps. In some embodiments, the method for detecting a target in a sample may be performed in a single fluid chamber (e.g., the assay chamber may be a single fluid chamber). In some embodiments, the method for detecting a target in a sample may be performed on a test strip that does not include multiple fluid chambers. In some embodiments, the complex comes into contact with the catalytic substrate (and optionally also with the mediator, if present) only upon application of a magnetic field (e.g., activation or generation) and movement of the complex to a second position.

[0031] As described above, in the test strip described herein, the test strip may be configured such that the introduction of a sample into the assay chamber through the sample inlet causes the sample to move or flow across the first position in a direction away from the second position.

[0032] Without being constrained by theory, this configuration of the test strip is particularly suitable for minimizing the risk of cross-contamination and / or background signaling due to the movement (e.g., diffusion) of unbound / uncomposited catalyst toward the catalyst substrate at the second position (e.g., within the detection zone) when the test strip is used in the various methods described herein. By positioning the sample inlet between the first and second positions, after the introduction of the sample, the flow of the sample across the first position means that any target-capturing portions conjugated to the catalyst, resuspended in the sample fluid, are less likely to move and / or diffuse toward the catalyst substrate located at the second position (because the fluid flow moves away from the second position). Thus, the risk of background signaling associated with unbound / uncomposited catalysts can be reduced.

[0033] Background signaling is a particular problem when using sandwich complexes formed from a target analyte, magnetically movable particles, and a catalyst, such as those described herein, because any signals associated with unbound / uncomplexed catalysts are also subject to amplification. Therefore, in these types of assays, it is crucial to isolate unbound / uncomplexed catalysts from any catalyst substrates (and optionally mediators) placed in the detection zone. To minimize the background signaling from such unbound / uncomplexed catalysts, prior art methods typically employ numerous fluid chambers, washing steps, and / or complex strip designs. Surprisingly, however, we have found that by positioning the sample inlet between first and second positions and moving the fluid flow of the sample across the first position and away from the second position, it is possible to minimize the background signaling associated with unbound / uncomplexed catalysts and avoid the need for such complex and expensive test strips.

[0034] Furthermore, the inventors have identified that these configurations can reduce the distance between the first and second positions, allowing for a reduction in the overall size of the test strip and / or the required sample volume. Typically, unidirectional fluid flow paths (e.g., unidirectional fluid flow after sample introduction, from the sample inlet through the reagent zone to the detection zone) require longer flow paths, numerous fluid chambers, and / or washing steps, which means that a relatively larger sample volume is required.

[0035] Therefore, as described above, in the test strip described herein, the test strip may be configured such that the introduction of a sample into the assay chamber through the sample inlet causes the sample to move or flow across the first position in a direction away from the second position.

[0036] In some embodiments, the test strip is configured such that the introduction of a sample into the assay chamber through the sample inlet causes the sample to move or flow away from a second position and / or toward the periphery and / or opposite end of the assay chamber. For example, by positioning the sample inlet between the first and second positions, multidirectional fluid flow of the sample into the assay chamber through the sample inlet is facilitated.

[0037] In some embodiments, the sample inlet causes substantially bidirectional fluid flow upon introduction of the sample, with one portion of the sample flowing away from the sample inlet to a first position away from a second position, and a second portion of the sample flowing away from the sample inlet to a second position away from the first position.

[0038] In some embodiments, the sample inlet is positioned substantially equidistant from or substantially midpoint between the first and second positions. In some embodiments, the sample inlet may be positioned asymmetrically (e.g., asymmetrically on or within the test strip with respect to the first and second positions). In some embodiments, the sample inlet may be positioned closer to the first position than to the second position. In some embodiments, the sample inlet may be positioned closer to the second position than to the first position.

[0039] In some embodiments, the sample may move toward and across a first position containing a target capture portion conjugated to the catalyst. In some embodiments, the sample fluid may further move toward and across a second position containing the catalyst substrate. In some embodiments, the flow of the liquid from the sample inlet branches in two or more different directions (e.g., bifurcates). The result of this substantially bidirectional (or multidirectional) fluid flow may be to push the respective components away from the first and second positions (the introduction of the sample fluid may be considered to wash the components resuspended toward the opposite direction and / or opposite end of the assay chamber). In some embodiments, the flow of the liquid from the sample inlet bifurcates in two or more different directions such that diffusion between the first and second positions is substantially reduced or eliminated. The test strip configurations described herein help to minimize the amount of unbound / non-compounded catalyst in contact with the substrate at the second position.

[0040] Further examples of the present disclosure are described below. It will be recognized that the following methods, systems, kits of parts, and uses may also be used with the test strip designs and systems of the second and third embodiments outlined above. Accordingly, any examples described below are equally applicable to the embodiments described above.

[0041] This disclosure describes a method for detecting a target in a sample using a system: (i) Bringing the mixture into contact on or within a test strip, wherein the mixture: (a) A target binding portion anchored to a magnetically movable particle, (b) A target-capturing portion conjugated to the catalyst, (c) Sample and The system includes, and component (b) is placed on or within the test strip in the first position, and the system further: (d) Catalytic substrate The mixture includes, and component (d) is placed on or within the test strip at the second position, (ii) If a target is present, incubate (a), (b), and (c) for a certain period of time so that a complex is formed which includes components (a) and (b) that are independently bound to the target, (iii) Activating or generating a magnetic field to move the complex to a second position so that the complex is separated from one or more other components of the mixture, and so that the catalyst catalyzes the conversion of the catalytic substrate to another product, (iv) Steps to detect catalytic activity and The present invention provides a method including the following: As described above, the test strip may be a test strip according to the first embodiment.

[0042] Detection of catalysis may be direct or indirect. For example, detection may include a step of detecting a detectable product formed during and / or as a result of the catalytic step. In some examples, the detectable product may be formed by the conversion of a catalytic substrate to another product. Thus, the detectable product may be another product. In some examples, the detectable product may be an inert or converted mediator, as described above in this specification. Thus, as used herein, and unless otherwise indicated in the background, the detectable product may refer to another product and / or an inert or converted mediator.

[0043] The catalyst may be a redox catalyst. In this case, the system may further include a redox mediator located on or within the test strip at a second position. In these cases, activation or generation of a magnetic field moves the complex to the second position so that the complex is separated from one or more other components of the mixture, and so that the redox mediator can transfer electrons to or from the redox catalyst so that the redox catalyst catalyzes the conversion of the redox catalytic substrate to another product and the conversion of the redox mediator from an active state to an inactive state. Detection of catalysis may include the step of detecting an inactive redox mediator.

[0044] (iv) Detection is: (i) The working electrode is positioned at a third position that is at least close to the second position and away from the first position, (ii) Applying electrical stimulation to the working electrode, (iii) sensing an electrical reaction It may include.

[0045] This disclosure provides a method for separating a complex from one or more other components of a mixture on or within a test strip using a system, wherein the complex is: (a) A target binding portion anchored to a magnetically movable particle, (b) A target-capturing portion conjugated to the catalyst and The system further comprises a catalytic substrate positioned at a second location, wherein component (b) is located on or within a test strip at a first position, and components (a) and (b) each independently bind to a target to form a complex, and the system further comprises a catalytic substrate positioned at a second position. The above method: (i) Activating or generating a magnetic field to move the complex to a second position so that the complex is separated from one or more other components of the mixture and the catalyst catalyzes the conversion of the catalytic substrate to another product, (ii) To detect catalytic activity and This provides a method that includes [something].

[0046] The catalyst may be a redox catalyst. In this case, the system may further include, at a second position, a redox mediator located on or within the test strip.

[0047] Activation or generation of a magnetic field on or within the test strip moves the complex to the second position so that the complex is separated from one or more other components of the mixture, and so that the redox mediator can transfer electrons to or from the redox catalyst so that the redox catalyst catalyzes the conversion of the redox catalytic substrate to another product and the conversion of the redox mediator from an active state to an inactive state.

[0048] Detection of catalytic activity may include a step of detecting an inactive redox mediator.

[0049] (ii) detection is: (i) Applying electrical stimulation to a working electrode located at a third position that is at least close to the second position and away from the first position, (ii) A step of sensing an electrical reaction and It may include.

[0050] This disclosure further states that the system is: (a) Test strip and (b) At the first position, a target capture portion conjugated to a catalyst placed on or within the test strip, (c) A magnetic field generator configured to activate or generate a magnetic field, (d) A target binding portion anchored to a magnetically movable particle, (e) catalytic substrate and Includes, The system is configured such that when component (e) is placed in a second position and a magnetically mobile particle moves to the second position when the magnetic field is activated or generated. We provide the system.

[0051] When a sample containing the target comes into contact with (b) and (d), a complex is formed, and the complex contains components (b) and (d), each independently binding to the target. In this case, when a magnetic field is activated or generated, the complex moves to a second position so that it is separated from one or more other components of the mixture, and the catalyst catalyzes the conversion of the catalytic substrate to another product.

[0052] The catalyst may be a redox catalyst, and the system may further include a redox mediator positioned in a second position such that the redox mediator can transfer electrons to or from the redox catalyst when a magnetic field is activated or generated, so that the redox catalyst catalyzes the conversion of the redox catalytic substrate to another product and the conversion of the redox mediator from an active state to an inactive state.

[0053] The system may further include a detector, which may be a working electrode located at a third position, the third position being at least close to the second position and away from the first position, such that when an electrical stimulus is applied to the working electrode, electrons are transferred between the working electrode and either a catalytic substrate or an inactive redox mediator.

[0054] This disclosure is a kit of parts, consisting of separate components: (a) A test strip, (i) A target capture portion conjugated to the catalyst, (ii) catalytic substrate and A test strip comprising, wherein component (i) is positioned in the first position and component (ii) is positioned in the second position, (b) A target binding portion anchored to a magnetically movable particle, (c) an electromagnet, wherein the activation of the electromagnet can be configured to generate a magnetic field capable of moving a magnetically movable particle to a second position, or a permanent magnet, wherein the permanent magnet can be configured to generate a magnetic field capable of moving a magnetically movable particle to a second position. Further kits of parts are offered, including...

[0055] The catalyst may be a redox catalyst, and the kit of parts may further include a redox mediator positioned in a second position.

[0056] The kit may further include a detector, which may be a working electrode positioned at a third location that is at least close to the second location and away from the first location.

