Method and device for detecting a concentration of at least one analyte in a sample

Abstract

The invention relates to a method for detecting a concentration of an analyte (10) in a sample (20), the method comprising: providing a device (100) having a channel (40) with a substrate (42), the substrate (42) having surface binder elements (44) and / or a surface test analyte (47); mixing assaying elements (30) into the sample (20), the assaying elements (30) having capture binder elements (35) and / or the assaying elements (30) having an element test analyte (38); flowing the sample (20) through the channel (40); moving the assaying elements (30) to the substrate (42); moving the assaying elements (30) along / on the substrate (42) and changing the velocity of at least one assaying element (30) by an interaction comprising one or more of: an interaction between at least one surface binder element (44), the analyte (10) and at least one capture binder element (35); an interaction between the surface test analyte (47) and at least one of the capture binder elements (35); and / or an interaction between the element test analyte (38) of said at least one assaying element (30) and at least one of the surface binder elements (44); and determining a concentration of the analyte (10) by determining a change of velocity of the assaying elements (30) and / or by determining a depletion of the assaying elements (30), and / or determining binding kinetics by determining a change of velocity of the assaying elements (30). The invention further relates to a device (100).

Description

[0001] Method and device for detecting a concentration of at least one analyte in a sample

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for detecting a concentration of at least one analyte in a sample, and to a device for detecting a concentration of at least one analyte in a sample.

[0004] BACKGROUND OF THE INVENTION

[0005] In clinical chemistry, the detection and quantification of extracellular vesicles (EVs) as well as other vesicular analytes are challenging tasks. Manual workload, missing standardization, ultracentrifugation and large capital invest for central laboratory equipment are required to perform these assays. However, these assays cannot be performed with unprocessed patient samples, e.g., whole blood. They usually employ expensive signal readout devices based on optical labeling, such as for fluorescence flow cytometry, and, due to the dependence on workflow breaking centrifugation, cannot be integrated into point-of-care applicable workflows. Additionally, the typical dynamic concentration range of reported assays is in the order of 2-3 concentration log scales, which is insufficient to cover multiple vesicular biomarkers for multiplexed analysis at the same time.

[0006] Such assays may be classified as heterogeneous, homogeneous or competitive assays.

[0007] Heterogeneous assays are a type of biochemical analysis that involves the separation of unbound components from the bound analyte of interest. In these assays, the target molecule, such as a protein or nucleic acid, is immobilized on a solid support, such as microbeads or a microplate well, while the unbound components are washed away. The detection of the bound analyte is achieved by using a labeled probe, such as an antibody or a fluorescent molecule, which specifically binds to the immobilized target. This binding event can be quantified through various detection methods, such as fluorescence, luminescence, or absorbance measurements. These workflows always require a sequence with washing steps.

[0008] Homogeneous assays do not require a separation step to detect the target analyte. These assays are conducted in a homogeneous solution without the need for washing or removing unbound components. Instead, the detection signal is generated through specific interactions between the target analyte and two antibodies in close distance for a fluorescence resonance energy transfer (FRET) readout. However, sufficient optical transparency is required such that no distortion occurs of the FRET effect.

[0009] Competitive assays are a type of biochemical analysis that involve the competition between a labeled analyte and an unlabeled analyte for binding to a limited number of binding sites on a capture molecule. In these assays, the analyte of interest is typically labeled with a detectable marker, such as a fluorescent molecule or an enzyme. The labeled analyte competes with an unlabeled analyte in the sample for binding to the capture molecule. The amount of labeled analyte bound to the capture molecule is inversely proportional to the concentration of the unlabeled analyte present in the sample. By measuring the signal generated by the labeled analyte, the concentration of the unlabeled analyte can be quantified.

[0010] A method and device for detecting a concentration of an analyte is disclosed in WO 2006 / 134546 A2. The method of WO 2006 / 134546 A2 is a competitive assay. There, magnetic capture beads may have binder elements to bind to analytes in a sample. Further, the analytes bound by the capture beads may further bind to surface binder elements of a substrate.

[0011] Known immunoassays for vesicular analytes require a labelling step and need multiple antibodies per analyte for binding at different sites to discriminate from unspecific binding and background effects. Further, such immunoassays are usually not qualified for Point-of- Care-Testing (POCT). Additionally, known immunoassays cannot analyze unprocessed patient samples with low optical transparency, require washing steps before analysis, do not cover multiple biomarkers (e.g., vesicles, protein, nucleic acid, cells) on a single read-out platform and employ expensive, fluorescence labelling and complex readout systems to discriminate from complex matrix effects. Moreover, known immunoassays do not allow for multiplexing without sensor arrays or serialized workflow solutions, thereby driving consumable costs.

[0012] SUMMARY OF THE INVENTION

[0013] The drawbacks of the known assays are overcome by a method according to claim 1 and by a device according to claim 28. The dependent claims relate to particularly favorable embodiments of the invention.

[0014] A first aspect of the invention relates to a method for detecting a concentration of an analyte in a sample, the method comprising: providing a device having a channel, the channel having a substrate, the substrate having surface binder elements configured to bind to the analyte in the sample and / or the substrate having a surface test analyte; mixing assaying elements, preferably magnetic assaying elements, into the sample, the assaying elements having capture binder elements configured to bind to the analyte in the sample and / or the assaying elements having an element test analyte configured to bind to the surface binder elements; flowing the sample through the channel; moving the assaying elements to the substrate with an external force, preferably with a magnetic force; moving the assaying elements along / on the substrate and changing the velocity of at least one assaying element by an interaction, wherein the interaction comprises one or more of:

[0015] (i) an interaction, preferably a binding interaction, between at least one surface binder element of the substrate, the analyte and at least one capture binder element of said at least one assaying element;

[0016] (ii) an interaction, preferably a binding interaction, between the surface test analyte of the substrate and at least one of the capture binder elements of said at least one assaying element; and / or

[0017] (iii) an interaction, preferably a binding interaction, between the element test analyte of said at least one assaying element and at least one of the surface binder elements of the substrate; and determining a concentration of the analyte using a sensor unit by determining a change of velocity of the assaying elements along the channel and / or by determining a depletion of the assaying elements along the channel, preferably by differential counting of the assaying elements, and / or determining binding kinetics, preferably binding constants, between the analyte and the capture binder elements and / or between the analyte and the surface binder elements, wherein the binding kinetics are determined using the sensor unit by determining the change of velocity of the assaying elements along the channel.

[0018] By moving the assaying elements to the substrate and moving the assaying elements along / on the substrate, the method allows to differentiate assaying elements bound to the analyte from assaying elements not bound to the analyte. Thus, the analyte concentration in the sample may be determined. The method further allows for non-optical determination of the concentration, so that a preprocessing may not be necessary. The concentration of analyte in opaque samples may be determined. Moreover, the concentration may be determined over a large range. Further, the present invention allows for multiplexing, and / or heterogeneous assays, such that analytes of different type and / or kind and / or class in the sample may be analyzed, e.g., proteins, vesicles / vesicular material, cells / cellular material, chemicals, DNA, RNA and / or antibodies (serology).

[0019] Note that, in contrast to this, in the above mentioned WO 2006 / 134546 A2, the sample is not flown through a channel. Further, WO 2006 / 134546 A2 does not disclose an external force, e.g., a magnetic force, for moving assaying elements to the substrate, nor discloses moving of the assaying elements along / on the substrate, nor determining the concentration of the analyte by determining a change and / or a decrease of velocity of the assaying elements along the channel, and / or by determining a depletion of the assaying elements along the channel. Rather, WO 2006 / 134546 A2 determines the concentration by relating a binding rate of the analyte to the substrate to changes in a magnetic field which are registered when the magnetic (capture) bead with the analyte binds to surface binder elements.

[0020] In the invention, the sample may be or may comprise a liquid sample. The sample may be or may comprise blood and / or urine. The analyte may be or may comprise a vesicle, a vesicle structure, one or more exosomes, mitochondria, and the like. In some embodiments, the sample may comprise or may contain more than one kind and / or type of analyte, at least one of which maybe of interest for detecting its concentration. In other words, one or more of the kinds and / or types of analytes may be analytes for which the concentration in the sample is to be determined and / or detected. The analyte may comprise a ligand, e.g., an antigen. Different types and / or kinds of analyte may have different ligands and / or antigens.

[0021] The device may be or may comprise a device according to an aspect of the invention as further detailed below.

[0022] Moving the assaying elements along and / or on the substrate may comprise rolling the assaying elements along and / or on the substrate.

[0023] When the assaying elements are mixed in the sample, and when at least one (or all) of the assaying elements comprises one or more capture binder elements, analyte may bind to at least one of the assaying elements via a respective capture binder element such that at least one analyte bonded assaying element is formed. An assaying element to which an analyte is bound by a respective capture binder element maybe called an analyte bonded assaying element. A capture binder element and / or a surface binder element may be or may comprise one or more antibodies, respectively. A capture binder element may be or may comprise the same antibody as a surface binder element. An antibody may bind to the analyte, and / or to a ligand, e.g., an antigen, of the analyte. Different types and / or kinds of capture binder elements may comprise e.g., different antibodies, and / or may be configured to bind to different kinds and / or types of analyte. Different types and / or kinds of surface binder elements may comprise, e.g., different antibodies, and / or may be configured to bind to different kinds and / or types of analyte. In some embodiments, it maybe provided that the capture binder elements of at least one, multiple or all assaying elements are of the same type and / or kind as at least some, multiple or all of the surface binder elements. Alternatively or additionally, the capture binder elements of at least one, multiple or all assaying elements may match at least some, multiple or all of the surface binder elements, such that the respective capture binder elements and the surface binder elements form matched antibody pairs. In some embodiments, a capture binder element is or comprises a tetraspanin, e.g., CD81, CD63 and / or CD9. In some embodiments, a surface binder element is or comprises a tetraspanin, e.g., CD81, CD63 and / or CD9. In some embodiments, a capture binder element is or comprises an anti-Her2neu antibody and / or an anti-IL6 antibody. In some embodiments, an analyte is or comprises Her2neu+ EV, IL6, and / or biotin. In some embodiments, a substrate binder element is or comprises an anti- Her2neu antibody, an anti-IL6 antibody, and / or avidin.

[0024] An assaying element having a capture binder element may be a capture element or may correspond to a capture element. An assaying element having a capture binder element may be called “capture element”. An assaying element having a capture binder element may capture an analyte.

[0025] An interaction between a surface binder element of the substrate, the analyte and at least one capture binder element may comprise a binding interaction between the analyte bound by the capture binder element and the surface binder element. The same analyte may be bound to both the surface binder element and the capture binder element. The analyte may connect or may link the surface binder element and the capture binder element. An interaction between a surface binder element of the substrate, the analyte and at least one capture binder element may comprise an interaction between the analyte and the the capture binder element, and another interaction between the analyte and the surface binder element.

[0026] An interaction between a surface binder element and the analyte bound by a surface binder element may comprise a binding interaction between the respective surface binder element and the analyte. The capture binder elements and / or the surface binder elements may be chosen such that they may bind to a specific, chosen and / or desired analyte. Particularly, if the sample comprises more than one type and / or kind of analyte, the capture binder elements and / or the surface binder elements may be chosen such as to bind to one of the analytes for which the concentration maybe determined and / or tested, and / or which shall be assayed.

[0027] An interaction between the surface test analyte and a capture binder element may comprise a binding interaction between the surface test analyte and the capture binder element.

[0028] An interaction between the element test analyte and a surface binder element may comprise a binding interaction between the element test analyte and the surface binder element.

[0029] Mixing the assaying elements into the sample may comprise or may be equal to introducing the assaying elements into the sample. In some embodiments, multiple or all assaying elements may be bond to analyte in the sample, e.g. depending on the concentration of analyte in the sample, the number of assaying elements, the kind and / or type of analyte, and / or the kind and / or type of the capture binder elements of the respective assaying elements.

[0030] The binding kinetics, e.g. binding constants, between the analyte and binder elements, e.g. the capture binder elements or the surface binder elements, can be determined by determining the change of velocity of the assaying elements along the channel. The larger the change of velocity and / or the depletion, the stronger the binding interaction and / or the binding rate. Thus, the method allows for measuring and / or determining binding interactions, e.g. binding constants, of different analytes. In some embodiments, the method maybe used to analyze, determine or tabulate the binding kinetics of analytes, e.g. analytes for which the binding kinetics are not known or only tentatively known. Further, the method may be used for checking, validating or verifying known binding kinetics.

[0031] The assaying elements maybe or may comprise beads. The beads maybe or comprise magnetic beads. The assaying elements and / or the beads be magnetic. The assaying elements and / or beads may be at least partially made of or comprise a magnetic material. In some embodiments, moving the magnetic assaying elements and / or the magnetic beads to the substrate comprises pulling or attracting the magnetic assaying elements and / or the magnetic beads to the substrate. Alternatively or additionally, moving the magnetic assaying elements and / or the magnetic beads to the substrate may comprise pushing the magnetic assaying elements and / or the magnetic beads to the substrate. The external force may be a magnetic force provided by a magnetic field. In some embodiments, the magnetic field is a static magnetic field. Alternatively, the magnetic field may be varied, e.g., over time and / or along the channel. In some embodiments, the external force comprises an acoustic force and / or gravity. In these cases, the assaying elements do not necessarily need to be magnetic. The external force may be configured to move the assaying elements towards the substrate. Additionally, assaying element movement from the external force may dominate over Brownian motion and movement by diffusion. Thus, the assaying elements may be reliably moved in a direction specified by the external force, e.g., reliably pulled or attracted to (a wall of) the channel. Because of the external force, the movement and / or attraction of the assaying elements towards (a wall of) the channel may be substantially directed, instead of, e.g., a random walk due to diffusion. Thereby, the path traveled by the assaying elements when moving towards (a wall of) the channel may be shorter, and / or at least shorter on average, than compared to the prior art as, e.g., of WO 2006 / 134546 A2. A contact between the assaying elements and the substrate, and / or a contact force, may be substantially due to the external force. In some embodiments, the external force is generated by an external force generator.

[0032] In some embodiments, multiple or all assaying elements are moved, pushed and / or pulled to the channel and / or the substrate. It may be preferred that all assaying elements are moved, pushed and / or pulled to the channel and / or the substrate. The external force may be configured suitable for moving all assaying elements to the channel and / or the substrate. When one or more assaying elements is / are moved to the channel and / or the substrate, the assaying element(s) may contact the channel and / or the substrate.

[0033] It may be provided that the assaying elements are detected by the sensor unit. In some embodiments, the assaying elements comprise or consist of a metal or an alloy. Alternatively or additionally, the assaying elements may comprise or may consist of glass or plastic. It may be provided that the assaying elements are coated or plated. For instance, the assaying elements may have a core comprising a first material, e.g., a suitable plastic, glass or metal, and may be coated or plated with another material, e.g., a suitable plastic, glass or metal. The plastic maybe or may comprise a polymer. Alternatively or additionally, the assaying elements may comprise or consist of a biological material. For instance, the assaying elements may have an inner material comprising a first material, e.g., suitable polymers, glass, oil, protein, vesicular material, cellular material or metal, and may have an outer material comprising a second material, e.g., suitable polymers, glass, oil, protein, vesicular material, cellular material or metal.

[0034] The assaying elements maybe or may comprise beads. For instance, the beads maybe metallic, and / or at least partially or fully made of a metal. It may be provided that the assaying elements are spherical, but their form is not necessarily limited thereto. In some embodiments, the assaying elements comprise or consist of cellular and / or vesicular material. Additionally or alternatively, the assaying elements may be or comprise stabilized droplets and / or cells. In some embodiments, the capture binder elements are or correspond to an outer surface of the assaying element, e.g. to a wall of a droplet and / or a cell. Alternatively or additionally, the capture binder elements may be or may correspond to the outer material of the assaying elements.

[0035] The flow through the channel, e.g., the flow with which the assaying elements and / or the sample is flown through the channel, may be a laminar flow. However, the flow is not necessarily limited thereto. Depending on, e.g., the dimensions of the channel, the flow velocity, the viscosity of the flow and / or of the sample, the smoothness of the surface of the channel, and / or other relevant parameters, the flow maybe at least partially turbulent or may undergo a turbulent transition.

[0036] In some embodiments, multiple or all assaying elements move along / on the substrate. When an assaying element moves along / on the substrate, it may roll along / on the substrate. Rolling on or rolling along the substrate does not necessarily mean that a respective assaying element rolls on / along the full length of the substrate. Rolling on or rolling along the substrate may mean that a respective assaying element rolls on / along some length of the substrate, which may be very short or minimal compared to a length of the substrate in some embodiments. Additionally or alternatively, rolling on or rolling along the substrate may be facilitated with, but not necessarily only with, the external force and / or the laminar flow. In some embodiments, the rolling is facilitated, supported, aided and / or caused by the external force. It may be provided that the movement and / or the speed of the assaying element, in particular the rolling on the substrate, depend(s) on the external force, e.g., on its amplitude, amount and / or power. Alternatively or additionally, the rolling may be facilitated, supported, aided and / or caused by the flow through the channel. It may be provided that the movement and / or the speed of the assaying element, in particular the rolling on the substrate, depend(s) on the flow, e.g., on its flow velocity, e.g., its mean flow velocity and / or flow rate. In some embodiments, the length along which the assaying element rolls or may roll on / along the substrate, and / or its traveling path on / along the substrate, depend(s) on the external force and / or on the flow, and / or is specified or determined by the external force and / or by the flow. The movement of an assaying element on or along the substrate may depend on the external force and / or on the flow, or may be specified or determined by the external force and / or by the flow. When rolling on or rolling along the substrate, an assaying element may at least partially contact the substrate. When rolling on or rolling along the substrate, the assaying element may rotate while at least partially contacting the substrate. When rolling on or rolling along the substrate, the assaying element maybe rotating while moving on / along the substrate. A rolling movement may consist of or may comprise a translational movement and a rotational movement. A rolling movement may be or may correspond to a superposition of a translational movement and a rotational movement. When an assaying element is rolling on or along the substrate, a sliding of the assaying element maybe negligible.

[0037] It may be provided that the velocity of at least one, multiple or all of the assaying elements is changed and / or reduced when moving or rolling on or along the substrate. Changing the velocity of the assaying elements may comprise decreasing a velocity of at least one, of multiple or of all assaying elements. It may be provided that the velocity of assaying elements, which are not bound to analyte, is not changed and / or reduced, or at least not substantially changed and / or reduced, when moving or rolling on or along the substrate.

[0038] Changing and / or reducing the velocity may include that an assaying element may be stuck or immobilized on the substrate. Changing and / or reducing the velocity of at least one of the assaying elements may comprise immobilizing said at least one assaying element, e.g. by bonding the surface binder element to the analyte bound by the capture binder element. Alternatively or additionally, changing and / or reducing the velocity of at least one of the assaying elements may comprise immobilizing said at least one assaying element e.g. by bonding the surface test analyte to the capture binder element of the respective assaying element. Alternatively or additionally, changing and / or reducing the velocity of at least one of the assaying elements may comprise immobilizing said at least one assaying element e.g. by bonding the element test analyte of the respective assaying element to a surface binder element of the substrate.

[0039] The element test analyte maybe of the same kind and / or type as the analyte in the sample. The surface test analyte maybe of the same kind and / or type as the analyte in the sample.

[0040] The sensor unit may comprise at least one sensing element. In a preferred embodiment, determining the concentration of the analyte comprises determining a time-of-roll between a first sensing element and a second sensing element. In some embodiments, the first sensing element is arranged upstream of the substrate. The second sensing element may be arranged downstream of the substrate and / or downstream of the first sensing element. It may be provided that the first sensing element is or comprises a magnetic field sensor, and / or a magnetic sensor half bridge. The second sensing element maybe or may comprise a magnetic field sensor, and / or a magnetic sensor half bridge.

[0041] The time-of-roll may be defined as the time required for an assaying element, or a group of assaying elements, to move or roll along / on the substrate between two defined points or regions on and / or upstream and / or downstream of the substrate, e.g., between two sensing elements (e.g. between the first sensing element and the second sensing element), and / or between entering and leaving a region on the substrate.

[0042] The sensor unit and / or the sensing elements maybe or may comprise a device configured for impedance sensing and / or magnetoresistive sensing. The sensor unit and / or the sensing elements may detect and / or may register an assaying element passing over, along and / or through the sensor unit and / or sensing elements. It may be provided that the sensor unit and / or the sensing elements measure / measures and / or determine / determines the size of an assaying element. Alternatively or additionally, the sensor unit and / or the sensing elements maybe or may comprise a coulter counter.

[0043] In a preferred embodiment, the external force is a magnetic force generated by a static magnetic field, and the sensor unit may comprise at least one sensor configured to detect and / or to determine a magnetic field. When an assaying element, particularly a magnetic assaying element and / or a magnetic bead, passes the sensor unit, it may be provided that the magnetic field as detected and / or determined by the sensor unit changes, such that the (e.g., magnetic) assaying element maybe detected and / or its velocity may be determined.

[0044] In a preferred embodiment, the sample is flown or driven by a syringe, a syringe pump, and / or a metering pump. The syringe, the syringe pump and / or the metering pump maybe fluidically connected to the channel, e.g., at or to an inlet of the channel. The syringe, the syringe pump and / or the metering pump may exert a force onto the sample such that the velocity of the sample is substantially constant when flowing into or through the channel. Thus, a velocity of the assaying elements upstream and / or downstream of the substrate, and / or at the inlet of the channel, may be known. The velocity of the assaying elements may be determined, detected and / or measured by the sensor unit downstream of the substrate. Then, the change and / or decrease of velocity of the assaying elements may be determined by comparing the velocity as imposed by the syringe, the syringe pump and / or the metering pump, e.g., at the inlet, and the velocity as determined by the sensor unit. In a preferred embodiment, determining a depletion of the assaying elements comprises differential counting of the assaying elements, or is carried out by differential counting of the assaying elements. Differential counting maybe carried out by determining a difference of the number of assaying elements counted and / or registered by two sensing elements. Additionally or alternatively, differential counting may be carried out by determining a difference of the number of assaying elements counted and / or registered by one sensing element and a known assaying element count before the sample with the assaying elements reaches the substrate. A depletion of assaying elements may occur when one or more assaying elements are stuck or immobilized on the substrate. In some embodiments, a single sensing element is provided and / or is sufficient for carrying out the differential counting, as the number of assaying elements mixed into the sample may be known. Then, the difference between assaying elements mixed into the sample and assaying elements registered by the sensing element may be determined, such that the number of assaying elements stuck or immobilized on the substrate upstream of the sensing element maybe determined.

[0045] Thus, in some embodiments a non-optical determination of the analyte concentration is possible. Hence, the method and the device according to the invention allow for assays where the sample does not need to be processed before carrying out the method. Further, the sample maybe opaque.

[0046] In a preferred embodiment, determining the change of velocity comprises determining a first distribution function of the assaying elements at a first position in the channel and a second distribution function of the assaying elements at a second position in the channel. Alternatively or additionally, determining the time-of-roll may comprise determining a first distribution function of the assaying elements at a first position in the channel and a second distribution function of the assaying elements at a second position in the channel.

[0047] The first distribution function may be a distribution function of the time difference between the timepoint of a respective assaying element crossing the first position and a first reference timepoint. The second distribution function may be a distribution function of the time difference between the timepoint of a respective assaying element crossing the second position and a second reference timepoint.

[0048] A distribution function may be or may comprise a histogram, a density function and / or a cumulative function. A density function may be or may correspond to a normalized histogram. For instance, a density function maybe or may comprise a histogram normalized such that an integral or a sum over the histogram is equal to unity. A density function may be or may correspond to a probability density function.