[0057] This disclosure further provides the use of either the systems described above or a kit of parts for detecting the presence or absence of a target in a sample. [Brief explanation of the drawing]

[0058] [Figure 1] Formal drawing of a test strip usable for electrochemical detection of magnetic separation of a magnetically movable particle anchored to a target binding portion capable of binding to the target itself, and a target capture portion conjugated to a catalyst. Lengths are shown in mm. RE is the reference electrode, WE is the working electrode, and CE is the counter electrode. [Figure 2a]This photograph shows the magnetic accumulation of magnetic beads coated with horseradish peroxidase (HRP) enzyme. Urea peroxide (UHP) is used as the enzyme substrate, and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is used as the redox mediator. As the HRP-labeled beads react with the substrate and mediator, oxidizing the ABTS, a color change occurs. This color change can be detected simultaneously using an electrode-potentiostat system. The photograph on the right shows a negative sample, where no magnet is present on the working electrode. [Figure 2b] A histogram showing the current detected by the electrode-potentiostat system using the system shown in the photograph in Figure 2a. n=5, and the error bars indicate the standard deviation. The electrochemical reaction of the magnetic separation system coupled with the redox signal amplified by the enzyme is measured, and significant differences may be observed between positive (magnet in place) and negative (magnet absent) samples. [Figure 3a] The figure shows magnetic beads coated with IL-6 antibody, and the capture of the IL-6 antigen by further IL-6 antibody conjugated to biotin. Streptavidin in polyhorse radish peroxidase (HRP) conjugated to streptavidin can bind to the biotin in the antibody. As shown in the center of the figure, the solution containing the complex is applied to the reagent zone of a dumbbell strip. The complex can be transported along the strip to the electrode zone on the right side of the strip, where the substrates of HRP (shown as reagents A+B) can interact with HRP to produce an electrochemical signal that can be detected in the electrode zone. [Figure 3b] A photograph of a dumbbell strip inside a magnetic rig. [Figure 3c] Graphs of atomic unit charge (auC) detected using the setup shown in Figures 3a and 3b, as a function of IL-6 antigen concentration. [Figure 4a]The next diagram shows the capture of a portion of the nucleic acid target by a biotin-conjugated nucleic acid probe strand bound to a streptavidin-conjugated magnetic bead. Another portion of the nucleic acid target is captured by the detection strand, which then binds to the enzyme. [Figure 4b] Graph of the current detected when targeting a nucleic acid target (Oxa1 - oxacillin resistance gene) as a function of the target concentration in the presence of another nucleic acid (tetA - tetracycline resistance). Horseradish peroxidase (HRP) was used as the enzyme, hydrogen peroxide as the substrate, and 3,3',5,5'-tetramethylbenzidine (TMB) as the redox mediator. Electrochemical detection was performed using chronoamperometry. [Figure 4c] Graph of the current detected when targeting a nucleic acid target (Oxa1 - oxacillin resistance gene) as a function of the target concentration in the presence of another nucleic acid (tetA - tetracycline resistance). Alkaline phosphatase was used as the enzyme and p-nitrophenyl phosphate (pNPP) as the substrate. Electrochemical detection was performed via linear sweep voltammetry (LSV). [Figure 5] A labeled line drawing of a test strip suitable for use in the method disclosed herein. [Figure 6] Graphs showing the charge densities derived from (i) plasma samples containing 1000 ng / mL of PCT antigen and (ii) plasma samples containing 0 mg / mL of PCT antigen, as determined using test strips as shown in Figure 5. [Modes for carrying out the invention]

[0059] In the following discussion, several terms are used, and unless otherwise indicated by the context, they have the meanings provided below. The nomenclature used herein to define compounds, in particular the compounds described in this invention, is generally based on the rules of the IUPAC organization for chemical compounds, in particular the "IUPAC Compendium of Chemical Terminology (Gold Book)".

[0060] The term “comprising” or its variations shall be understood to indicate the inclusion of an element, integer, or step, or a group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or a group of elements, integers, or steps.

[0061] The term "consisting" or its variations shall be understood to indicate the inclusion of the elements, integers, or steps, or groups of elements, integers, or steps, and the exclusion of any other elements, integers, or steps, or groups of elements, integers, or steps.

[0062] In this specification, the term "about" is used to modify a number or value, meaning a value within ±5% of the specified value. For example, if the third position is at least about 1.0 cm away from the first position, this includes a range of 0.95 cm to 1.05 cm.

[0063] As described above, a method for detecting a target in a sample using a system including a test strip, wherein the test strip is: Assay chamber and The assay chamber and the sample inlet with fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, the catalyst substrate and are placed on or inside the assay chamber. Includes, The above sample inlet is positioned between the first and second positions. The system further: Target binding portion anchored to a magnetically movable particle Includes, Method: (a) The step of introducing a sample into the assay chamber through a sample inlet so that the sample moves or flows across the first position in a direction away from the second position, (b) Incubating a mixture of the sample, a target-capturing portion conjugated to the catalyst, and a target-binding portion anchored to a magnetically movable particle at the first position for a certain period of time so that a complex is formed, comprising the target-capturing portion conjugated to the catalyst and the target-binding portion anchored to a magnetically movable particle, each independently binding to the target if a target is present. (c) The step of moving the complex to a second position by activating or generating a magnetic field so that the complex is separated from one or more other components of the mixture and the catalyst catalyzes the conversion of the catalytic substrate to another product, (d) detecting catalytic activity and The above method is provided, including the following.

[0064] The test strip may include a base and a lid. To avoid doubt, the first position may be located anywhere on the base or the lid. In some embodiments, the first position is within the test strip and may be sealed by positioning the base with respect to the lid, for example. In some embodiments, the first position is within the test strip on the base and may be sealed by the lid of the strip, for example on the base. As used herein, the base (e.g., the base of the test strip) may also be considered a test substrate, and the two terms may be used interchangeably within the context of this disclosure.

[0065] The assay chamber may be located on or within a test strip (or, for example, on or within the base of the test strip). The assay chamber can define a volume on or within the test strip and / or base into which a sample may be introduced and / or held during assays and methods as described herein. The assay chamber may include a single fluid chamber. The assay chamber may include multiple zones (e.g., at least two zones) that are in fluid contact with one another. The assay chamber may include a reagent zone in a first position. The assay chamber may include a detection zone in a second position. The first position and / or the reagent zone may be at the first end of the assay chamber. The second position and / or the detection zone may be at the second end of the assay chamber. The first end and the second end of the assay chamber may be opposite ends of the assay chamber.

[0066] The assay chamber may optionally be sealed by a strip lid. The assay chamber may be partially sealed (for example, a portion of the assay chamber, e.g., the reagent zone and / or the first position, may be sealed, while another portion of the assay chamber, e.g., the detection zone and / or the second position, may not be sealed). In some embodiments, the lid may be transparent, and / or at least a portion of the lid may be transparent. For example, the portion of the lid that seals the detection zone and / or the second position may be transparent.

[0067] The assay chamber may include any suitable dimensions and / or shape that facilitate the movement or flow of the sample across the first position in a direction away from the second position (and optionally also facilitate the movement or flow of the sample across the second position in a direction away from the first position). In some embodiments, the assay chamber may include a first expanded end (including the first position and / or reagent zone) and / or a second expanded end (including the second position and / or detection zone). The first and second expanded ends may be connected by a connecting channel. The connecting channel may be narrower than the first and second expanded ends. The first expanded end of the assay chamber may taper toward the connecting channel. The second expanded end of the assay chamber may taper toward the connecting channel. In some embodiments, the first and / or second ends (e.g., expanded ends) may taper toward the sample inlet. Such configurations may facilitate and / or promote the desired flow of the sample after it has been introduced into the assay chamber.

[0068] The test base and / or assay chamber may be dumbbell-shaped. The first position may be one end of the dumbbell shape. The second position may be the second end of the dumbbell shape. The base and / or assay chamber may include an hourglass shape. For example, an hourglass shape including an elongated central portion. The first position may be one end of the hourglass shape. The second position may be the second end of the hourglass shape.

[0069] As described above, the sample inlet has fluid communication with the assay chamber and is located between the first and second positions. The placement of the sample inlet between the first and second positions is sometimes referred to herein as a “centre-fill” design or configuration. However, the sample inlet does not need to be located exactly midway between the first and second positions. In some embodiments, the sample inlet may be located away from the first and / or second positions. In some embodiments, the sample inlet may be located approximately midway between the first and second positions. In some embodiments, the sample inlet is positioned or located so that the sample is introduced into the connecting channel of the assay chamber.

[0070] The sample inlet may optionally be positioned on the base of the test strip, adjacent to the assay chamber. In some embodiments, the sample inlet may be positioned so that it is substantially in the same plane as the assay chamber. In such configurations, the sample is introduced into the assay chamber such that the sample is delivered substantially parallel to the base surface and / or moves or flows across the base surface in a direction substantially parallel to the base surface.

[0071] The sample inlet may be located on the lid, positioned on the lid, or defined by the lid. In some embodiments, the sample inlet may be positioned such that the inlet is substantially above the assay chamber. In such configurations, the sample is delivered from a first direction where the sample is substantially perpendicular to the base surface (e.g., from above if the test strip is held in a horizontal configuration). In some embodiments, the sample may also strike and / or collide with the base surface before moving or flowing across the first position in a direction away from the second position (and optionally also facilitating the movement or flow of the sample across the second position in a direction away from the first position).

[0072] The sample may be deposited in the sample inlet. The inventors have found that, in the configuration described, it is preferable to place the sample in the sample inlet to minimize the risk of the target binding portion, which is anchored to a magnetically movable particle, and / or the target capturing portion, which is conjugated to the catalyst, potentially diffusing to a second location upon sample deposition.

[0073] When using the sample, it may be applied to a sample inlet located between the first and second positions. For example, the sample may be applied to a sample inlet located between the two ends of the base.

[0074] As described above, the target capture portion conjugated to the catalyst is positioned at a first position on or within the test strip (e.g., on or within the assay chamber). The target capture portion may be positioned, for example, by attaching it to the first position by any suitable contact means. In some cases, the target capture portion conjugated to the catalyst may be attached to the strip as a solution or suspension, for example by spraying or dripping, and the strip is dried. In some embodiments, the target capture portion conjugated to the catalyst is printed onto the test strip at a first position. As described above, the first position may be one end of the test strip, for example, one end of the base.

[0075] In some embodiments, the target binding portion, anchored to a magnetically movable particle, is positioned on or within a test strip. As will be recognized, this component may be positioned in any suitable position at the start of the method as described herein, as long as it is in the first position during the incubation stage. In some embodiments, the target binding portion, anchored to a magnetically movable particle, is positioned on or within a test strip by any suitable contact means. In some embodiments, the target binding portion, anchored to a magnetically movable particle, is attached to or within a strip as a solution or suspension, for example by spraying or dripping, and the strip is dried. In some embodiments, the target binding portion, anchored to a magnetically movable particle, is printed on a test strip, for example, on a base.