[0049] A cumulative function may be or may comprise a cumulative sum and / or a running sum. A cumulative function may be or may comprise a cumulative distribution function. In some embodiments, the cumulative function may be or may comprise an empirical cumulative distribution function (ecdf).

[0050] The distribution function may be determined and / or measured by the sensing unit, and / or by a sensing element. Alternatively or additionally, the distribution function may be determined optically, e.g. by using a camera device.

[0051] The first position may be a position of the first sensing element, and / or may coincide with the position of the first sensing element. In particular, the first position may correspond to the location of the first sensing element in the channel. The second position may be a position of the second sensing element, and / or may coincide with the position of the second sensing element. In particular, the second position may correspond to the location of the second sensing element in the channel.

[0052] In a preferred embodiment, the first distribution function comprises a density function or a histogram and the second distribution function comprises a density function or a histogram, and the change of velocity is determined by a time difference between a peak of the second distribution function and a peak of the first distribution function. The peak of the first distribution function may be or may correspond to a maximum value of the first distribution function. The peak of the second distribution function may be or may correspond to a maximum value of the second distribution function. Since in some embodiments the peak may be easily identified, the change of velocity (and / or the time of roll) may be easily determined.

[0053] In a preferred embodiment, the first distribution function comprises a cumulative function and the second distribution function comprises a cumulative function, and the change of velocity is determined by a time difference between an inflection point of the second distribution function and an inflection point of the first distribution function. In some embodiments, it may be easier to determine the cumulative function than a histogram or the corresponding density function. In some embodiments, the cumulative function may be determined more accurately than a corresponding density function. In a preferred embodiment, the change of velocity is determined by a distance metric of the second distribution function and the first distribution function. Alternatively or additionally, the change of velocity may be determined by a change of shape of the second distribution function compared to the first distribution function. In some embodiments, it may be easier and / or more accurate to determine a distance metric and / or a change of shape instead of calculating a time difference between peaks and / or inflection points. For instance, when a distribution has more than one peak and / or more than one inflection point, the distance metric and / or change of shape maybe more accurate and / or less arbitrary.

[0054] A distance metric maybe or may comprise a Kolmogorov-Smirnov test, a Wasserstein metric, a divergence metric, an entropy metric such as e.g. the Kullback-Leibler divergence, or some other suitable function such as e.g. but not limited to an f-divergence, the Jenson-Shannon divergence, the Bhattacharyya distance, or the like.

[0055] The change of shape may be determined by fitting the first distribution function and the second distribution function to the same candidate distribution function, and comparing the respective fitting parameters. The candidate distribution function may be a normal distribution function, a gamma distribution function, a lognormal distribution function or a Weibull distribution function, but is not limited thereto. In some embodiments, the distribution function may be a distribution function having support [o, x), where x is a positive number. In some embodiments, the distribution function may be a distribution function having support [o, oo).

[0056] In a preferred embodiment, the first reference timepoint is equal to the second reference timepoint. Thereby, the time difference can be accurately determined, since both distributions are functions of time relative to the same reference point or zero point.

[0057] In a preferred embodiment, the first reference timepoint is equal to the timepoint of a first crossing of the first position by an assaying element. Alternatively or additionally, the second reference timepoint may be equal to the timepoint of a first crossing of the second position by an assaying element. Thereby, the first distribution function and the second distribution function maybe moved to the left such that they start from the origin. Thereby, determining a distance metric and / or a change of shape maybe facilitated and / or maybe more accurate. For instance, moving the first distribution function and / or the second distribution function to the left may lead to an increased overlap of the first and second distribution function. In a preferred embodiment, determining the concentration of the analyte comprises an optical detection. The optical detection may comprise capturing a movement and / or a position of the assaying elements spatially and / or temporally resolved in a field of view. By determining the position and / or the movement of assaying elements on the substrate, the time-of-roll and / or the differential counting maybe determined optically. The optical detection maybe carried out by one or more optical devices, e.g. by one or more cameras. Each optical device and / or camera may have a field of view. In some embodiments, more than one optical device and / or more than one camera is provided, such as to have a larger total field of view at sufficient resolution. The fields of view of at least two optical devices and / or cameras may at least partially overlap. In some embodiments, the fields of view of at least two optical devices and / or cameras don’t overlap. It may be provided that a first optical device and / or camera is arranged such that its field of view may cover a region upstream of the substrate, and a second optical device and / or camera is arranged such that its field of view covers a region downstream of the substrate. The optical detection may detect one or more of color, size, luminescence and granularity of the assaying elements. Thereby, in some embodiments, multiplexing is enabled.

[0058] In a preferred embodiment, the external force, e.g. a magnetic force provided by a magnetic field, and a flow velocity of the sample are adjusted such that at least one of the assaying elements moves, rolls and / or is rolled along / on the substrate. For instance, in some embodiments, the assaying elements, when moved towards the substrate, contact the channel and / or the substrate at substantially similar positions, or at least in a defined region on the channel and / or the substrate. Thereby, e.g., the length and / or the distance of the rolling on the substrate may be fixed, or at least may be specified more accurately. In some embodiments, the flow velocity is chosen such that the assaying elements are only slowed down when moving or rolling along / on the substrate, but not stuck or immobilized.

[0059] In a preferred embodiment, the external force, e.g., a magnetic force provided by a magnetic field, is adjusted such that the assaying elements are pushed away from the substrate and / or sorted. In some embodiments, the external force is adjusted such that the assaying elements are pushed away from the substrate and / or sorted, preferably after determining the concentration of the analyte. Thereby, the channel and / or the substrate may be prepared for another analysis or assay, and / or the assaying element used for further analysis with different methods. For instance, assaying elements stuck on the substrate may be released from the substrate, which assaying elements may then be used, e.g., for PCR (polymerase chain reaction). Alternatively or additionally, the temperature can be increased to minimize unspecific binding on the substrate or to achieve dissociation of the assaying elements. In particular, when the binding is a DNA-DNA, RNA-DNA or DNA-PNA binding, the strength of the binding may be varied and / or decreased by increasing the temperature. In some embodiments, the temperature may be increased such that it approaches a corresponding melting temperature of the binding or is larger than a corresponding melting temperature of the binding, e.g., a corresponding melting temperature of a respective DNA-DNA binding, RNA-DNA binding and / or DNA-PNA-binding.

[0060] In a preferred embodiment, in the channel a laminated flow is flown between the sample and the substrate such as to separate the sample from the substrate. The assaying elements may be moved by the external force, e.g., by a magnetic force generated by a magnetic field, from the sample through the laminated flow to the substrate. Viewed in a longitudinal section of the channel, a height of the laminated flow may be larger than the largest dimension, e.g., the diameter, of the assaying elements. In some embodiments, more than one laminated flow is flown over the substrate, wherein the laminated flows may be substantially layered above each other, e.g., in a direction perpendicular to the surface of the substrate. The laminated flow may prevent debris to obstruct or to hinder the moving or rolling of the assaying elements along / on the substrate. The laminated flow, e.g., may minimize interference from hematocrit. In some embodiments, the laminated flow maybe or may comprise a sheath flow.

[0061] In a preferred embodiment, at least one, at least two, multiple or all of the assaying elements has / have at least two different capture binder elements each. The different capture binder elements may be configured to bind to different analytes. Thus, the method allows for multiplexing. At least two, multiple or all of the assaying elements may have different sizes, different magnetic moments, different acoustic properties, different electrical properties, and / or different optical properties. It may be provided that the assaying elements may be distinguished or grouped according to their different sizes, different magnetic moments, different acoustic properties, different electrical properties, and / or different optical properties.

[0062] The optical property may comprise a color or a marker, such that e.g. an assaying element may be colored or marked assaying element, but is not necessarily limited thereto. The acoustic property may comprise a compressibility and / or a density, such as e.g. a compressibility and / or density of an assaying element, but is not necessarily limited thereto. The electric property may comprise an electrical impedance and / or an electrical charge, such as e.g. an electrical impedance and / or electrical charge of an assaying element, but is not necessarily limited thereto.

[0063] Alternatively or additionally, the different capture binder elements may be configured to bind to the same analyte. For instance, the analyte maybe or may comprise a vesicle having different proteins, such as e.g. in case of extracellular vesicles such as CD9, CD63 or CD81. These proteins may bind to different capture binder elements. Thereby, the binding of the analyte to the assaying element may be facilitated. Alternatively or additionally, different capture binder elements may comprise different antibodies, which may be configured to bind to different proteins of the same analyte.

[0064] In a preferred embodiment, at least two of the assaying elements have different sizes and different capture binder elements, wherein the different capture binder elements may be configured to bind to different analytes. Alternatively or additionally, at least two of the assaying elements may have different magnetic moments and different capture binder elements, wherein the different capture binder elements maybe configured to bind to different analytes. Alternatively or additionally, at least two of the assaying elements may have different optical properties and different capture binder elements, wherein the different capture binder elements may be configured to bind to different analytes. Alternatively or additionally, at least two of the assaying elements may have different acoustic properties and different capture binder elements, wherein the different capture binder elements may be configured to bind to different analytes. Alternatively or additionally, at least two of the assaying elements may have different electrical properties and different capture binder elements, wherein the different capture binder elements may be configured to bind to different analytes. In some embodiments, it may be provided that the assaying elements comprise at least two groups of assaying elements and the assaying elements of each group may have the same capture binder elements. At least two assaying elements, and / or the at least two groups of assaying elements may differ in at least one or multiple of size, color, luminescence, granularity and magnetic moment. Thus, the method allows for multiplexing.

[0065] In a preferred embodiment, the substrate has at least two different surface binder elements, wherein the different surface binder elements maybe configured to bind to different analytes. In some embodiments, it may be provided that the different surface binder elements are grouped into homogeneous groups, preferably separated by sensing elements. Thus, the method allows for multiplexing.

[0066] In some embodiments, the capture binder elements of at least one or all assaying elements are of the same type and / or kind as at least one or all surface binder elements.

[0067] The method allows for multiplexing, and / or heterogeneous assays. With the method, analytes of different type and / or kind and / or class in the sample may be analyzed, e.g., proteins, vesicles / vesicular material, cells / cellular material, chemicals, DNA, RNA, antibodies (serology), and / or the concentration of multiple analytes in the sample may be determined. Different assaying binder elements may differ in type and / or kind, e.g., may comprise or may be different antibodies. Different surface binder elements may differ in type and / or kind, e.g., may comprise or may be different antibodies. In some embodiments, different capture binder elements differ in number of capture binder elements arranged on respective assaying element surfaces.

[0068] It maybe provided that an orientation and / or a rolling direction of the assaying elements are / is varied when the assaying elements are moving or rolling on or along the substrate. Thus, a larger fraction of the surface of the assaying elements is coming into contact with the surface binder elements when moving or rolling along / on the substrate, facilitating the bonding of surface binder elements to analytes bound to capture binder elements of the rolling assaying elements. Thereby, the accuracy of the method may be improved, since e.g. assaying elements bound to analytes may be better differentiated from those not bound to analyte.

[0069] Alternatively or additionally, it may be provided that an orientation and / or a rolling direction of the assaying elements are / is varied when the assaying elements are moving or rolling downstream of the substrate or downstream of the sensing unit. For instance, the orientation and / or a rolling direction may be varied by a pattern. The pattern may be or may comprise a mechanic pattern. The mechanic pattern may guide the assaying elements by a mechanical interaction, e.g. by one or more guiding elements. Alternatively or additionally, the pattern may be or may comprise a magnetic pattern. The magnetic pattern may guide the assaying elements by a magnetic field and / or a magnetic force acting on the assaying elements.

[0070] Alternatively or additionally, the external force may vary an orientation and / or a rolling direction of the assaying elements. It may be provided that the external force may be controlled such that the orientation and / or the rolling direction of the assaying elements may change and / or may vary.

[0071] In a preferred embodiment, a second external force acts on the assaying elements when the assaying elements are moving downstream of the substrate and / or downstream of the sensing unit. The second external force may sort the assaying elements. In some embodiments, the assaying elements may be sorted or grouped according to their different properties, such as e.g. according to their different magnetic moments, different sizes, different acoustic properties, different electrical properties, and / or different optical properties. In some embodiments, the second external force may singulate at least one, multiple or all of the assaying elements. The second external force may be a second magnetic force, but is not necessarily limited thereto. The second external force may be different from the external force, e.g. may differ in amplitude, power and / or frequency. It may be provided that the second external force is controlled, and / or may vary, e.g. overtime and / or locally. Thereby, the sorting and / or singulation of the assaying elements may be controlled. Sorting and / or singulating the assaying elements maybe useful for re-using the device and / or the assaying elements.

[0072] In some embodiments, the second external force may vary an orientation and / or a rolling direction of the assaying elements when the assaying elements are moving or rolling on the substrate, or downstream of the substrate or downstream of the sensing unit.

[0073] A second aspect of the invention relates to a device for detecting a concentration of an analyte in a sample, the device comprising a channel, wherein the channel has a substrate, the substrate having surface binder elements and / or the substrate having a surface test analyte. The channel is configured such that a sample comprising an analyte and assaying elements, the assaying elements having capture binder elements and / or having element test analyte, can flow therethrough. The device is configured such that, when the sample that comprises the analyte and assaying elements flows through the channel, an external force moves the assaying elements to the substrate. The channel is configured such that, when the sample flows through the channel, the assaying elements move along / on the substrate. The device is further configured such that when assaying elements move along / on the substrate, the velocity of at least one assaying element is changed by an interaction, the interaction comprising one or more of:

[0074] (i) an interaction, preferably a binding interaction, between at least one surface binder element of the substrate, the analyte and at least one capture binder element of said at least one assaying element;

[0075] (ii) an interaction, preferably a binding interaction, between the surface test analyte of the substrate and at least one of the capture binder elements of said at least one assaying element; and / or

[0076] (iii) an interaction, preferably a binding interaction, between the test analyte of said at least one assaying element and at least one of the surface binder elements of the substrate.

[0077] The device further comprises a sensor unit configured to determine a change of velocity of the assaying elements along the channel, and / or to determine a depletion of the assaying elements along the channel, preferably by differential counting of the assaying elements. Alternatively or additionally, the device is configured to determine binding kinetics, preferably binding constants, between the analyte and the capture binder elements and / or between the analyte and the surface binder elements. The device may be further configured to determine the concentration of the analyte, and / or the binding kinetics, by the determined change of velocity.

[0078] It may be provided that when the assaying elements are flown through the channel, multiple or all of the assaying elements roll along / on the substrate. “Moving” an assaying element along / on the substrate may comprise rolling the assaying element along / on the substrate.

[0079] The device may be configured to carry out a method according to the first aspect of the invention. The device may be used to determine an analyte concentration in a sample. The device may be used to determine binding kinetics. The device may have one, multiple or all advantages of the inventive method as described herein.

[0080] In some embodiments, the device may comprise the sample. In some other embodiments, the device may not comprise the sample. In some embodiments, the device may comprise the assaying elements. In some other embodiments, the device may not comprise the assaying elements.

[0081] In some embodiments, the device comprises an external force generating device which is configured to generate the external force. The external force generating device maybe or may comprise a magnetic field generating device configured to generate a magnetic field, such that the external force maybe a magnetic force. The external force, e.g., the magnetic force, maybe configured to move the assaying elements to the substrate. The assaying elements may be or may comprise magnetic beads, and / or may be magnetic.

[0082] The sensor unit may comprise at least one sensing element. In a preferred embodiment, the sensor unit comprises a first sensing element and a second sensing element, wherein the sensor unit may be configured to determine the concentration of the analyte by determining a time- of-roll between the first sensing element and the second sensing element. In some embodiments, the first sensing element is arranged upstream of the substrate. The second sensing element may be arranged downstream of the first sensing element and / or the substrate. The first sensing element may be or may comprise a magnetic field sensor, and / or a magnetic sensor half bridge. The second sensing element maybe or may comprise a magnetic field sensor, and / or a magnetic sensor half bridge.

[0083] In a preferred embodiment, the external force generating device is a magnetic field generating device, e.g., a permanent magnet or the like. The external force may be a magnetic force. The magnetic field generating device may generate a static magnetic field. The sensor unit may comprise at least one sensor configured to detect and / or to determine a magnetic field and / or a magnetic field change. When an assaying element, particularly a magnetic assaying element and / or magnetic bead, passes the sensor unit, it may be provided that the magnetic field as detected and / or determined by the sensor unit changes, such that the (e.g., magnetic) assaying element maybe detected and / or its velocity maybe determined.

[0084] In a preferred embodiment, the device and / or the wherein the sensing unit is configured to determine the change of velocity by determining a first distribution function of the assaying elements at a first position in the channel and a second distribution function of the assaying elements at a second position in the channel. The first distribution function may be a distribution function of the time difference between the timepoint of a respective assaying element crossing the first position and a first reference timepoint. The second distribution function may be a distribution function of the time difference between the timepoint of a respective assaying element crossing the second position and a second reference timepoint.

[0085] The first position may be position of the first sensing element, e.g. a position or location of the first sensing element in the channel. The second position may be a position of the second sensing element, e.g. a position or location of the first sensing element in the channel.

[0086] In a preferred embodiment, the first distribution function comprises a density function or a histogram and the second distribution function comprises a density function or a histogram. The change of velocity may be determined by a time difference between a peak, preferably a maximum value, of the second distribution function and a peak, preferably a maximum value, of the first distribution function.

[0087] In a preferred embodiment, the first distribution function comprises a cumulative function and wherein the second distribution function comprises a cumulative function. The change of velocity may be determined by a time difference between an inflection point of the second distribution function and an inflection point of the first distribution function.

[0088] In a preferred embodiment, the change of velocity is determined by a distance metric of the second distribution function and the first distribution function, and / or by a change of shape of the second distribution function compared to the first distribution function.

[0089] In a preferred embodiment, the sensor unit is or comprises an optical device. The optical device may be configured to determine the concentration of the analyte by capturing a movement and / or a position of the assaying elements spatially and / or temporally resolved in a field of view. The optical device may comprise one or more cameras. Each optical device and / or camera may have a field of view. In some embodiments, more than one optical device and / or more than one camera is provided, such as to have a larger total field of view at sufficient resolution. The fields of view of at least two optical devices and / or cameras may at least partially overlap. In some embodiments, the fields of view of at least two optical devices and / or cameras don’t overlap. It maybe provided that a first optical device and / or camera is arranged upstream of the substrate and / or such that its field of view may cover a region upstream of the substrate, and a second optical device and / or camera is arranged downstream of the substrate and / or such that its field of view may cover a region downstream of the substrate. The optical device may be configured to detect one or more of color, size, luminescence and granularity of the assaying elements. Thereby, in some embodiments, multiplexing is enabled.

[0090] In a preferred embodiment, the substrate has at least two different surface binder elements, wherein the different surface binder elements maybe configured to bind to different analytes. It may be provided that the different surface binder elements are grouped into homogeneous groups separated by sensing elements.

[0091] Alternatively or additionally, the substrate may have at least two different surface binder elements, wherein the different surface binder elements may be configured to bind to the same analyte. For instance, the analyte may be or may comprise a vesicle having different proteins, such as e.g. in case of extracellular vesicles such as CD9, CD63 or CD81. These proteins may bind to different surface binder elements. Thereby, the binding of the analyte to the substrate may be facilitated. Alternatively or additionally, different substrate binder elements may comprise different antibodies, which may be configured to bind to different proteins of the same analyte.

[0092] In a preferred embodiment, the device is configured to vary an orientation and / or a rolling direction of the assaying elements when and / or before the assaying elements are rolling along / on the substrate or downstream of the substrate or downstream of the sensor unit.

[0093] In a preferred embodiment, the channel and / or the substrate have / has a mechanical and / or magnetic pattern. The mechanical and / or magnetic pattern may be or may comprise a mechanical and / or magnetic chevron pattern. The mechanical and / or magnetic pattern may be or may comprise a mechanical and / or a magnetic structure. The mechanical and / or magnetic pattern maybe configured to vary an orientation and / or a moving or rolling direction of the assaying elements when the assaying elements are moving or rolling along / on the substrate. Thus, a likelihood or chance of analyte bound to capture binder elements for contacting and / or interacting with surface binder elements is increased.

[0094] In a preferred embodiment, the channel may be tapered. When the channel is tapered, its cross-section may vary along its length. When the cross-section increases, the flow velocity may decrease. The channel may be tapered such that its cross-section increases in flow direction. When the channel is tapered, a driving force imparted by the flow on the assaying elements may decrease, and the velocity of an assaying element may be further decreased. In some embodiments, the likelihood of an assaying element to be immobilized or stuck may be increased. In some embodiments, the assaying elements are guided and / or directed to the sensing unit by the tapering of the channel.

[0095] In a preferred embodiment, the device comprises a second external force generating device which is configured to generate a second external force. The second external force generating device is / may be arranged downstream of the sensor unit. Alternatively or additionally, the second external force generating device may be arranged at a position along the substrate. In some embodiments, the second external force generating device is or comprises a second magnetic field generating device and the second external force is a magnetic force. The second magnetic field generating device may be or may comprise a permanent magnet. The second external force generating device may be configured to generate the second external force varying in amplitude and / or spatial direction. The second external force may vaiy over time. It maybe provided that the second external force generating device is a sorting unit for sorting assaying elements. For instance, the second external force may be used to sort assaying elements, e.g., sorting the assaying elements downstream of the sensor unit. By sorting, the assaying elements may be sorted into different groups. Assaying elements of different groups may be used for further analysis or diagnostics.

[0096] The second external force may be a second magnetic force, but is not necessarily limited thereto. The second external force may be different from the external force, e.g. may differ in amplitude, power and / or frequency. It may be provided that the second external force is controlled, and / or may vaiy, e.g. overtime and / or locally.

[0097] Alternatively or additionally, the second external force generating device and / or the second external force may be configured to vary an orientation and / or a rolling direction of the assaying elements. Alternatively or additionally, the second external force generating device and / or the second external force may be configured to singulate one or more of the assaying elements. The second external force generating device may comprise the external force generating device. Alternatively, the external force generating device may comprise the second external force generating device.

[0098] SHORT DESCRIPTION OF THE FIGURES

[0099] The invention is further disclosed and detailed in view of the following figures:

[0100] Fig. 1: a schematic view of a device according to an embodiment of the invention;

[0101] Fig. 2: another schematic view of a device according to an embodiment of the invention;

[0102] Fig. 3: a schematic view of assaying elements with capture binder elements;

[0103] Fig. 4: a schematic view of an assaying element and a substrate according to another embodiment of the invention;

[0104] Fig. 5: a schematic view of an assaying element and a substrate according to another embodiment of the invention;

[0105] Fig. 6: a flow diagram showing a method according to an embodiment of the invention;

[0106] Fig. 7: another flow diagram showing a method according to an embodiment of the invention;

[0107] Fig. 8: a schematic view of a device according to an embodiment of the invention, at a first time ti;

[0108] Fig. 9: the embodiment of Fig. 8, at a second time t2;

[0109] Fig. 10: an exemplary output of the sensor unit of the embodiment of Figs. 8 and 9;

[0110] Fig. 11: a comparison of the embodiment of Figs. 8 to 10 to conventional assays;

[0111] Fig. 12: an exemplary output of a sensor unit for a single assaying element;

[0112] Fig. 13: an example of distribution functions;

[0113] Fig. 14: another example of distribution functions;

[0114] Fig. 15: another example of distribution functions;

[0115] Fig. 16: an exemplary curve of a shape factor of a distribution function as function of the analyte concentration.