[0076] In some embodiments, the target binding portion, anchored to a magnetically movable particle, is positioned on or within the test strip at a first position. In such embodiments, the target binding portion, anchored to the magnetically movable particle, is already in the first position before the incubation stage. In some embodiments, the target binding portion, anchored to the magnetically movable particle, is positioned on or within the test strip at a position different from the first position. For example, the target binding portion, anchored to the magnetically movable particle, may be positioned between the first and second positions, or at the second position. In such embodiments, the target binding portion, anchored to the magnetically movable particle, may be moved to the first position before the incubation stage. For example, the target binding portion, anchored to the magnetically movable particle, may be moved magnetically to the first position. Furthermore, or / or, the target binding portion, anchored to the magnetically movable particle, may be moved to the first position after the introduction of the sample (for example, as the sample moves or flows across the first position and away from the second position).

[0077] In some embodiments, the target binding portion anchored to a magnetically movable particle and the sample may be brought into contact on or within a test strip by any suitable means. For example, each may be attached separately to or within the test strip. Alternatively, both components may be added to the base simultaneously. In some cases, the target binding portion anchored to a magnetically movable particle and the sample may be attached independently to or within the strip as a solution or suspension, for example by spraying or dripping.

[0078] The target binding portion, anchored to a magnetically movable particle, and the target capturing portion, conjugated to a catalyst, may be attached to or within a strip as a solution or suspension, for example by spraying or dripping, and the strip is dried. The target binding portion, anchored to a magnetically movable particle, and the target capturing portion, conjugated to a catalyst, may be printed to or within a test strip, for example, at a first position, either separately or simultaneously.

[0079] The target binding portion, anchored to a magnetically movable particle, may be located at one end of the test strip, for example, one end of the base, or the same end as the target capture portion conjugated to the catalyst. If the test strip includes a dumbbell-shaped assay chamber and / or base, both the target capture portion conjugated to the catalyst and the target binding portion anchored to the magnetically movable particle may be located at the same one end of the dumbbell shape.

[0080] The inventors have found that it is possible to induce aggregation of the target binding portion, which is anchored to a magnetically movable particle, and the target capturing portion, which is conjugated to the catalyst, before they are attached to the test strip, thereby increasing the likelihood of false positive results. Therefore, the target binding portion, which is anchored to a magnetically movable particle, is typically attached to or within the strip separately from the target capturing portion, which is conjugated to the catalyst. In some embodiments, the target binding portion, which is anchored to a magnetically movable particle, is positioned on or within the test strip in a location that does not overlap with the target capturing portion, for example, in a section of the first position. In some embodiments, the target binding portion, which is anchored to a magnetically movable particle, and the target capturing portion, which is conjugated to the catalyst, are each located (e.g., attached) in different sections of the reagent zone (e.g., so that they do not overlap or substantially overlap). During the test assay, these components may be resuspended as the sample moves or flows across the first position.

[0081] As described above, the catalyst substrate is placed at a second position on or within the test strip (e.g., on or within the assay chamber). As described above, and to avoid doubt, the second position may be located anywhere on the base or lid. In some embodiments, the second position is within the test strip, and for example, the second position may be sealed by the placement of the base with respect to the lid. In some embodiments, the second position is on the base and within the test strip, for example, on the base and sealed by the lid of the strip.

[0082] In some embodiments, the catalyst substrate (and optionally the redox mediator) is placed in a second position on or within a test strip, for example, on or within an assay chamber, by any suitable contact means. In some embodiments, one or both of these components are attached to or within the strip as a solution or suspension, for example, by spray or drip, and the strip is dried. In some embodiments, the catalyst substrate (and optionally the redox mediator as well) is printed onto the test strip, for example, on a base. In some embodiments, the redox catalyst and the redox mediator may be pre-mixed and attached or printed simultaneously onto the test strip, for example, on a base.

[0083] A catalytic substrate is a chemical species that can be acted upon by a catalyst and can be catalytically converted by the catalyst into one or more different products. For example, a catalytic substrate can be cleaved at one or more positions by the catalyst to produce one or more different products. Any one type of catalyst may have more than one suitable catalytic substrate that can be catalytically converted into one or more different products. Any suitable substrate may be used in combination with the corresponding catalyst.

[0084] The catalyst substrate is placed at a second position on or within the test strip (e.g., on or within the assay chamber). The second position is located away from the first position, i.e., the first and second positions do not overlap. In some embodiments, the first and second positions are separated by at least about 0.1, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, or 3 cm. For example, the first and second positions may be separated by at least about 1 cm. In some embodiments, the first and second positions are separated by a distance ranging from about 0.5 cm to about 2 cm. In other examples, the catalyst substrate is placed at the second position on or within the test strip, and at other locations on or within the test strip, for example, across or within a portion or the entire surface of the base.

[0085] In some embodiments, the catalyst is a redox catalyst. To avoid any doubt, in these embodiments, the catalyst substrate is a redox catalyst substrate. In these embodiments, the test strip may further include, in a second position, a redox mediator placed on or within the test strip (e.g., on or within the assay chamber).

[0086] A redox mediator is a chemical species that acts as an electron shuttle between the oxidized and reduced forms of a redox catalyst. As described above, when the method of the present invention is applied by the inventors, the redox mediator may be used in an "active" state, i.e., an oxidized state that allows for the transfer of electrons to or from the redox catalyst, so that the redox catalyst catalyzes the conversion of the redox catalytic substrate to another product. Typically, the redox mediator is introduced into the system in an active state.

[0087] Both the redox mediator and the redox catalyst substrate may be placed at the second position on or within the test strip (e.g., on or within the assay chamber). The redox mediator may also be placed at other positions on the test strip, for example, overlapping with the second position, or across or within a portion or the entire surface of the base including the second position. Alternatively, the redox mediator may be placed only at the second position, and the redox catalyst substrate may be placed at the second position and other positions on the test strip. In this case, the redox catalyst substrate may overlap with the second position, or across or within a portion or the entire surface of the base including the second position.

[0088] The second position may be one end of the test strip, for example, one end of the base. For example, if the base is dumbbell-shaped, the first position may be one end of the dumbbell shape, and the second position may be the other end.

[0089] In certain embodiments, both the target-capturing portion conjugated to the catalyst and the target-binding portion anchored to magnetically movable particles are each located at the same end of the test strip, for example, at the same end of the base, and the catalyst substrate (and optionally a redox mediator) is located at the other end. For example, if the base is dumbbell-shaped, the target-capturing portion conjugated to the catalyst and the target-binding portion anchored to magnetically movable particles may each be located at the same one end of the dumbbell shape, and the catalyst substrate (and optionally a redox mediator) may be located at the other end of the dumbbell shape.

[0090] When used, after introducing the sample onto the test strip, a mixture is formed containing a target binding portion anchored to a magnetically movable particle, a target capture portion conjugated to a catalyst, and the sample. The mixture on the test strip, containing the target binding portion anchored to a magnetically movable particle, the target capture portion conjugated to a catalyst, and the sample, may be a liquid. For example, a sample containing a target may be a liquid that, when added to or within the strip, forms a mixture, i.e., a mixture containing the target binding portion anchored to a magnetically movable particle, the target capture portion conjugated to a catalyst, and the sample.

[0091] As described herein, a method of the first embodiment involves incubating a sample and a target binding portion anchored to a magnetically movable particle, a target capture portion conjugated to a catalyst, for a certain period of time so that a complex is formed comprising the target binding portion anchored to a magnetically movable particle and the target capture portion conjugated to a catalyst, each independently binding to the target if a target is present. If a target is present, the complex is often formed within minutes or seconds. The incubation time is typically in the range of about 0.5 seconds to about 10 minutes, about 2 seconds to about 5 minutes, about 5 seconds to about 60 seconds, or about 10 to about 30 seconds. In some embodiments, the incubation time is in the range of about 2 minutes to about 5 minutes.

[0092] A method of the first embodiment further includes activating or generating a magnetic field to move the complex to a second position such that the complex is separated from one or more other components of the mixture and moves to or near the catalytic substrate and, in some cases, the redox mediator. The magnetic field can be activated or generated by activating an electromagnet or by mechanically activating a permanent magnet, for example, by moving it near the second position.

[0093] The magnetic field strength is strong enough to attract and move the magnetically mobile particles of the composite toward a second location on or within the strip, thereby separating the composite from one or more other components of the mixture. In some embodiments, the composite is separated from at least any unbound target-trapping portions conjugated to the redox catalyst in the mixture. The magnetic field typically has an intensity of about 0.05 mT to about 5 T, e.g., about 0.1 mT to about 2 T, about 1 mT to about 500 mT, or about 10 mT to about 50 mT. In some embodiments, the magnetic field has an intensity of about 1.8 Tesla.

[0094] Magnetically movable particles may be paramagnetic particles that are made movable by a magnetic field. Paramagnetic particles may be paramagnetic beads, for example, "magnetic beads." Magnetic beads are well known in the art and may contain any suitable paramagnetic material, such as iron oxide, for example, magnetite (Fe3O4).

[0095] If the second location is physically separated from the first location (e.g., they do not overlap), then the shape and exact location of the magnetic field at the second location do not need to be restricted. To reduce the possibility of any unbound target-trapping fraction conjugated to the redox catalyst diffusing to the second (or third) location, it may be beneficial to position the first location as far away as practically possible from the second (or third) location on or within the test strip.

[0096] In some embodiments, activation or generation of a magnetic field moves the composite laterally, for example, along a test strip, to a second position, so that the composite is separated from one or more other components of the mixture (e.g., from any unbound target capture portions conjugated to the catalyst in the mixture). Lateral movement means that the composite moves across the horizontal plane rather than the vertical plane of the test strip. For example, if the strip includes a substrate which is a thin sheet of material, the composite may move horizontally across the surface with the largest surface area.

[0097] In certain embodiments, the test strip includes a base containing one or more capillary channels such that, upon activation or generation of a magnetic field, the composite can move from a first to a second position through one or more capillary channels.

[0098] If the catalyst is a redox catalyst and requires a redox mediator to catalytically convert the redox catalyst substrate, then both the redox mediator and the redox catalyst substrate are positioned at a second location. In the absence of a redox mediator, the redox catalyst may be capable of converting one redox catalyst substrate to another product. However, after conversion, the redox catalyst is unable to act on any further redox catalyst substrates until it is oxidized or reduced again to its active state (the active state being the state in which the redox catalyst is capable of converting the redox catalyst substrate to another product). To catalytically convert the redox catalyst substrate to one or more other products and to amplify the electrochemical signal generated per target, the redox mediator and redox catalyst substrate must be available to the redox catalyst at a second location. In some embodiments, the redox mediator and redox catalyst substrate are present in excess (e.g., molar excess) with respect to the redox catalyst constituting the complex.

[0099] If the catalyst does not require a redox mediator to catalytically convert the catalytic substrate, the catalytic substrate is placed at the second position, but the redox mediator may not be present. To catalytically convert the catalytic substrate to one or more other products and to amplify the electrochemical signal generated per target, the catalytic substrate must be available to the redox catalyst at the second position. In some cases, the catalytic substrate is present in excess (e.g., molar excess) with respect to the redox catalyst constituting the complex.

[0100] The target-capturing portion conjugated to the catalyst is positioned on or within the test strip at a first position, away from the second position. Activation or generation of a magnetic field moves the target-capturing portion conjugated to the catalyst, which forms part of a complex (including the target and the target-binding portion anchored to a magnetically movable particle), to the second position. In some embodiments, the second position is close to at least a detector, such as a working electrode.

[0101] In some embodiments, the second position overlaps at least partially with a detector, such as a working electrode. In such embodiments, at least a portion of the catalyst substrate, and in some cases, the redox mediator, may be positioned on the detector, such as a working electrode, by adsorption or deposition, for example, on the detector surface. Alternatively, one of the redox catalyst substrate and the redox mediator may be positioned on the detector, such as a working electrode, and the other on the non-overlapping portion of the second position. In some embodiments, the catalyst substrate, and in some cases, the redox mediator, are adsorbed on the surface of the detector, such as a working electrode, and the second position overlaps with the entirety of the third position. In some embodiments, the detector is the working electrode.

[0102] In a further embodiment, the second position partially overlaps with the detector, and the catalytic substrate is positioned within the detector. When a magnetic field is activated or generated, the magnetically movable particles and therefore (if a target exists) the composite move to the second position. The magnetically movable particles may move to a portion of the second position that is close to but does not overlap with the detector, i.e., the magnetically movable particles do not need to overlap with the detector in order to detect the catalytic activity.

[0103] When both the redox mediator and the redox catalytic substrate are located at the second position, they are unavailable to any unbound target-capturing moiety, and therefore, any unbound target-capturing moiety conjugated to the redox catalyst cannot convert the redox catalytic substrate to another product, nor can it convert the redox mediator from an active to an inactive state. In contrast, the redox mediator and redox catalytic substrate are available to the complex that moves to the second position upon activation or generation of a magnetic field. As a result, the redox catalyst contained within the complex at the second position can catalytically convert the redox catalytic substrate to one or more other products, and simultaneously convert the redox mediator from an active to an inactive state, thus amplifying the signal per target.

[0104] If both the redox mediator and the redox catalytic substrate are available to any complex that moves to a second position upon activation or generation of a magnetic field, then the redox catalyst contained within the complex at the second position can catalytically convert the redox catalytic substrate into one or more other products, and simultaneously convert the redox mediator from an active state to an inactive state, thus amplifying the signal per target.

[0105] If a catalyst does not require a redox mediator to catalytically convert the catalytic substrate, then when the catalytic substrate is located at the second position, it is unavailable to any unbound target-capturing moiety, and therefore, any unbound target-capturing moiety conjugated to the catalyst cannot convert the catalytic substrate to another product. In contrast, upon activation or generation of a magnetic field, the catalytic substrate is available to the complex that moves to the second position. As a result, the catalyst contained within the complex at the second position catalytically converts the catalytic substrate to one or more other products, thus amplifying the signal per target.

[0106] If the catalytic substrate is available to any complex that moves to a second position upon activation or generation of a magnetic field, then at the second position, the catalyst contained within the complex will catalytically convert the catalytic substrate into one or more other products, and simultaneously convert the redox mediator from an active state to an inactive state, thus amplifying the signal per target.

[0107] In some embodiments, the method of the first embodiment further includes positioning a detector, e.g., a working electrode, at a third position, the third position being at least close to the second position and away from the first position. The third position may further be located within the detection zone of the assay chamber. The second position may at least partially overlap with the position of the detector, e.g., the working electrode, so that activation or generation of a magnetic field moves at least some of the complex to the detector. The third position is away from the first position. In some embodiments, the third position is slightly away from the first position so that any diffusion of any unbound target capture portion conjugated to the catalyst to the third position is negligible, i.e., does not contribute to a measurable signal. In some embodiments, the third position is at least about 0.1, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, or 3 cm away from the first position. For example, the third position may be at least about 1 cm away from the first position. In some embodiments, the first and third positions are separated by a distance ranging from about 0.5 cm to about 2 cm.

[0108] The third position may be at one end of the test strip. In some examples, the first position may be at the first end of the test strip, and the second and third positions may be at the second end (e.g., the opposite end) of the test strip. For example, if the test strip is dumbbell-shaped, the first position may be at one end of the dumbbell shape, and the second and third positions may be at the other end.

[0109] In certain embodiments, both the target-capturing portion conjugated to the catalyst and the target-binding portion anchored to magnetically movable particles are each located at the same end of the test strip, while the catalyst substrate and working electrode are located at the other end. For example, if the test base is dumbbell-shaped, the target-capturing portion conjugated to the catalyst and the target-binding portion anchored to magnetically movable particles may each be located at the same end of the dumbbell shape, while the catalyst substrate and working electrode may be located at the other end of the dumbbell shape.

[0110] When the detector is a working electrode, the method of the first embodiment also includes applying an electrical stimulus to the working electrode to sense an electrical reaction. When the catalyst is a redox catalyst and a redox mediator is required to catalytically convert the redox catalytic substrate, the system may be configured such that when an electrical stimulus is applied to the working electrode, electrons are transferred between the working electrode and an inactive redox mediator (i.e., an inactive redox mediator). In such cases, the electrical reaction is an indicator of the concentration of the inactive redox mediator. Alternatively, when an electrical stimulus is applied to the working electrode, the system may be configured such that electrons are transferred between the working electrode and a detectable product resulting from the conversion of the redox catalytic substrate. In such cases, the electrical reaction is an indicator of the concentration of a detectable product produced during the conversion of the redox catalytic substrate.

[0111] If the catalyst does not require a redox mediator to catalytically convert the catalytic substrate, the system may be configured such that when an electrical stimulus is applied to the working electrode, electrons are transferred between the working electrode and the detectable product resulting from the conversion of the catalytic substrate. In such cases, the electrical reaction is an indicator of the concentration of the detectable product produced during the conversion of the catalytic substrate.

[0112] The electrical stimulation is applied after the activation or generation of the magnetic field. The time between the activation or generation of the magnetic field and the application of the electrical stimulation does not need to be limited, as long as at least some of the complex (if a target exists) is able to move to a second position on or within the test strip, and either an inactive redox mediator or a detectable product is able to be generated before the electrical stimulation is applied. The inventors have found that the movement of the complex to the second position induced by the magnetic field is surprisingly rapid. Therefore, in some embodiments, the electrical stimulation is applied about 0.1 s to 20 s after the activation or generation of the magnetic field. However, it may be beneficial to have more time between the activation or generation of the magnetic field and the application of the electrical stimulation to allow for a greater increase in the amount of inactive redox mediator or a detectable product, thereby allowing a larger reaction to be sensed. In some embodiments, the method includes a first incubation step in which the complex is formed and a second incubation step after the complex has moved to a second position. In embodiments including a second incubation step, the method further includes the step of incubating a complex comprising a catalytic substrate (and optionally a redox mediator) in a second location and / or detection zone. This second incubation step may be a time that allows an increased amount of inactive redox mediator or detectable product to grow. In some embodiments, electrical stimulation is applied a few minutes after activation of the magnetic field. Electrical stimulation may be applied about 0.5 seconds to about 15 minutes, about 1 second to about 10 minutes, about 2 seconds to about 5 minutes, about 5 seconds to about 60 seconds, or about 10 to about 30 seconds after activation of the magnetic field.

[0113] The electrochemical signal generated when electrons are transferred between the working electrode and the inactive redox mediator is amplified when the conversion of the redox mediator from the active to the inactive state is catalyzed by the redox catalyst. Activation or generation of a magnetic field moves the target-trapping portion, conjugated to the redox catalyst and forming part of the complex (which includes the target and the target-binding portion anchored to a magnetically mobile particle), to a second position.

[0114] If the detector is the working electrode, the second position is at least very close to the working electrode, and therefore the magnetic field moves at least a portion of the complex to the vicinity of the electrode, thereby separating the complex containing the target-capturing portion from any unbound target-capturing portion. The proximity of the complex and the working electrode is such that when an electrical stimulus is applied to the working electrode, electrons are transferred between the working electrode and the inactive redox mediator or detectable product, thereby producing a sensed reaction. If the second position does not overlap with the working electrode, the redox mediator or detectable product may be diffusible to the working electrode.

[0115] When a target is present, the reaction intensity detected by the detector is directly proportional to the amount of redox mediator and / or catalytic substrate converted by the catalyst, which depends on the concentration of the catalyst at the second position (if the redox mediator and / or catalytic substrate is in excess), which in turn depends on the concentration of the target present in the sample. Thus, in some embodiments, the reaction intensity is proportional to the amount of target in the sample.

[0116] Typically, the redox mediator and / or catalytic substrate are present in the system in molar excess relative to each of the target binding moieties anchored to the magnetically movable particles and the target capture moieties conjugated to the catalyst. Often, the target binding moieties anchored to the magnetically movable particles and the target capture moieties conjugated to the catalyst are present in substantially equal amounts (i.e., their average molar concentrations are within ±10% of each other). Typically, the redox mediator and / or catalytic substrate are present in a molar excess relative to each of the target binding moieties anchored to the magnetically movable particles and the target capture moieties conjugated to the catalyst. 1 ~10 20 times, for example, 10 3 ~10 18 double, 10 5 ~10 15 It is present in twice as many molars. In some embodiments, the redox mediator and / or catalytic substrate is at least 10 times greater than each of the target binding portion anchored to a magnetically movable particle and the target capture portion conjugated to the catalyst. 10 It exists in twice the molar amount.

[0117] The test strip may include a base. The base may include a film. The film may include a hydrophilic or hydrophobic polymer, or a hydrophilic or hydrophobic treatment of the polymer, to either promote or hinder wetting or fluid movement or diffusion. Hydrophilic polymers, such as some polyesters, may be beneficial for improving wetting of the base by the sample and for improving wicking of the sample across the base surface.