[0116] Fig. 17: a schematic view of a device according to another embodiment of the invention;

[0117] Fig. 18: a comparison of the embodiment of Fig. 17 to conventional assays;

[0118] Fig. 19: a schematic view of a device according to yet another embodiment of the invention;

[0119] Fig. 20: an exemplary output of the sensor unit of the embodiment of Fig. 19;

[0120] Fig. 21: a comparison of the embodiment of Figs. 19 and 20 to conventional assays; Fig. 22: a schematic view of a device according to an embodiment of the invention with sheath flow;

[0121] Fig. 23: a schematic view of a device according to an embodiment of the invention with multiplexing;

[0122] Fig. 24: a schematic view of a device according to another embodiment of the invention with multiplexing;

[0123] Fig. 25: a schematic view of a device according to yet another embodiment of the invention with multiplexing;

[0124] Fig. 26: a schematic view of a device according to an embodiment of the invention, with a pattern for varying a direction and / or an orientation of the assaying elements;

[0125] Fig. 27: a schematic view of a device according to another embodiment of the invention, with a pattern for varying a direction and / or an orientation of the assaying elements;

[0126] Fig. 28: a schematic view of a device according to yet another embodiment of the invention, with a pattern for varying a direction and / or an orientation of the assaying elements;

[0127] Fig. 29: a schematic view of a device according to an embodiment of the invention, with a tapered channel; and

[0128] Fig. 30a: exemplary concentrations of analyte, as determined by methods according to the invention as function of change of velocity of the assaying elements;

[0129] Fig. 30b: exemplary concentrations of analyte, as determined by methods according to the invention as function of the ratio the ratio of immobilized and / or stuck assaying elements;

[0130] Fig. 31a: a comparison of exemplary concentrations of analyte, as determined by different methods according to the invention as function of the ratio of immobilized and / or stuck assaying elements;

[0131] Fig. 31b: a comparison of exemplary concentrations of analyte, as determined by different methods according to the invention as function of the ratio of immobilized and / or stuck assaying elements; and

[0132] Fig. 32: an exemplary curve of the velocity decrease as function of the analyte concentration.

[0133] DESCRIPTION OF PREFERRED EMBODIMENTS

[0134] It is to be understood that both the foregoing general description and the following description are exemplary and explanatory only and are not restrictive of the methods and devices described herein. In this application, the use of the singular may include the plural unless specifically state otherwise. Also, the use of "or" means "and / or" where applicable or unless stated otherwise. Those of ordinary skill in the art will realize that the following description is illustrative only and is not intended to be in any way limiting. Other embodiments will readily suggest themselves to such skilled persons having the benefit of this disclosure. Reference will now be made in detail to various implementations of the example embodiments as illustrated in the accompanying drawings. Same reference numerals may denote same objects in the figures.

[0135] Figs. 1 and 2 show exemplary embodiments of a device too according to the invention. The device too may be considered a test device for carrying out an assay. The device too may be used to determine a concentration of an analyte to in a sample 20. The device too may be configured to cariy out a method according to the invention.

[0136] The sample 20 may comprise a liquid or may be a liquid sample 20. For example, the sample 20 may be or may comprise blood or urine. The sample 20 may contain one or more types and / or kinds of analyte 10 to be analyzed. The analyte 10 maybe or may comprise Interleukin 6 (IL-6). The analyte 10 may be or may comprise a vesicle. The analyte 10 may be or may comprise a vesical structure, one or more exosomes, mitochondria, and the like. However, other and / or different analytes, and / or types and / or kinds of analyte 10, may be analyzed and / or detected.

[0137] The device too comprises a channel 40. A sample 20 with analyte 10 and with at least one analyte bonded assaying element 30, and / or with assaying elements 30, may be flown through the channel 40. In particular, a flow 110 of the sample 20 with the assaying elements 30 mixed therein may be flown through the channel 40. The flow 110 may be laminar. The assaying elements 30 may be magnetic. The assaying elements 30 may be or may comprise magnetic beads.

[0138] The channel 40 may be or may comprise a microfluidic channel. The channel 40 may have an inlet and an outlet for flowing the sample 20 through the channel 40. The channel 40 may have a cross-diameter in the z-y-plane (e.g., Figs. 1 and 2), through which cross-diameter the flow 110 may flow. The cross-diameter may be substantially, rectangular, oval, circular, or have any other suitable shape. For instance, a flow direction through the channel 40 may be in x- direction. In some embodiments, the channel 40 maybe arranged and / or maybe oriented such that its length-wise extension is substantially perpendicular to gravity. For instance, gravity may act in negative z-direction. The channel 40 has a substrate 42. The substrate 42 may be arranged at an inner side wall of the channel 40, or may be part of an inner side wall of the channel 40. The substrate 42 may be arranged at a bottom of the channel 40. The substrate 42 may extend in a flow direction through the channel 40 (e.g., in x-direction in Figs. 1 and 2). The substrate 42 may be or may comprise a plane or a surface, and / or may extend in a direction perpendicular to its lengthwise extension (e.g., in y-direction in Fig. 1 and 2). The substrate 42 maybe arranged such that the flow 110 flows over / above the substrate 42. Gravity may act in negative z-direction (cf. e.g. Figs. 1 and 2), and the substrate 42 may be arranged such that it supports the flow 110. However, different arrangements and / or orientations of the channel 40, and / or the substrate 42, are possible.

[0139] The substrate 42 comprises surface binder elements 44. The surface binder elements 44 are arranged on a surface of the substrate 42 along which the sample 20 flows and / or along / on which the assaying elements 30 may move or roll. The surface binder elements 44 may extend from the substrate 42. The surface binder elements 44 may bind to the analyte 10, and / or the analyte 10 may bind to the surface binder elements 44.

[0140] The surface binder elements 44 may be or may comprise antibodies for binding to the analyte 10. The analyte 10 may be or may comprise a ligand and / or an antigen to bind to the surface binder elements 44 and / or respective antibodies. The surface binder elements 44 may be chosen such that they may bind to a specific, chosen, and / or desired analyte 10. A capture binder element 35, 36, 37 may have the same antibody as a surface binder element 44, 45, 46. In some embodiments, a surface binder element 44, 45, 46 is or comprises a tetraspanin, e.g., CD81, CD63 and / or CD9. In some embodiments, a capture binder element 35, 36, 37 is or comprises an anti-Her2neu antibody and / or an anti-IL6 antibody. In some embodiments, an analyte 10 is or comprises Her2neu+ EV, IL6, and / or biotin. In some embodiments, a substrate binder element 42 is or comprises an anti-Her2neu antibody, an anti-IL6 antibody, and / or avidin.

[0141] The device too may further compromise an external force generating device 120. The external force generating device 120 may be or may comprise a magnetic field generating device 120. The external force generating device 120 may be configured to generate an external force 60. The external force 60 may be or may comprise a magnetic force, e.g., generated by a magnetic field, and / or an acoustic force. The magnetic field generating device may be configured to generate a magnetic field and / or a magnetic force. In some embodiments, the external force generating device 120 and / or the magnetic field generating device is arranged at the channel 40. The magnetic field generating device may be or may comprise a magnet, e.g., a permanent magnet. For instance, in the embodiment of Fig. 1, the magnetic field generating device comprises a magnetic north pole and a magnetic south pole. It may be provided that the magnetic field is a static magnetic field.

[0142] Alternatively or additionally, the magnetic field generating device may be or may comprise an electromagnet. However, the magnetic field generating device is not necessarily limited thereto. In some embodiments, e.g., when the magnetic field generating device is or comprises an electromagnet, it may be provided that the magnetic field is varied, e.g. locally or over time.

[0143] When the assaying elements 30 are magnetic and / or comprise magnetic beads, the magnetic field and / or the magnetic field generating device may be configured to move the assaying elements 30 to the substrate 42, e.g., by attracting the assaying elements 30.

[0144] In some embodiments, the device too comprises more than one or multiple external force generating devices 120 and / or magnetic field generating devices, as shown in the embodiment of Fig. 2. In some embodiments, when the device too comprises more than one external force generating device 120 and / or more than one magnetic field generating device, the external force6o, e.g., a magnetic force, generated by the respective external force generating devices 120 differs, e.g., in strength and / or orientation.

[0145] It may be provided that external force 60, e.g., the magnetic field and / or magnetic force, is varied or reversed. In some instances, the external force 60 is varied or reversed to release stuck and / or immobilized assaying elements 30 from the substrate 42. In some embodiments, the channel 40 and / or the substrate 42 are / is freed from stuck and / or immobilized assaying elements 30 when the analyte concentration has been determined and / or after completion of the analyte concentration analysis.

[0146] In some embodiments, e.g., in those of Figs. 1 and 2, the external force generating device 120, e.g., a magnetic field generating device 120, is arranged below the substrate 42, e.g., below the substrate 42 in z-direction. The external force generating device 120 may exert a pulling force onto the assaying elements 30 flowing within the channel 40 and / or the assaying elements 30 above the substrate 42. Thus, the assaying elements 30 flowing in the channel 40 may be moved or maybe moving to the substrate 42.

[0147] Additionally or alternatively, in some embodiments, one or more of the external force generating devices 120, e.g. one or more magnetic field generating device 120, is / are arranged above the substrate 42, e.g. at or above an upper sidewall of the channel 40 or maybe arranged opposite the substrate 42. The external force generating device 120 may exert a pushing force onto the assaying elements 30 flowing within the channel 40 and / or the assaying elements 30 above the substrate 42. Thus, the assaying elements 30 flowing in the channel 40 may be moved or may be moving to the substrate 42. Alternatively or additionally, the external force generating device 120 may exert a pushing force onto the assaying elements 30 such that they may be sorted.

[0148] In some embodiments, the external force 60 is or comprises gravity. In some embodiments, it may be provided that the external force 60 is superposed by gravity, and / or includes gravity besides another force. If the external force 60 is or comprises gravity, in some embodiments the device too does not comprise an external force generating device 120. However, in some other embodiments the device too comprises an external force generating device 120 even in cases when the external force 60 is or comprises gravity. In some embodiments, the flow velocity and / or the assaying elements 30 are chosen and / or configured such, e.g., the assaying elements 30 may comprise a suitable material and / or may have a suitable size, that gravity is sufficient to move the assaying elements 30 towards the substrate 42. In some other embodiments, gravity is not sufficient to cause or to provide said movement of the assaying elements 30.

[0149] It maybe provided that the external force 60, e.g., a magnetic field and / or a magnetic force, is adjusted, chosen or varied in combination with a flow velocity of the flow 110 such that the assaying elements 30 may move or roll along / on the substrate 42. In particular, the flow velocity of the flow 110 and the external force 60 may be adjusted, chosen or varied such that all assaying elements 30, or a chosen or specific fraction of the assaying elements 30, may move or roll along / on the substrate 42. The flow velocity of the flow 110 and the magnetic field 60 may be adjusted, chosen or varied such that all assaying elements 30, or a chosen or specific fraction of the assaying elements 30, may contact the substrate 42 at or near a specified point along the flow direction (e.g., along the x-direction in the figures).

[0150] In some embodiments, the flow velocity of the flow 110 is chosen such that the analyte bonded assaying elements 30 are only slowed down when moving or rolling along / on the substrate 42, but not stuck or immobilized. In some embodiments, the flow velocity of the flow 110 is controlled or varied, e.g., depending on the size of the assaying elements 30 and / or the strength of the binding interaction. The external force 60 and / or the external force generating device 120 may be configured and / or arranged such that some, most or all of the assaying elements 30 may contact an inner side wall of the channel 40 and may move or roll along / on said inner side wall, before moving or rolling onto the substrate 42.

[0151] The device too comprises a sensor unit 70 (not shown in Figs. 1 and 2). Exemplary embodiments of the sensor unit 70 are described below with reference to Figs. 8 to 29. For instance, the sensor unit 70 of the embodiments of Figs. 1 and 2 (not shown in those figures) may correspond to or may be equal to one or more of the sensor units 70 as described hereafter and / or shown in Figs. 8, 10, 11, 19, 26, 27, 28, or 29.