[0118] The test strip, for example, the base, may be further treated with a polymer suitable for inhibiting the diffusion of macromolecules (e.g., molecules with a molecular weight greater than about 100 Da, in some embodiments, molecules with a molecular weight greater than about 4000 Da, in some embodiments, molecules with a molecular weight in the range of 1 MDa) across the base surface. In this manner, the diffusion of any unbound target-capturing moieties conjugated to the catalyst to a second or third position on the base may also be inhibited. To avoid doubt, the polymer suitable for inhibiting macromolecule diffusion may also inhibit the movement of the complex to the second position upon activation or generation of a magnetic field, but to a much lesser degree. In some embodiments, the polymer is positioned at least at the first position. The polymer may contain, or consist of, one or more of the group consisting of carboxymethylcellulose, polyethylene glycol, dextran, dextrin, and polystyrene.

[0119] Polymers suitable for inhibiting the diffusion of macromolecules can also inhibit the diffusion of redox mediators and / or catalytic substrates, so as to inhibit the diffusion of redox mediators and / or catalytic substrates to the first position.

[0120] The first and second positions on or within the test strip may be covered at least partially or completely by the mixture. In some embodiments, the first and third positions on or within the strip may be covered at least partially or completely by the mixture. In some embodiments, the first, second and third positions may be covered at least partially or completely by the mixture. In certain embodiments, the first, second and third positions on or within the strip are covered at least partially or completely by the mixture, and the mixture contains a single liquid, i.e., the mixture is not separated.

[0121] The target-capturing portion may be conjugated to the catalyst through a linker, i.e., a molecule that binds to both the target-capturing portion and the catalyst. The linker may be a particle, such as a latex particle. Alternatively, the linker may be formed by conjugation techniques known in the art, such as biotin-streptavidin or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS). For example, the catalyst may be conjugated to biotin, and the target-capturing portion may be conjugated to streptavidin. Biotin and streptavidin can bind to each other, thereby linking the catalyst and the target-capturing portion.

[0122] Similarly, the target binding portion may be conjugated to the magnetically mobile particle through a linker, i.e., a molecule that binds to both the target binding portion and the magnetically mobile particle. The linker may be a particle, such as a latex particle. Alternatively, the linker may be formed by conjugation techniques known in the art, such as biotin-streptavidin or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS). For example, the magnetically mobile particle may be conjugated to streptavidin, and the target binding portion may be conjugated to biotin. Biotin and streptavidin can bind to each other, thereby linking the magnetically mobile particle and the target binding portion.

[0123] In some embodiments, the catalyst is an enzyme. To avoid doubt, in such embodiments, the catalyst substrate is an enzyme substrate. A suitable catalyst (e.g., enzyme) substrate will be selected according to the type of catalyst or enzyme chosen. Thus, the enzyme substrate may be any substrate of the enzymes described herein. In some examples, the enzyme substrate may be glucose or hydrogen peroxide (e.g., urea peroxide).

[0124] In some embodiments, if the catalyst is an enzyme, the enzyme is any one selected from the group consisting of horseradish peroxidase, alkaline phosphatase, glucose dehydrogenase, glucose oxidase, glutathione reductase, xanthine oxidase, laccase, glutaredoxin, cytochrome c oxidase, alcohol dehydrogenase, pyruvate dehydrogenase, and sorbitol dehydrogenase.

[0125] In some embodiments, the catalyst is a redox catalyst, such as a redox enzyme. In some embodiments, the redox enzyme is a reductase. When the enzyme is a reductase, the redox mediator is typically added to the system as a reduction mediator. In this manner, the redox mediator can reduce the oxidized enzyme formed after the reduction of the enzyme substrate, thereby reforming the reduced form of the enzyme. The reduced form of the enzyme can then convert another molecule of the substrate, and the cycle is repeatable.

[0126] In certain embodiments, the enzyme is a reductase, and the redox mediator is a reducing mediator. The reductase may be glutathione reductase.

[0127] In another embodiment, the redox enzyme is an oxidase. In such embodiments, the redox mediator is typically added to the system as an oxidized mediator. The oxidase may be any one selected from the group consisting of oxidases, peroxidases, and dehydrogenases, such as horseradish peroxidase, glucose dehydrogenase, glucose oxidase, xanthine oxidase, laccase, cytochrome c oxidase, alcohol dehydrogenase, pyruvate dehydrogenase, and sorbitol dehydrogenase.

[0128] The redox mediator may be any one selected from the group consisting of ferricyanides / ferrocyanides; ruthenium(II) and (III) complexes (e.g., hexaamineruthenium(III) chloride); ferrocene and ferrocenium derivatives; cobaltocene, rhodocene and other metallocenes; p-nitrophenyl phosphate (pNPP); quinones; nicotinamide adenine dinucleotide; nicotinamide adenine dinucleotide phosphate; flavin adenine dinucleotide; methylene blue and derivatives; 2,6-dichlorophenoline dopheno; and phenylenediamine and derivatives).

[0129] In some embodiments, the redox enzyme is an oxidase, and the redox mediator is added to the system as a reduced mediator. Alternatively, the redox enzyme may be a reductase, and the redox mediator may be deployed to the system as an oxidized mediator.

[0130] Those skilled in the art can also evaluate which redox catalysts a redox mediator is compatible with. For example, horseradish peroxidase is compatible with at least 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), diaminobenzidine, and phenylenediamine. Glucose dehydrogenase is compatible with at least ferricyanide, hexamineruthenium(III) (and its salts, e.g., chloride salts), tris(bipyridine)ruthenium(III) chloride, ferrocene, and ferrocene derivatives.

[0131] The target-capturing portion and the target-binding portion do not need to be limited as long as they are capable of binding to the target at separate binding sites. For example, in certain embodiments, the target-binding portion and the target-capturing portion each independently include: at least one of the following: nucleic acids, e.g., single-stranded DNA, RNA, PNA or LNA molecules, antibodies or their antigen-binding fragments, cell surface receptors or their ligands, or biologically active fragments of cell surface receptors or their ligands, aptamers, molecularly imprinted polymers, enzymes, lipids, glycans, glycoproteins, glycolipids, or proteoglycans. In some embodiments, the target-capturing portion and the target-binding portion include a fusion of one or more of the above portion types. Furthermore, the target-capturing portion and the target-binding portion may be fused to or otherwise bound to a magnetically movable particle or redox catalyst using techniques known in the art.

[0132] Nucleic acid sequence tagging is well-known in the art and can be performed using standard bioconjugate chemistry (see, for example, O. Koniev and A. Wagner, Chem. Soc. Rev., 2015, 44, 5495; and C. Sornay et al., R. Soc. Open Sci., 2022, 9:211563). For example, N-hydroxysuccinimidyl (NHS) esters that couple with amines, carbodiimides that couple with amines (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), or diisopropylcarbodiimide (DIC)), biotin that couple with streptavidin (see, for example, CM Dundas et al., Appl. Microbiol. Biotechnol., 97, 9343-9353 (2013)); or maleimides that couple with the thiol moiety (see, for example, SS Ghosh et al., Bioconjug. Chem., 1990, 1:71-6).

[0133] In some embodiments, the target binding portion and the target capture portion each independently comprise one or more selected from the group consisting of proteins, DNA, and RNA. The target binding portion and the target capture portion each independently comprise one or more selected from the group consisting of antibodies, antigen-binding antibody fragments, antibody mimetics, and nucleic acid chains (e.g., DNA or RNA chains). Often, the target binding portion and the target capture portion each independently comprise one or more selected from the group consisting of antibodies, antibody-binding antibody fragments, and antibody mimetics.

[0134] As used herein, the phrase “its antigen-binding fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. The antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments included in the term “its antigen-binding fragment” include: (i) a Fab fragment, a monovalent fragment consisting of VH, VL, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide crosslink at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VH and VL domains of a single arm of the antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544 546); and (vi) an isolated complementarity-determining region (CDR) or (vii) a combination of two or more isolated CDRs that may be linked by a synthetic linker, at the option of choice. Furthermore, although the two domains of the Fv fragment, VH and VL, are encoded by separate genes, they may be linked using recombination via a synthetic linker, thereby enabling them to be produced as a single protein chain in which the VH and VL regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423 426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 5883). Such single-chain antibodies are also intended to be included within the term "their antigen-binding fragment." These antigen-binding fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for use in the same manner as intact antibodies.

[0135] The inventors have found that the method of the present invention can detect the presence of a target in a sample at a concentration of less than 50 pg / mL, for example, as low as 10 pg / mL or 1 pg / mL. In certain embodiments, the method can detect the presence of a target at a concentration of 1 pM, 5 pM, or 10 pM in a sample, for example, a target at a concentration of 1 pM or 10 pM or higher in a sample.

[0136] When the detector is the working electrode, the working electrode is typically part of an electrode system that includes at least one further electrode; that is, the method of the present invention typically includes an electrode system that includes two or more electrodes. In some embodiments, the electrode system is electrically coupled to a sensor such as a potentiostat. In certain embodiments, the electrical stimulus is a potential and the electrical response is an electric current. In such cases, the electrode system may be any system suitable for applying a potential difference to the working electrode. To supply the potential difference, the electrode system includes at least two electrodes. For example, the working electrode can apply a desired potential to an inert redox mediator to transport charge to (thereby acting as an anode and reducing the inert redox mediator) or from the mediator (thereby acting as a cathode and oxidizing the inert redox mediator). A second electrode of known potential is needed for use as a reference and to complete the circuit and balance the charge. For example, if the working electrode acts as the anode and reduces the redox mediator, the second electrode balances the charge by oxidizing the components of the system, thereby acting as the cathode. The second electrode acts as both the reference electrode and the counter electrode.

[0137] Typically, electrode systems include two to four electrodes, often three or four. Generally, electrode systems include three electrodes. When an electrode system includes three electrodes, it includes a working electrode, a reference electrode (with a known potential and used as a reference), and a counter electrode (to complete the circuit and balance the charge). In such an electrode system, the change in potential difference at the working electrode is measured independently of the change in potential difference at the counter electrode. This setup is preferable to a two-electrode system because the analysis of the electrical response is simpler.

[0138] When an electrode system includes four electrodes, these include a working electrode, a reference electrode, a counter electrode, and a working sensing electrode. In this setup, the potential difference is measured at the working sensing electrode (relative to the reference electrode) and is independent of the electrochemical reaction occurring at the working electrode, i.e., the effect of the applied current; the system itself is being measured. Such a setup is useful for measuring the impedance across the system (discussed below).

[0139] The electrodes of the electrode system may be made of any material suitable for conducting electrons. It is preferable that the material is corrosion-resistant and capable of conducting a suitable current load. Sometimes, the required current load is in the range of ±1nA to ±1mA; ±10nA to ±0.1mA; or ±100nA to ±0.01mA (with an error of ±1%). Suitable materials include any one or a selection from the group consisting of gold, silver, platinum, palladium, titanium, graphite, carbon, brass, tungsten, ruthenium, iridium, titanium, nickel, aluminum, tin, or their oxides. Often, the electrodes of the system are made from any one or a selection from the group consisting of gold, silver, platinum, palladium, titanium, graphite, and carbon. Typically, the electrodes are made from any one or a selection from the group consisting of gold, silver, platinum, and palladium, preferably gold and silver. Generally, electrodes are made from one type of material; that is, electrodes are not made from a selection of materials.

[0140] Electrode systems may be manufactured through additional printing processes, such as 3D printing or screen printing. These are suitable approaches for the cost-effective manufacture of electrode systems and sensors (Tan, C., Nasir, MZM, Ambrosi, A., Pumera, M., 2017. Anal. Chem. 89, 8995-9001). Electrode systems may also be microfabricated and manufactured by depositing a desired material, patterning the material with desired fine features (e.g., by UV photolithography), and removing or etching the material if necessary. Preferably, electrode systems are screen printed. Screen-printed electrodes (SPEs) offer many advantages over more traditional electrodes, including ease of manufacture and cleaning, reliability, low cost, repeatability, and rapid time to results. SPEs are suitable for mass production and can be manufactured at a relatively low cost compared to traditional macro or micro electrodes (Hayat, A., Marty, JL, 2014. Sensors. 14, 10432-10453). Due to these advantages, SPEs are excellent for prototyping and the development of novel sensing technologies, as reported herein.

[0141] The electrodes may be electrically connected to the potentiostat. The electrical connection may be any suitable connection that carries an electrical signal between the potentiostat and the electrode system. Typically, such an electrical connection is a wire. Each electrode in the electrode system is electrically connected to the potentiostat to enable the application and detection of electrical signals to and from each electrode.

[0142] In some embodiments, electrical stimuli are applied and electrical responses are sensed using amperometry (e.g., chronoamperometry), electrochemical impedance spectroscopy (EIS), open-circuit potentiometry, chronocoulometry, pulsed voltammetry, cyclic voltammetry, and / or linear sweep voltammetry.

[0143] In some embodiments, amperometry (e.g., chronoamperometry) applies an electrical stimulus and senses an electrical response using electrochemical impedance spectroscopy (EIS), open-circuit potentiometry, chronocoulometry, pulsed voltammetry, and / or cyclic voltammetry. The potentiostat of the system may be suitable for cyclic voltammetry, open-circuit potentiometry, chronoamperometry, EIS, square wave voltammetry (SWV), and differential pulsed voltammetry (DPV).

[0144] DPV may be used to investigate electron transfer to or from an electrode surface with high sensitivity. The potential is measured between the working electrode and the reference electrode, while the current is measured between the working electrode and the counter electrode. The potential is increased or decreased linearly over time to a set potential (linear potential sweep) or increased or decreased incrementally over time (step waveform). A series of normal voltage pulses are superimposed on the linear potential sweep or step waveform. The current is measured before (initial current) and after (final current) the voltage pulses, and the difference between the final and initial currents is plotted as a function of the applied potential. In this method, the effect of non-Faraday charged currents is minimized, i.e., only Faraday currents (currents generated by the reduction or oxidation of system components) are measured, and therefore electron transfer can be analyzed more accurately.

[0145] The peak current (I) measured in the DPV plot pk) corresponds to the current generated upon oxidation of the components of the system. The change in peak current reflects the change in complex concentration at the working electrode: as the amount of complex increases, the peak current increases. Thus, measurement of the peak current over time gives an indication of the rate of increase of the complex at the working electrode.

[0146] When, the electrical signal applied to the electrode system is a series of normal voltage pulses superimposed on a potentiodynamic sweep or staircase waveform, and the applied potential ranges from about -1.0 V to 1.0 V. In other cases, the applied potential ranges from about -0.5 V to 0.7 V or from about -0.3 V to 0.5 V.

[0147] In one embodiment, the electrical stimulus includes the potential applied between the working electrode and the reference electrode, and the electrical response consists of the current between the working electrode and the counter electrode. The type of electrical response detected from the electrode system depends on the measurement. If the measurement is DPV, the electrical response is direct current (DC). The current is measured before (initial current) and after (final current) the voltage pulse. Thus, when measuring DPV, the electrical signal applied is a series of normal voltages superimposed on a potentiodynamic sweep or staircase waveform, and the electrical response is the DC measured before and after the voltage pulse.

[0148] EIS enables real-time data capture. Using an alternating potential difference over a range of frequencies, the impedance of the working electrode-structure interface may be studied to establish information regarding the interface, its electron transfer properties and the surrounding diffusional behavior. The term "impedance", as used herein, is a measure of the frequency-dependent resistance of a first substance to the current of a circuit and is calculated according to the following equation, where Eω is equal to the frequency-dependent potential and I ω is equal to the frequency-dependent current.

Number

[0149] The change in impedance reflects the change at the electrode surface as a function of time: as the number of complexes at the working electrode increases, the frequency-dependent resistance (i.e., impedance) at the working electrode surface to the current increases.

[0150] Those skilled in the art know that an electrical stimulus may be applied to a working electrode, and the electrical response may be sensed from the working electrode at multiple points in time. For example, the response may be measured before and after the activation or generation of a magnetic field, and the two responses may be compared to assess whether a target is present in the sample.

[0151] To avoid any doubt, each embodiment described in relation to the first embodiment of the method can be applied to any other method, test strip, system, kit and use described above and herein, with modifications where necessary.

[0152] A second aspect of the present invention is a test strip: Assay chamber and The assay chamber and the sample inlet with fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, the catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is located between the first and second positions. A test strip is provided. The test strip may be configured such that the introduction of a sample into the assay chamber through the sample inlet causes the sample to move or flow across the first position in a direction away from the second position.

[0153] A third aspect of the present invention is a system: (a) Test strip: Assay chamber and The assay chamber and the sample inlet with fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, the catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is positioned between the first and second positions such that the introduction of the sample into the assay chamber through the sample inlet causes the sample to move or flow across the first position, away from the second position. Test strip and, (b) A target binding portion anchored to a magnetically movable particle, (c) A magnetic field generator configured to activate or generate a magnetic field Includes, When a magnetic field is activated or generated, a magnetically mobile particle is configured to move to a second position. We provide the system.

[0154] When a sample containing a target is brought into contact with a target-capturing portion conjugated to a catalyst and a target-binding portion anchored to a magnetically movable particle, a complex is formed, each independently containing a target-capturing portion conjugated to a catalyst and a target-binding portion anchored to a magnetically movable particle that binds to the target. In such cases, when a magnetic field is activated or generated, the complex moves to a second position so as to separate the complex from one or more other components of the mixture, and so as to catalyze the conversion of the catalytic substrate to another product.

[0155] The catalyst may be a redox catalyst, and the system may further include a redox mediator positioned at a second position such that the redox mediator can transfer electrons to or from the redox catalyst so that when a magnetic field is activated or generated, the redox catalyst catalyzes the conversion of the redox catalytic substrate to another product and catalyzes the conversion of the redox mediator from an active state to an inactive state.

[0156] The system may further include a detector. This may be any detector suitable for sensing reactions characteristic of the catalytic action, such as inactive redox mediators or active products formed by the transformation of the catalytic substrate. For example, the detector may be suitable for detecting the absorption wavelength of inactive redox mediators or active products formed by the transformation of the catalytic substrate, and may be, for example, a UV-vis spectrometer.

[0157] Alternatively, the detector may be a working electrode positioned at a third location, which is at least close to the second location and away from the first location, such that when an electrical stimulus is applied to the working electrode, electrons are transferred between the working electrode and an inactive redox mediator or active product formed by the transformation of the catalytic substrate.

[0158] In some embodiments, the system of the third embodiment further includes a sample which may or may not include a target. The system of the third embodiment may further include a sensor which senses an electrical response.

[0159] A fourth aspect of the present invention is a kit of parts, comprising separate components: (a) Test strip: Assay chamber and The assay chamber and the sample inlet with fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, the catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is located between the first and second positions. Test strip and, (b) A target binding portion anchored to a magnetically movable particle, (c) an electromagnet, wherein the activation of the electromagnet is such that it generates a magnetic field capable of moving a magnetically movable particle to a second position, or a permanent magnet, wherein the permanent magnet is such that it generates a magnetic field capable of moving a magnetically movable particle to a second position. The above kit of parts is provided, including the above.

[0160] The catalyst may be a redox catalyst, and the kit of parts may further include a redox mediator positioned in a second location.

[0161] The kit may further include any detector suitable for sensing reactions characteristic of the catalytic result, such as inactive redox mediators or active products formed by the transformation of the catalytic substrate. For example, the detector may be suitable for detecting the absorption wavelength of inactive redox mediators or active products formed by the transformation of the catalytic substrate, and may be, for example, a UV-vis spectrometer.

[0162] Alternatively, the detector may be a working electrode positioned at a third location on the test strip, where the third location is at least close to the second location and away from the first location.

[0163] In some embodiments, the redox mediator and / or catalyst substrate are each placed on the working electrode.

[0164] The kit of parts is: (i) at least one additional electrode; (ii) Sensors such as potentiostats; and (iii) One or more electrical connections that can complete the circuit between the electrode and the sensor It may include one or more of the more selected options.

[0165] A fifth aspect of the present invention provides the use of a test strip of the second aspect, a system of the third aspect, or a kit of parts of the fourth aspect for detecting the presence or absence of a target in a sample. For example, the test strips of the second, third, and fourth aspects may further contain a sample that may or may not contain a target. The sample may be added to the test strip to form a mixture as defined in the first aspect. The first, second, and third positions may be at least partially or completely covered by the mixture.

[0166] To avoid any doubt, each embodiment described in relation to the method of the first embodiment is applied to the test strip of the second embodiment, the system of the third embodiment, the kit of parts of the fourth embodiment, and the use of the fifth embodiment, with modifications where necessary. For example, both the redox mediator and the redox catalyst substrate may be placed on the working electrode; the redox catalyst may be a redox enzyme; and / or the test strip may further include a polymer suitable for hindering the diffusion of macromolecules, for example, across the surface of the test strip, where the polymer is placed in at least the first position.

[0167] The methods, test strips, systems, kits, and uses described herein are intended for use in detecting the presence of a target in a sample. The target may be an analyte. In particular, the target may be a component found (or potentially found) in a biological sample. In other examples, the target may be a component (e.g., a contaminant) found (or potentially found) in a sample taken from a manufacturing process.

[0168] In some examples, the target may be a protein, peptide, nucleic acid, metabolite, sugar or polysaccharide, lipid, drug or drug metabolite, etc.

[0169] The sample may be a biological fluid. For example, a biological fluid may be blood, plasma, serum, urine, sweat, feces, cerebrospinal fluid, interstitial fluid, saliva, sputum, nasal fluid, or any other body fluid.