[0152] The device too may comprise a control unit (not shown in the figures). The control unit may be or may comprise a computer unit. The control unit may be configured to control the external force generating device 120, the external force 60 and / or the flow velocity of the flow 110. The control unit may be configured to determine the concentration of the analyte 10 in the sample 20.

[0153] In Fig. 3, exemplary assaying elements 30 are shown. The assaying elements 30 may be spheres, or at least substantially spherical. However, the assaying elements 30 are not necessarily limited thereto. Other shapes or forms may be employed. In some embodiments, the assaying elements 30 may have diameters between 2 - too pm. In some embodiments, the assaying elements 30 are magnetic. It may be provided that the assaying elements 30 are made of, consist of or comprise at least one or more of a metal, alloy, plastic and glass. It may be provided that a core of an assaying element 30 is made of, consists of or comprises a first material, and that a surface of the assaying element 30 is made of, consists of or comprises a second material. The first material may be different from the second material. In some embodiments, the assaying elements 30 are coated and / or plated.

[0154] The assaying elements 30 may have one or more capture binder elements 35, to which an analyte 10 may bind or may be bound. It may be provided that at least one assaying element 30, or several, multiple or all assaying elements 30, has different kinds and / or types of capture binder elements 35, 36, 37. For instance, an assaying element 30 may have a first type of capture binder elements 35, a second type of capture binder elements 36, and / or a third type of capture binder elements 37. The number of types and / or kinds of capture binder elements 35? 36? 37 is not necessarily limited to three. In some embodiments, there may be more or less different types and / or kinds of capture binder elements 35, 36, 37. In some embodiments, it may be provided that at least one capture binder element, and / or multiple or all capture binder elements, has / have only one and / or a single type and / or kind of capture binder elements 35. In some embodiments, a capture binder element 35, 36, 37 is or comprises a tetraspanin, e.g., CD81, CD63 and / or CD9.

[0155] In some embodiments, the number of capture binder elements 35, 36, 37 per assaying element 30 differs for different assaying elements 30.

[0156] It may be provided that the number of capture binder elements 35, 36, 37 for each type and / or kind is larger than one, cf., e.g., Fig. 3. The capture binder elements 35, 36, 37 may extend from a surface of the assaying element 30. The capture binder elements 35, 36, 37 maybe arranged and / or may be distributed on the assaying element 30 and / or its surface symmetrically, isotropically, and / or substantially over the full surface area of the assaying element 30.

[0157] The capture binder elements 35, 36, 37 may be chosen with respect to the analyte 10 to be detected and / or to be analyzed. In some embodiments, it may be provided that when the assaying element 30 has two or more types and / or kinds of capture binder elements 35, 36, 37, the method and / or the device too may be configured for multiplexing and / or heterogeneous assays, as described below.

[0158] The capture binder elements 35, 36, 37 may be or may comprise antibodies for binding to an analyte 10. When an analyte 10 is bound to at least one of the capture binder elements 35, 36, 37 of an assaying element 30, an analyte bonded assaying element 30 is formed. The analyte 10 maybe or may comprise a ligand and / or an antigen to bind to a capture binder element 35, 36, 37 and / or a respective antibody. The capture binder element 35, 36, 37 may be chosen such that it may bind to a specific, chosen, to be determined or desired analyte 10. Particularly, when the sample 20 comprises more than one type and / or kind of analyte 10, the capture binder elements 35, 36, 37 may be chosen such as to bind to one of the analytes 10 for which the concentration may be determined or tested, and / or which shall be assayed. It may be provided that when the sample 20 comprises more than one type and / or kind of analyte 10, different kinds and / or types of capture binder elements 35, 36, 37 may be chosen such as to selectively bind to analytes 10 for which the concentration may be determined or tested, and / or which shall be assayed. In some embodiments, multiplexing for multiple analytes 10, e.g. multiple kinds and / or types of analyte 10, is provided, as described below.

[0159] In Fig. 4, an exemplary embodiment of an assaying element 30 according to the invention is shown. The assaying element 30 has element test analyte 38. The element test analyte 38 may be arranged at an outer surface of the assaying element 30. The element test analyte 38 may be configured and / or selected such that it may interact with surface binder elements 44 of the substrate 42. In particular, the element test analyte 38 may be bound to surface binder elements 44. When the element test analyte 38 of the assaying element 30 binds to one or more surface binder elements 44 (or vice versa), the velocity of the respective assaying element 30 is changed, in particular reduced.

[0160] As the analyte 10 in the sample 20 may also bind to the surface binder elements 44 of the substrate 42, there is a competition between the binding interaction of analyte 10 to surface binder elements 44 and of the element test analyte 38 to the surface binder elements 44. In particular, it may be provided that when analyte 10 is bound to a surface binder element 44, binding of an element test analyte 38 to said surface binder element 44 is reduced or inhibited, and vice versa. In some embodiments, the assaying element 30 may be immobilized by an interaction between the element test analyte 38 and one or more surface binder elements 44 (or vice versa).

[0161] Hence, the higher the concentration of analyte 10 in the sample 20, the more analyte 10 is bound to the surface binder elements 44 of the substrate 42, and the chance or occurrence of binding of an element test analyte 38 to surface binder element 44 is more reduced. Thus, the higher the concentration of analyte 10 in the sample 20, the less is the velocity of the assaying element 30 having element test analyte 38 changed and / or reduced.

[0162] By providing assaying elements 30 having element test analyte 38, a competitive assay may be performed.

[0163] In Fig. 5, another exemplary embodiment of an assaying element 30 according to the invention is shown. The assaying element 30 has capture binder elements 35. The capture binder elements 35 may be arranged at an outer surface of the assaying element 30. The capture binder elements 35 may be configured such that analyte 10 may be bound thereto. The substrate 42 has substrate test analyte 47. The substrate test analyte 47 may be arranged on a surface of the substrate 42. The substrate test analyte 47 may be arranged on the substrate 42 such that it may be in contact with the sample 20. The substrate test analyte 47 may be arranged on the substrate 42 such that it may extend into the channel 40.

[0164] The substrate test analyte 47 may be configured and / or selected such that it may interact with the capture binder elements 35 of the assaying element 30. In particular, the substrate test analyte 47 and the capture binder elements 35 may bind to each other. When substrate test analyte 47 of the substrate 42 binds to a binder element 35 (or vice versa), the velocity of the respective assaying element 30 is changed, in particular reduced. In some embodiments, the assaying element 30 may be immobilized by an interaction between the surface test analyte 47 and one or more surface capture binder elements 35 (or vice versa).

[0165] As the analyte 10 in the sample 20 may also bind to the capture binder elements 35 of the assaying element 30, there is a competition between the binding interaction of analyte to capture binder elements 35 and of the surface test analyte 47 of the substrate 42 to the capture binder elements 35. In particular, it may be provided that when analyte 10 is bound to a capture binder element 35, binding of an surface test analyte 47 to said capture binder element 35 is reduced or inhibited, and vice versa.

[0166] Hence, the higher the concentration of analyte 10 in the sample 20, the more analyte 10 is bound to the capture binder elements 35 of the assaying element 30, and the chance or occurrence of binding of an capture binder elements 35 to surface test analyte 47 is more reduced. Thus, the higher the concentration of analyte 10 in the sample 20, the less is the velocity of the assaying element 30 having element test analyte 38 changed and / or reduced.

[0167] By providing a substrate 42 having surface test analyte 47, a competitive assay may be performed.

[0168] The disclosure and the invention is not necessarily limited to cases where the substrate 42 and all assaying elements 30 are configured such as shown in figures 3, 4 and 5, and / or as discussed with reference to these figures. In particular, it maybe provided that in some embodiments, at least one, multiple or all assaying elements 30 have capture binder elements 35 and element test analyte 38 (not shown in the figures). Alternatively or additionally, there may be some assaying elements 30 having only capture binder elements 35, and some other assaying elements 30 having only element test analyte 38. Alternatively or additionally, a substrate 42 may have both surface binder elements 44 and surface test analyte 47 (not shown in the figures). Alternatively or additionally, there may a substrate 42 having only surface binder elements 44, and some other substrate 42 having only surface test analyte 47. For instance, the embodiments as discussed and shown with reference to any of figs. 1 to 3 may comprise element test analyte 38 and / or surface test analyte 47, but not necessarily so.

[0169] In Fig. 6, steps of an exemplary embodiment of a method 1000 according to the invention are shown. The method may comprise one or more steps of a method disclosed herein. In step 1010, a device 100 having a channel 40 is provided. The channel 40 has a substrate 42. The substrate 42 has surface binder elements 44, 45, 46 configured to bind to the analyte 10 in the sample 20, and / or the substrate 42 has a surface test analyte 47. The device 100 may be or may comprise any device 100 as disclosed herein.

[0170] In step 1020, assaying elements 30 are mixed into a sample 20. The assaying elements 30 may be or may comprise any assaying element 30 as disclosed herein. The sample 20 comprises an analyte 10. The assaying elements 30 have capture binder elements 35, 36, 37 configured to bind to the analyte 10, and / or the assaying elements 30 have an element test analyte 38 configured to bind to the surface binder elements 44, 45, 46. When the substrate 42 includes surface test analyte 47, the surface test analyte 47 is configured to interact with the capture binder elements 35, 36, 37, in particular to bind thereto.

[0171] When the assaying elements 30 have capture binder elements 35, 36, 37, the analyte 10 in the sample 20 may bind to capture binder elements 35, 36, 37 when or after mixing the assaying elements 30 into the sample 20. The capture binder elements 35, 36, 37 may be or may comprise one or more antibodies, respectively. The antibodies may bind to the analyte 10, and / or a ligand, e.g., an antigen, of the analyte 10. The capture binder element 35, 36, 37 may be chosen such that it may bind to a specific, chosen, or desired analyte 10.

[0172] When the assaying elements 30 have element test analyte 38, the element test analyte 38 may have similar characteristics as the analyte 10 in the sample 20. The element test analyte 38 may be of the same kind and / or type as the analyte 10 in the sample 20. When the substrate 42 has surface test analyte 47, the surface test analyte 47 may have similar characteristics as the analyte 10 in the sample 20. The surface test analyte 38 may be of the same kind and / or type as the analyte 10 in the sample 20.

[0173] In step 1030, the sample 20 with the assaying elements 30 mixed therein is flown through the channel 40. For instance, a pump, a syringe device or the like may be used to flow the sample 20 through the channel 40.

[0174] In step 1040, the assaying elements 30 are moved to the substrate 42. In some embodiments, the assaying elements 30 are magnetic, and the assaying elements 30 are moved by a magnetic field 60 to the substrate 42. The magnetic field 60 may exert a magnetic force onto the assaying elements 30, such that they are moved towards the substrate 42. The magnetic field 60 may be a static magnetic field. The assaying elements 30 may be pulled or pushed towards the substrate 42. In step 1050, the assaying elements 30 are moved along / on the substrate 42. In some embodiments, the assaying elements 30 are moved by the flow of the sample 20. Moving an assaying element 30 along / on the substrate 42 may comprise that the assaying element 30 rolls along / on the substrate 42. In some embodiments, the assaying elements 30 may be moved by the magnetic field 60, or a different magnetic field, along / on the substrate 42. The assaying elements 30 may be moved by an external force, such as e.g. a magnetic force, along / on the substrate 42. Alternatively or additionally, the magnetic field and / or the external force may support the movement of the assaying elements 30 along / on the substrate 42.