[0170] The sample may be a liquid sample. The sample may be diluted with a liquid (e.g., a solution or buffer) and / or mixed with a liquid before being introduced into the sample inlet. Therefore, in some examples, the sample may include a biological fluid that has been diluted with a liquid (e.g., a solution or buffer) and / or mixed with a liquid.

[0171] As described above, the test strips and systems described herein allow for a reduction in the distance between the first and second positions, and also allow for minimizing the overall size of the test strip and / or the required volume of sample. In particular, the required volume of biological sample can be minimized. In some embodiments, the method may also be suitable for detecting the presence of a target in 1 μL to 1 mL of biological sample (e.g., about 1 μL to about 100 μL, or about 1 μL to about 10 μL, e.g., about 5 μL to about 10 μL of biological sample). Such biological samples may be diluted with and / or mixed with a biologically compatible liquid or buffer before introduction into the sample inlet.

[0172] Each and all patent and non-patent references referenced herein are incorporated herein by reference in their entirety, as if the entire content of each reference were presented herein in whole.

[0173] The present invention can be further understood by referring to the following examples. [Examples]

[0174] The magnetic accumulation of magnetite beads coated with horseradish peroxidase (HRP) enzyme was demonstrated using the test strips of the present invention.

[0175] Urea peroxide was used as the enzyme substrate, and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was used as the redox mediator. Dextran and polyethylene glycol were used to adhere them to the surface of the working electrode, and they were air-dried.

[0176] As the magnetic field moves the HRP-labeled beads to a second position on the top of the strip, reacting with the substrate and mediator, it causes an electrochemical change (i.e., oxidation of ABTS) that is detectable using the electrode-potentiostat system, accompanied by a color change. The color change appears between t=0 (left) and t=60s (center) (see Figure 2a). A significant difference in the detected current may be observed between positive (in-place magnet) and negative (no magnet) samples (see Figure 2b).

[0177] A negative sample, photographed 300 seconds later and without a magnet on the working electrode, is shown in the right-hand image of Figure 2a.

[0178] Interleukin-6 (IL-6) assay A wash-free IL-6 chronoamperometry magnetic assay was performed using a magnetic rig (shown in Figure 3b), dumbbell strips (shown in Figure 3a), and IL-6 reagent in a wet configuration.

[0179] Apparatus and materials -PolyHRP, Lot P366115 (Catalog No. 898387, manufactured by RD Systems): Polyhorseradish peroxidase conjugated with streptavidin. -IL-6 conjugate, lot P355923 (catalog number 898934, manufactured by RD Systems): A polyclonal antibody specific to human IL-6 and conjugated to biotin. - IL-6 antigen, lot P318690 (catalog number 840115, manufactured by RD Systems): Human IL-6 standard - Reagent substrate A, lot P361118 (catalog number 895000, manufactured by RD System): Stabilized hydrogen peroxide - Reagent substrate B, lot P361121 (catalog number 895001, manufactured by RD Systems): Stabilized tetramethylbenzidine -Tween 20 - Phosphate-buffered saline (PBS) - Dumbbell strips with lids - Palmsense connector - Magnetic rig

[0180] The dumbbell strip contained a reagent zone (see Figure 3a), to which a solution containing magnetic beads coated with anti-IL-6 antibody, IL-6 antigen, biotin-conjugated IL-6 detection antibody, and biotin-conjugated HRP enzyme was added. Before adding to the dumbbell strip, the solution was incubated for 20 minutes to allow the formation of a complex containing magnetic beads-IL-6 antibody-IL-6 antigen-biotin-conjugated IL-6 antibody-streptavidin-conjugated polymer HRP.

[0181] The dumbbell strip also includes a detection zone (e.g., electrode zone) (see Figure 3a) which contains a working electrode on which the substrates for the HRP enzyme (hydrogen peroxide and tetramethylbenzidine) are placed.

[0182] Tween20 and phosphate-buffered saline (PBS) solutions were added to the dumbbell strip at the positions shown in Figure 3a. These act as stacker solutions, meaning they are suitable for facilitating molecular diffusion and are particularly suitable for larger molecules, such as magnetic beads-IL-6 antibody-IL-6 antigen-biotin conjugated IL-6 antibody-streptavidin conjugated polymer HRP, or magnetic beads-IL-6 antibody-conjugated complexes.

[0183] Once the reagent was added to the dumbbell strip, the magnet was moved along the magnetic rig to pull the magnetic beads (both complexed and uncomplexed) to the electrode zone where they were mixed with the HRP substrate.

[0184] Chronoamperometry was used to measure the electrochemical reactions induced by HRP acting on the substrate.

[0185] Formal method steps A) The formation of immune complexes between magnetic beads coated with anti-IL-6 antibody, biotinylated detection antibody, streptavidin polyHRP, and IL-6 antigen was performed as follows: 1) 1% coated magnetic beads were pre-diluted 1:1 in RD System reagent diluent. 2) 0.5% of coated beads, 5 μL, were added to a low-protein binding test tube. 3) Next, 2.5 μL of biotin-conjugated detection IL-6 antibody from the HS IL-6 RD system kit was added to a test tube. 4) Next, 7.5 μL of streptavidin-conjugated polyHRP from the HS IL-6 RD system kit was added to a test tube. 5) The IL-6 antigen was dissolved in deionized water at a final concentration of 60 ng / mL, and further dilution was performed using RD System reagent diluent. 6) One of several IL-6 antigen solutions was added to a test tube in 5 μL, or a reagent diluent was added as a blank sample. The IL-6 solutions prepared were at concentrations of 4 ng / mL, 2 ng / mL, 400 pg / mL, and 200 pg / mL. 7) The test tube was quickly vortexed to mix the reagents, then rotated to allow all volume to collect at the bottom of the test tube, and allowed to stand and incubate for 20 minutes. 8) After 20 minutes, the test tube was placed on a magnet to separate the beads, and 15 μL of the supernatant was removed.

[0186] B) The magnetic assay was performed as follows: 1) A dumbbell strip with a lid was inserted into the Palmsense connector and embedded on the magnetic rig. 2) The magnetic rig was connected to the laptop, and the magnets were placed at the top of the strip (reagent zone). 3) 5 μL of a 1:1 mixed solution of RD System reagent substrates A and B was added to the bottom of the strip (electrode zone). 4) 8 μL of Tween 20 0.5% PBS was added to the electrode zone, and the strip area shown in Figure 3a was also filled with it. 5) A) was prepared by adding 5 μL of the solution to the top of the strip (reagent zone). 6) To activate the magnet movement, press the magnetic rig button; the magnet pulled the bead into the electrode zone. 7) Chronoamperometry measurements were performed at -0.1V and a 20-second scan. Two rigs and two potentiostats were used. For each IL-6 concentration level, the following replicates were performed: 5 replicates for the blank, 4 replicates for 50 pg / mL, and 3 replicates for 100, 500, and 1000 pg / mL.

[0187] result A linear correlation was observed between the IL-6 antigen concentration and the charge detected by the working electrode via chronoamperometry as described in step B)7) above, in the range of 50 to 1000 pg / mL (see Figure 3c) (R square = 0.995).

[0188] The limit of blank (LoB) was calculated to be equal to the mean blank + (1.645xSD blank), resulting in a value of 29.8 pg / mL.

[0189] The detection limit was calculated as =LoB + (1.645xSD), resulting in a minimum detectable analyte concentration of IL-6 of 83.7 pg / ml.

[0190] The exemplary methods described above were successfully applied to detect IL-6 targets using antibody-coated magnetic beads and biotin-conjugated antibodies, along with streptavidin-conjugated polymer HRP and HRP substrates.

[0191] nucleic acid assay An exemplary method for detecting nucleic acid target strands is described.

[0192] method 1) The nucleic acid probe strands were ligated to magnetic beads via common conjugation techniques, such as biotin-streptavidin or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS) (see Figure 4a). 2) A nucleic acid target was bound to the probe strand, and an overhanging region on the target complementary to the detection strand was exposed. 3) Enzyme-labeled detection chains (e.g., HRP, glucose dehydrogenase, alkaline phosphatase, etc.) were bound to the overhanging region to form a complex on the magnetic beads. 4) The complex was added to one portion of the test strip. 5) Enzyme substrates (e.g., HRP = horseradish peroxidase substrate, TMB and ABTS mediator; glucose dehydrogenase = glucose substrate; alkaline phosphatase = pNPP substrate, etc.) were added to or near the working electrode on another part of the test strip. 6) The complex was pulled to the working electrode using magnetism, where the enzyme acted on the substrate. 7) The enzyme signal was electrochemically measured by incubation of the entire complex with a redox mediator (e.g., 3,3',5,5”-tetramethylbenzidine (TMB), p-nitrophenyl phosphate (pNPP), ferricyanide, hexamine ruthenium chloride, etc.). 8) Electrochemical detection was performed via chronoamperometry, DPV, SWV, linear sweep voltammetry, or cyclic voltammetry.

[0193] data A linear relationship was observed in both calibration curves obtained: (1) using HRP, hydrogen peroxide, and TMB as enzyme, substrate, and mediator combinations via chronoamperometry for specific target (Oxa1-oxacillin resistance gene) versus nonspecific target (tetA-tetracycline resistance); and (2) using alkaline phosphatase and pNPP as enzyme and substrate combinations via linear sweep voltammetry (LSV) for specific target (Oxa1-oxacillin resistance gene) versus nonspecific target (tetA-tetracycline resistance) (see Figure 4c).

[0194] conclusion With minimal signal interference observed from another gene sequence (tetA) used as a negative control sequence, it was possible to generate a dose-response curve for the specific gene sequence (oxa1).

[0195] Example of a test strip Figure 5 shows a line drawing of an exemplary test strip for use in the methods disclosed herein. The test strip includes a dumbbell-shaped base, one of which is a reagent zone, where a target binding portion anchored to a magnetically movable particle (such as a capture antibody anchored to a magnetic bead, as in Figure 5) and a target capture portion conjugated to a catalyst (such as a detection antibody conjugate anchored to an antibody, as in Figure 5) are arranged.

[0196] The other end of the base is the detection zone, where the catalyst substrate (in Figure 5, this is the enzyme substrate) is placed, and the redox mediator is placed therein when in use. In Figure 5, the enzyme substrate and redox mediator are pre-mixed before being attached to the test strip. During detection, the catalytic action includes (i) applying an electrical stimulus to a working electrode located at a third position that is at least close to the second position but away from the first position, and (ii) sensing the electrical reaction, the working electrode may be placed in the detection zone (shown as a carbon electrode in Figure 5). The base in Figure 5 further includes a silver / silver chloride reference electrode and a carbon pair electrode, both placed in the detection zone. The electrodes are electrically connected using a coupling pad. The electrodes may be connected to a potentiometer.

[0197] The base of Figure 5 further includes a sample inlet positioned between the reagent and the detection zone. The sample is placed in the sample inlet. While the sample may be placed in the reagent zone, the inventors have found it preferable to place and / or introduce the sample via the sample inlet. If the sample is applied to the reagent zone as a liquid, it may flow into and overlap with the detection zone, allowing target binding portions and / or target capture portions conjugated to the catalyst, which are anchored to magnetically movable particles, to move into the detection zone, potentially resulting in false positive results. By positioning the sample inlet between the first and second positions and introducing the sample through this inlet, the risk of target binding portions and / or target capture portions conjugated to the catalyst, which are anchored to magnetically movable particles, diffusing into the detection zone during sample application is minimized.

[0198] The reagent in the reagent zone and the sample in the sample inlet are controllably resuspendable, allowing the components to be mixed and, if a target is present, to form a complex. A magnetic field may then be activated or generated to move the complex to the detection zone so that the catalyst catalyzes the conversion of the catalytic substrate to another product. The catalytic activity can be detected by (ii) applying an electrical stimulus to a working electrode located in the detection zone and (ii) sensing the electrical reaction. The base of Figure 5 further includes two air escapes at either end of the strip to prevent an increase in air that could interfere with the mixing of the components and the movement of the complex.

[0199] The reference numbers shown in Figure 5 indicate the following features of the test strip: 5-vent; 10-magnetic bead-capture antibody; 15-enzyme-detection antibody conjugate; 20-reagent zone; 25-detection zone; 30-working electrode (carbon); 35-reference electrode (silver / silver chloride); 40-counter electrode (carbon); 45-vent; 50-connecting pad; 55-catalyst substrate and electrochemical redox mediator (pre-mixed before attachment); 60-sample inlet (centralized filling design allows for resuspension of reagents while preventing magnetic beads or enzyme conjugate from moving to the detection zone during sample application).

[0200] Assay example using a "central filling" test strip Materials used: [Table 1]

[0201] method: The substrate / mediator (pre-mixed), enzyme-detection antibody conjugate, and magnetic bead-capture antibody solution were deposited onto strips in the typical deposition pattern shown in Figure 5 and then dried.

[0202] An exemplary assay was performed on plasma samples using the test strip configuration shown in Figure 5. One sample was spiked with 1000 ng / mL of procalcitonin, while the other was left as a control (without procalcitonin). The procalcitonin (PCT) antigen stock was prepared in plasma rather than buffer to ensure that the plasma concentration on the strip remained at 100%. Sample (25 μL) was added to the test strip via sample inlet 60. The test strip was incubated for 2 minutes to form an immunocomplex, and then magnetic beads were magnetically transferred to the working electrode using a 0.5 T magnetic field, where the enzyme acted on the substrate, and incubated for 5 minutes. Electrochemical detection was performed using chronoamperometry with a potential of +0.25 V for 20 seconds.

[0203] Figure 6 shows the signals (charge density) generated from a positive plasma sample containing 1000 ng / mL of procalcitonin (PCT) antigen and from a control plasma sample (containing 0 ng / mL of PCT antigen). This assay did not require any washing steps after the sample was introduced onto the test strip.

Claims

1. A method for detecting a target in a sample using a system including a test strip, wherein the test strip is: Assay chamber and The assay chamber and the sample inlet having fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, a catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is positioned between the first and second positions. The aforementioned system further: It includes a target binding portion anchored to a magnetically movable particle, The aforementioned method: (i) Introducing the sample into the assay chamber through the sample inlet such that the sample moves or flows across the first position in a direction away from the second position, (ii) Incubating at the first position for a certain period of time such that a composite is formed, comprising the target-capturing portion conjugated to the catalyst and the target-binding portion anchored to a magnetically movable particle, each independently binding to the target if the target is present. (iii) Activating or generating a magnetic field to move the complex to the second position so that the complex is separated from one or more other components of the mixture and the catalyst catalyzes the conversion of the catalytic substrate to another product, (iv) To detect the catalytic action and The method, including the method described above.

2. The method according to claim 1, wherein the introduction of the sample into the assay chamber through the sample inlet is also carried out such that the sample moves or flows across the second position in a direction away from the first position.

3. The method according to claim 1 or 2, wherein the catalyst is an enzyme.

4. The method according to any one of claims 1 to 3, wherein the catalyst is a redox catalyst, and the system further comprises a redox mediator disposed on the test strip at the second position.

5. To detect the catalytic action: (a) Optionally, detect the detectable product formed by and / or as a result of the catalytic action by sensing an electrical response in response to the application of an electrical stimulus, or by detecting a color change. The method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

6. The aforementioned detection: (i) Applying electrical stimulation to a working electrode located at a third position that is at least close to the second position but away from the first position, (ii) Sensing electrical reactions and The method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

7. The method according to claim 6, wherein the catalyst substrate is placed on the working electrode, and optionally both the redox mediator and the redox catalyst substrate are placed on the working electrode.

8. The method according to any one of claims 6 to 7, wherein the working electrode is part of an electrode system comprising at least one further electrode, and optionally the electrode system is electronically coupled to a sensor such as a potentiostat.

9. The method according to any one of claims 6 to 8, wherein the electrical stimulus is an electric potential and the electrical response is an electric current.

10. The method according to any one of claims 6 to 9, wherein an electrical stimulus is applied and an electrical response is sensed using amperometry (e.g., chronoamperometry), pulsed voltammetry, electrochemical impedance spectroscopy, and / or cyclic voltammetry.

11. The method according to any one of claims 6 to 10, wherein the intensity of the electrical reaction is proportional to the amount of the catalyst substrate that is converted.

12. The method according to any one of the preceding claims, wherein the activation or generation of a magnetic field moves the complex to the second position laterally so that the complex is separated from one or more other components of the mixture.

13. The aforementioned magnetic field: (i) activate the electromagnet; or (ii) Mechanically activate the permanent magnet in the vicinity of the second position. The method according to any one of the prior claims, which is generated by...

14. The method according to any one of the preceding claims, wherein the target binding portion, which is anchored to the magnetically movable particle, is also positioned on or inside the assay chamber at the first position.

15. below: The above method is carried out in a single liquid; The aforementioned method does not involve the use of a washing step; The method does not involve contacting the sample at one or more fluid interfaces; and The assay chamber is a single-fluid chamber. The method according to any one of the prior claims, wherein at least one or all of the above.

16. The method according to any one of the preceding claims, wherein the complex comes into contact with the catalytic substrate and, optionally, a mediator if present, only in the event of activation or generation of the magnetic field and movement of the complex to the second position.

17. The method according to any one of the preceding claims, wherein the first and second positions are at least partially or completely covered by the sample after the introduction of the sample into the assay chamber.

18. The method according to any one of the preceding claims, wherein the target binding portion and the target capture portion each independently comprise an amino acid and / or nucleic acid.

19. The method according to any one of the preceding claims, wherein the target binding portion and the target capture portion each independently include one or more selected from the group consisting of proteins, DNA, and RNA.

20. The method according to any one of the preceding claims, wherein the target binding portion and the target capture portion each independently comprise one or more selected from the group consisting of antibodies, antigen-binding antibody fragments, and antibody mimetic bodies.

21. This is a test strip: Assay chamber and The assay chamber and the sample inlet having fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, a catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is positioned between the first and second positions such that the introduction of the sample into the assay chamber through the sample inlet causes the sample to move or flow across the first position in a direction away from the second position. Test strip.

22. It is a system: (a) A test strip: Assay chamber and The assay chamber and the sample inlet having fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, a catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is positioned between the first and second positions such that the introduction of the sample into the assay chamber through the sample inlet causes the sample to move or flow across the first position in a direction away from the second position. Test strip and, (b) A target binding portion anchored to a magnetically movable particle, (c) A magnetic field generator configured to activate or generate a magnetic field Includes, The magnetically movable particle is configured to move to the second position when the magnetic field is activated or generated. system.

23. The first position mentioned above is: (i) a reagent zone or including a reagent zone; (ii) located at or adjacent to the first end of the assay chamber; and / or (iii) further comprising the target binding portion anchored to the magnetically movable particle, The test strip according to claim 21 or the system according to claim 22.

24. The second position mentioned above is: (i) a detection zone or containing a detection zone; (ii) Displaced at or near the second end of the assay chamber, A test strip according to claim 21 or 23, or a system according to claim 22 or 23.

25. The test strip according to any one of claims 21 or 23-24, or the system according to any one of claims 22-24, wherein the assay chamber is a single fluid chamber.

26. A test strip according to any one of claims 21 or 23-25, or a system according to any one of claims 22-25, further comprising a detector, optionally the detector comprising a working electrode positioned at a third location, the third location being at least close to the second location and away from the first location.

27. A test strip according to any one of claims 21 or 23-26, or a system according to any one of claims 22-26, wherein the catalyst is a redox catalyst, and the test strip further comprises a redox mediator positioned at the second position.

28. The test strip or system according to claim 27, wherein both the redox mediator and the redox catalyst substrate are placed on the working electrode.

29. The test strip according to claim 21 or any one of claims 23 to 28, or the system according to any one of claims 22 to 28, wherein the introduction of the sample into the assay chamber through the sample inlet also causes the sample to move or flow across the second position in a direction away from the first position, the sample inlet is positioned between the first and second positions.

30. It is a kit of parts, and as separate components: (a) A test strip: Assay chamber and The assay chamber and the sample inlet having fluid communication, In the first position, a target capture portion conjugated to the catalyst is located on or inside the assay chamber, In the second position, a catalyst substrate and are placed on or inside the assay chamber. Includes, The sample inlet is located between the first and second positions. Test strip and, (b) A target binding portion anchored to a magnetically movable particle, (c) an electromagnet, wherein the activation of the electromagnet can be arranged to produce a magnetic field capable of moving a magnetically movable particle to the second position, or a permanent magnet, wherein the permanent magnet can be arranged to generate a magnetic field capable of moving a magnetically movable particle to the second position. A kit of parts, including...

31. The kit of parts according to claim 30, comprising a test strip as defined in any one of claims 21 or 23 to 29.

32. A kit of parts according to claim 31: (i) at least one additional electrode; (ii) Sensors such as potentiostats; and (iii) One or more electronic connections capable of completing the circuit between the electrode and the sensor A kit of parts that includes one or more of the selected items.

33. Use of a strip according to claim 21 or any one of claims 23-29, a system according to any one of claims 22-29, or a kit according to any one of claims 30-32 for detecting the presence or absence of a target in a sample according to the method described in any one of claims 1-20, optionally.

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