[0175] When moving along / on the substrate 42, the velocity of at least one assaying element 30 is changed via an interaction. The interaction may comprise or may be an interaction, preferably a binding interaction, between at least one surface binder element 44, 45, 46 of the substrate 42, the analyte 10 and at least one capture binder element 35, 36, 37 of said at least one assaying element 30. Alternatively or additionally, the interaction may comprise or may be an interaction, preferably a binding interaction, between the surface test analyte 47 of the substrate 42 and at least one of the capture binder elements 35, 36, 37 of said at least one assaying element 30. Alternatively or additionally, the interaction may comprise or may be an interaction, preferably a binding interaction, between the element test analyte 38 of said at least one assaying element 30 and at least one of the surface binder elements 44, 45, 46 of the substrate 42. The change of velocity may depend on the strength of the respective interaction.

[0176] The interaction may be or may comprise an interaction as shown in any of Figs. 1 to 5, and / or as described with reference to any of Figs. 1 to 5.

[0177] When the velocity is changed, it may be provided that an assaying element 30 may be immobilized or stuck on the substrate 42.

[0178] In step 1060, a concentration of the analyte 10 is determined by determining the change of velocity of the assaying elements 30 and / or by determining a depletion of the assaying elements 30. Alternatively or additionally, binding kinetics are determined by determining the change of velocity of the assaying elements 30 and / or by determining a depletion of the assaying elements 30.

[0179] In Fig. 7, steps of an exemplary embodiment of a method 1000 according to the invention are shown. The method according to the invention may be called a rolling bead sandwich assay. The method may comprise additional steps not shown in Fig. 7. The method may comprise one or more steps as disclosed herein, in particular of the method described above with reference to Fig. 6.

[0180] In step 1200, assaying elements 30 are mixed into a sample 20 comprising an analyte 10. The mixing may comprise introducing the assaying elements 30 into the sample 20. The assaying elements 30 may be or may comprise beads and / or magnetic beads.

[0181] In step 1025, at least one of the assaying elements 30 binds to the analyte 10 in the sample 20. For binding to the analyte 10, the assaying elements 30 have one or more capture binder elements 35, 36, 37. The capture binder elements 35, 36, 37 are configured to bind to the analyte 10. The analyte 10 may bind to the capture binder elements 35, 36, 37. The capture binder elements 35, 36, 37 may be or may comprise one or more antibodies, respectively. The antibodies may bind to the analyte 10, and / or a ligand, e.g., an antigen, of the analyte 10. The capture binder element 35, 36, 37 may be chosen such that it may bind to a specific, chosen, or desired analyte 10.

[0182] When the analyte 10 binds to an assaying element 30, an analyte bonded assaying element 30 may be formed, and / or an assaying element 30 with the analyte 10 bound thereto may be designated as analyte bonded assaying element 30. Not all assaying elements 30 may have or must have analytes 10 bound to capture binder elements 35, 36, 37. There may be assaying elements 30 in the sample 20, which are unbound to the analyte 10. However, in some embodiments, all assaying elements 30 are analyte bonded assaying elements 30 and / or have the analyte 10 bound to the capture binder elements 35, 36, 37.

[0183] In step 1030, the sample 20 is flown through a channel 40. The sample 20 flowing through the channel 40 contains or comprises the analyte 10, and at least one assaying element 30 to which the analyte 10 is bound.

[0184] The channel 40 has a substrate 42. The substrate 42 comprises surface binder elements 44. The surface binder elements 44 are arranged on a surface of the substrate 42 along / on which the sample 20 flows and / or along / on which the assaying elements 30 may move or roll. The surface binder elements 44 may bind to the analyte 10, and / or the analyte 10 may bind to the surface binder elements 44.

[0185] In step 1040, the assaying elements 30 in the sample 20 flowing through the channel 40 are moved towards a substrate 42 of the channel 40 by means of an external force 60, e.g. by means of a magnetic field. The external force 60 may act on the assaying elements 30, and may move the assaying elements 30 towards or to a substrate 42 of the channel 40. In some embodiments, the magnetic field 60 exerts a (magnetic) force on the assaying elements 30.

[0186] It may be provided that moving assaying elements 30 towards or to the substrate 42 comprises, is or corresponds to pulling or attracting the assaying elements 30 to the substrate 42. Alternatively or additionally, it maybe provided that moving assaying elements 30 towards or to the substrate 42 comprises, is or corresponds to pushing the assaying elements 30 to the substrate 42.

[0187] In step 1050, when the assaying elements 30 are moved to the substrate 42, the assaying elements 30 move or roll along / on the substrate 42. It may be provided that the assaying elements 30 are moved to the substrate 42 by the external force 60, e.g., by a magnetic force generated by a magnetic field, such as to contact the substrate 42 and / or to move or roll along / on the substrate 42. The assaying elements 30 maybe pushed on / onto the substrate 42 by the flow 110 of the sample 20, such that they may move or roll along / on the substrate 42. In some embodiments, all assaying elements 30 entering the channel 40 are moved by the external force 60, e.g., pulled to the substrate 42, such that they move or roll along / on the substrate 42 when flowing through the channel 40. In some embodiments, a majority or at least a fraction of the assaying elements 30 entering the channel 40 is moved by the external force 60, e.g., pulled to the substrate 42, such that they move or roll along / on the substrate 42 when flowing through the channel 40.

[0188] When an assaying element 30, to which an analyte 10 is bound, moves or rolls along / on the substate 42, the velocity of said assaying element 30 may be changed. When the velocity is changed, the velocity may be reduced. The reduction of velocity may be due to interactions, e.g., binding interactions, between the analyte 10 that is bound to the assaying element 30 and one or more surface binder elements 44. The binding interaction may be a so-called specific binding.

[0189] In some embodiments, changing and / or reducing the velocity comprises immobilizing at least one respective analyte bonded assaying element 30. In some embodiments, at least one analyte bonded assaying element 30 is slowed down, but not immobilized. It may be provided that the assaying elements 30 without an analyte 10 bound thereto are not substantially slowed down when moving or rolling along / on the substrate 42 (due to lack of any binding interaction described in the previous paragraph). The number and / or the ratio of respective analyte bonded assaying elements 30 immobilized and / or slowed down may depend, e.g., on the strength of the binding interaction between the surface binder elements 44 and the analyte 10 bonded to the assaying element 30, and / or on the density and / or the position of surface binder elements 44 on the substrate 42 and / or on the concentration of the analyte 10 in the sample 20. For instance, more than one capture binder element 35, 36, 37 of an assaying element 30 has an analyte 10 bound thereto, which - in some cases - may further reduce the velocity of said assaying element 30 along / on the substrate 42 as there may be multiple binding interactions between the respective bonded analytes 10 and the multiple surface binder elements 44.

[0190] In step 1060, a concentration of the analyte 10 in the sample 20 is determined, e.g., by determining a change and / or decrease of the velocity of the assaying elements 30 flowing within the channel 40. The higher the analyte concentration, the higher the change and / or decrease of velocity, since the assaying elements 30 moving or rolling along / on the substrate may be slowed down the more analyte 10 is bound to the assaying elements 30 due to an interaction, e.g., a binding interaction, between the surface binder elements 44 and the analyte 10 bound to the assaying elements 30. Alternatively or additionally, the concentration of the analyte 10 in the sample 20 may be determined e.g. by determining a depletion of the assaying elements 30 along the channel 40. A depletion of assaying elements 30 may comprise an immobilization of at least some of the assaying elements 30.

[0191] In an embodiment, the sample 20 is flown or driven by a syringe, a syringe pump, and / or a metering pump (not shown in the figures). The syringe, the syringe pump and / or the metering pump may be fluidically connected to the channel 40, e.g., at or to an inlet of the channel 40. The inlet may be arranged upstream of the substrate 42. The syringe, the syringe pump and / or the metering pump may exert a force onto the sample 20 such that the velocity of the sample 20 is substantially constant when flowing into or through the channel. Thus, a velocity of the assaying elements 30 upstream of the substrate 42, and / or at the inlet of the channel 40, may be known. The velocity of the assaying elements 30 may be determined, detected and / or measured by the sensor unit 70 downstream of the substrate 42. For instance, the velocity may be determined or measured by the sensor unit 70 as detailed below, e.g., by a sensing element 72, 74, 76 and / or by an optical device 78. Then, the change and / or decrease of velocity of the assaying elements 30 may be determined by comparing the velocity as imposed by the syringe, the syringe pump and / or the metering pump, e.g., at the inlet, and the velocity as determined by the sensor unit 70. In some embodiments, there is no sensing element 72, 74, 78 and / or optical device 78 upstream of the substrate 42, and / or a sensing element 72, 74, 78 and / or optical device 78 upstream of the substrate 42 is redundant. Figs. 8 to 11 show an exemplary embodiment of a device 100 and a method according to the invention. The device 100 comprises a channel 40 with a substrate 42, e.g., a channel 40 and a substrate 42 as described above.

[0192] The device 100 may comprise an external force generating device 120, e.g., a magnetic field generating device (not shown in Figs. 8 and 9), which is configured to generate an external force 60, e.g. a magnetic force generated by a magnetic field (not shown in Figs. 8 and 9). Further, the device 100 comprises a sensor unit 70. The sensor unit 70 comprises two sensing elements 72, 74. The second sensing element 74 is arranged downstream of the first sensing element 72, e.g., arranged downstream in flow direction (for instance downstream in x- direction in the Figs. 8 and 9). The embodiment as shown in Figs. 8 to 11 may have at least one, several or all features and / or advantages of at least one other embodiment according to the invention.

[0193] Fig. 8 shows a device 100 with the sample 20 comprising assaying elements 30 flowing through the channel 40 at a first time ti. Fig. 9 shows the device 100 with a sample 20 comprising assaying elements 30 flowing through the channel 40 at a second time t2. The second time t2 maybe later than the first time ti. Fig. 10 shows an output of the sensor unit 70, and / or of the first sensing element 72 and the second sensing element 74, as a function of time. Fig. 11 shows a comparison of analyte concentrations detected and / or determined by a method according to the invention to analyte concentrations detected and / or determined by methods of the prior art.

[0194] In some embodiments, the sensor unit 70 and / or the sensing elements 72, 74 are / is or comp rise / comp rises a device configured for impedance sensing and / or magnetoresistive sensing. For instance, the assaying elements 30 may be configured to be detected and / or sensed by impedance sensing and / or by magnetoresistive sensing. Alternatively or additionally, the sensor unit 70 and / or the sensing element 72, 74 may be or may comprise a coulter counter. In some embodiments, the first sensing element 72 and / or the second sensing element 74 are / is or comp rise / comp rises a magnetic field sensor, and / or a magnetic sensor half bridge, respectively. Thus, in some embodiments a non-optical determination of the analyte concentration is possible. Hence, the method and the device according to the invention allow for assays where the sample 20 does not need to be processed before carrying out the method. Further, the sample 20 may be opaque. The sensor unit 70 and / or the sensing elements 72, 74 may detect and / or may register an assaying element 30, e.g., a magnetic assaying element 30, passing over, along or through the sensor unit 70 and / or the sensing elements 72, 74. It may be provided that the sensor unit 70 and / or the sensing elements 72, 74 measure / measure and / or determine / determines the size of an assaying element 30.

[0195] The first sensing element 72 may be arranged upstream of the substrate 42 (cf., e.g., Figs. 8 and 9). In some embodiments, the first sensing element 72 and / or the second sensing element 74 are / is arranged over / above the substrate 42. The second sensing element 74 may be arranged downstream of the first sensing element 72 and / or downstream of the substrate 42 (cf., e.g., Figs. 8 and 9). The definition of “downstream” and “upstream” may be with respect to the flow direction of flow 110.

[0196] In some embodiments, e.g., in the embodiment of Figs. 8 to 11, the concentration of the analyte 10 and / or a decrease of the velocity of the assaying elements 30 may be determined by determining a time-of-roll of the assaying elements 30. The assaying elements 30 may be magnetic.

[0197] The time-of-roll maybe determined and / or may be measured between two sensing elements, e.g., between the first sensing element 72 and the second sensing element 74. However, it should be understood that, additionally or alternatively, a time-of-roll may be determined and / or may be measured between multiple sensing elements 72, 74, 76, and / or between two sensing elements 72, 74 separated by another sensing element 76. Multiple time-of-roll measurements and / or determinations may be carried out and alternatively or additionally at different temperatures. For instance, a first time-of-roll may be determined and / or may be measured between a first sensing element 72 and a second sensing element 74, and a second time-of-roll maybe determined and / or maybe measured between a third sensing element and a fourth sensing element. For instance, the first, second, third and fourth sensing element may be different sensing elements. However, it may be provided that, e.g., the third sensing element corresponds to or is equal to the first or the second sensing element 72, 74. Similarly, different arrangements of sensing elements 72, 74, 76, and / or time-of-roll measurements and / or determinations, are possible.

[0198] For instance, as depicted, e.g., in Fig. 8, a first sensing element 72 may detect and / or may register assaying elements 30, e.g. magnetic assaying elements 30, passing along and / or over the first sensing element 72, e.g. moving or rolling along / on the substrate 42. A second sensing element 74 may detect and / or may register assaying elements 30 passing along and / or over the second sensing element 72, e.g., moving or rolling along / on the substrate 42, cf.,e.g., Fig. 9. When one or multiple assaying elements 30 passes / pass along and / or over one of the sensing elements 72, 74, a corresponding signal may be outputted by the respective sensing element at a time of passing, cf., e.g., Fig. 10. The signal may be higher the more assaying elements 30 pass at one instance of time, and / or the closer assaying elements 30 are grouped or packed. Thus, at the first time ti, a first signal corresponding to at least one assaying element 30 or a group of assaying elements 30 passing the first sensing element 72 may be detected (cf. fig. 8 and fig. 10). Then, when the at least one assaying element 30 or the group of assaying elements 30 pass the second sensing element 74 downstream of the first sensing element 72, the second sensing element 74 may output a second signal at the second time t2 (cf., e.g., Fig. 9 and Fig. 10). The time difference At = t2- ti may be used together with a known distance between the first sensing element 72 and the second sensing element 74 to determine a velocity of the at least one assaying element 30 or the group of assaying elements 30. The time-of-roll may be or may correspond to the time difference At.

[0199] The higher the analyte concentration in the sample 20, the higher the time difference At and the higher the velocity decrease. Thus, the analyte concentration may depend on and / or maybe correlated with and / or may be a function of the velocity decrease, and / or the velocity decrease maybe employed to determine an analyte concentration in the sample 20.

[0200] In Fig. 11, the method according to the invention is compared to conventional assays. Whereas conventional assays may detect and / or may determine analyte concentrations over 2 to 3 orders of magnitude, the method and the device according to the invention may detect and / or may determine analyte concentrations over 3 to 4 orders of magnitude, or even more. Thus, the method and the device according to the invention allow for a significantly improved detection and / or determination of the analyte concentration over a wide range, and provide assays with higher range of detection. Another example of concentrations as determined by a change of velocity of assaying elements 30 is shown in Fig. 30a.

[0201] It may be provided that a reference velocity difference is given and / or determined, e.g., depending on a given flow velocity and / or for a given flow velocity. The reference velocity may be determined and / or given for a flow 110 without an analyte 10, i.e. for assaying elements 30 unbound to an analyte 10 and / or not slowed down when moving or rolling along / on the substrate 42. The velocity difference may be determined and / or given with respect to the reference velocity. In some embodiments, the reference velocity may be an average flow velocity of the flow 110. Alternatively or additionally, optical methods may be used to determine the concentration of the analyte to and / or to determine the velocity of assaying elements 30. The sensor unit 70 may be or may comprise one or more optical devices 78 (not shown in Figs. 8 and 9), e.g., one or more cameras. The sensor unit 70, the optical device 78 and / or the camera may take snapshots at specific times, and / or may capture photos or videos. The sensor unit 70, the optical device 78 and / or the camera may temporally and / or spatially capture the movement and / or the position of the assaying elements 30. The resolution of the sensor unit 70, of the optical device 78 and / or of the camera may be temporally, and / or spatially, resolved. The sensor unit 70, the optical device 78 and / or the camera may take snapshots at specific times, and / or may capture photos or videos in a field of view. The field of view (not shown in Figs. 8 and 9) may encompass the substrate 42, fully or at least part of it. The field of view may be along the flow direction, e.g., in x-direction of Figs. 8 and 9.

[0202] Then, the velocity of the assaying elements 30 may, e.g., be determined by comparing different positions of the assaying elements 30 at different times and alternatively or additionally at different temperatures.

[0203] In some embodiments, more than one optical device 78 and / or more than one camera is provided, such as to have a larger total field of view 79 at sufficient resolution. It may be provided that different optical devices 78 and / or cameras have different fields of view 79, respectively. In some embodiments, at least two, multiple or all different fields of view 79 at least partially overlap. Alternatively or additionally, it may be provided that at least two, multiple or all fields of view 79 do not overlap. Different and / or overlapping fields of view 79 may form a composite field of view and / or a total field of view. It may be provided that a first optical device 78 and / or camera is arranged upstream of the substrate 42 and / or such that its field of view 79 may cover a region upstream of the substrate 42. A second optical device 78 and / or camera may be arranged downstream of the substrate 42 and / or such that its field of view 79 may cover a region downstream of the substrate 42.

[0204] The optical device 78 and / or the camera may detect one or more of color, size, luminescence and granularity of the assaying elements 30. Thereby, in some embodiments, multiplexing is enabled.

[0205] In Fig. 12, an exemplary signal as detected by the sensor unit 70 for a single assaying element 30 is shown. For instance, the sensor unit 70 comprises a first sensing element 72 and a second sensing element 74. The first sensing element 72 is arranged upstream of the second sensing element 74. When the assaying element 30 passes the first sensing element 72 at a first timepoint, a signal is recorded by the sensing element 70, as indicated by the left spike in Fig. 12. Then, the assaying element 30 is transported downstream, e.g. by moving along / on a substrate 42, and / or by moving along / on the channel 40, until it passes the second sensing element 74 at a second timepoint. When the assaying element 30 passes the second sensing element 74, the second sensing element 74 records a signal, as indicated by the right spike in Fig. 12.

[0206] Alternatively or additionally, the respective signals may be recorded optically. For instance, each spike may correspond to a timepoint when the assaying element 30 is at a specific position along the channel 40 and / or the substrate 42, as identified e.g. by an optical device such as a camera.

[0207] The first timepoint and the second timepoint maybe determined and / or measured relative to the same reference timepoint. In some embodiments, the reference timepoint may be a timepoint previous to the first timepoint. In some embodiments, the reference timepoint may be the first timepoint.

[0208] The time difference between the second timepoint and the first timepoint may be determined to determine the velocity of the assaying element 30, and / or a change of velocity of the assaying element 30.

[0209] When more than one assaying element 30 is included in the sample 20, and / or when more than one assaying element 30 is flown or moved through the channel 40, a distribution function may be constructed from the recordings of the sensor unit 70, e.g. from the recordings of the first sensor unit 72 and the second sensor unit 74.

[0210] Fig. 13 shows distribution functions as constructed from the recordings of a sensor unit 70. In particular, the left distribution function 80 (left bell-shaped curve in Fig. 13) is constructed from the recordings of a first sensing element 72. The two distribution functions 82, 83 on the right (solid and dashed bell-shaped curves on the right in Fig. 13) are constructed from the recordings of a second sensing element 72. Alternatively or additionally, the distribution functions 80, 82, 83 may be determined optically, e.g. by counting the number of assaying elements 30 in a small area at either a specific timepoint or in a small time interval. Each distribution function may correspond to a specific position in or along the channel 40 and / or the substrate 42. Each distribution function 80, 82, 83 maybe determined at a specific position in or along the channel 40 and / or the substrate 42. The distributions functions 80, 82, 83 as shown in Fig. 13 may be or may correspond to histograms, and / or to density functions. A density function may be a normalized histogram. When the histogram is normalized such that the sum or the integral of the histogram over time is equal to unity, the density function may be interpreted as a probability density function. The density function may be normalized such that the integral or sum over the density function between two points in time yields the fraction of assaying elements 30 at the specific positions, e.g. at the first or the second sensing element 72, 74, in the time interval between the two points in time.

[0211] In Fig. 13, the solid distribution function 82 on the right and the dashed distribution function 83 on the right correspond to different concentrations of analyte 10 in the sample 20. On the other hand, in the example of Fig. 13, the left distribution function 80 is the same for the two different concentrations of analyte 10 in the sample 20. The distribution function 80 on the left may be a first distribution function or may correspond to a first distribution function, and any of the distribution functions 82, 83 on the right may be a second distribution function or may correspond to a second distribution function.

[0212] The time of roll and / or velocity of the assaying elements 30 can be determined by the difference in time At between the peaks of the respective distribution functions. For instance, for a sample 20 having a first concentration of analyte 10, a difference in time may be determined by calculating the difference in time between the peak of the solid distribution function 82 on the right and the peak of the distribution function 80 on the left in Fig. 13. For instance, for a sample 20 having a second concentration of analyte 10, a difference in time maybe determined by calculating the difference in time between the peak of the dashed distribution function 83 on the right and the peak of the distribution function 80 on the left in Fig. 13. The peaks may be or may correspond to respective maximum values.

[0213] The respective differences At may be indicative of the change of velocity. The difference between the two differences in time may be indicative of the difference in concentration of analyte. Similarly, the difference in time At may be compared to a reference difference in time for a reference concentration of analyte.

[0214] Fig. 14 shows distribution functions as constructed from the recordings of a sensor unit 70. In particular, the left distribution function 80 (left S-shaped curve in Fig. 14) is constructed from the recordings of a first sensing element 72. The two distribution functions 82, 83 on the right (solid and dashed S-shaped curves on the right in Fig. 14) are constructed from the recordings of a second sensing element 72. Alternatively or additionally, the distribution functions 80, 82, 83 maybe determined optically, e.g. by counting the number of assaying elements 30 in a small area at either a specific timepoint or in a small time interval. Each distribution function may correspond to a specific position in or along the channel 40 and / or the substrate 42. Each distribution function 80, 82, 83 may be determined at a specific position in or along the channel 40 and / or the substrate 42.

[0215] The distributions functions as shown in Fig. 14 may be or may correspond to cumulative functions. The distribution function may be or may correspond to an empirical cumulative distribution function (ecdf). A cumulative function may be a running sum of values of a histogram or of a density function. When the cumulative function is normalized such that its right- most value is equal to unity, the cumulative function may be interpreted as a cumulative probability distribution function. The cumulative function may be normalized such that difference of the cumulative function between two points in time yields the fraction of assaying elements 30 at the specific positions, e.g. at the first or the second sensing element 72, 74, in the time interval between the two points in time.

[0216] In Fig. 14, the solid distribution function 82 on the right and the dashed distribution function 83 on the right correspond to different concentrations of analyte 10 in the sample 20. On the other hand, in the example of Fig. 14, the left distribution function 80 is the same for the two different concentrations of analyte 10 in the sample 20. The distribution function 80 on the left may be a first distribution function or may correspond to a first distribution function, and any of the distribution functions 82, 83 on the right may be a second distribution function or may correspond to a second distribution function.

[0217] The time of roll and / or the velocity of the assaying elements 30 can be determined by the difference in time At between the inflection points of the respective distribution functions. For instance, for a sample 20 having a first concentration of analyte 10, a difference in time may be determined by calculating the difference in time between the inflection point of the solid distribution function 82 on the right and the inflection point of the distribution function 80 on the left in Fig. 14. For instance, for a sample 20 having a second concentration of analyte 10, a difference in time may be determined by calculating the difference between the inflection point of the dashed distribution function 83 on the right and the inflection point of the distribution function 80 on the left in Fig. 14.

[0218] The respective differences At may be indicative of the change of velocity. The difference between the two differences in time At may be indicative of the difference in concentration of analyte. Similarly, the difference in time At may be compared to a reference difference in time for a reference concentration of analyte.

[0219] Fig. 15 shows distribution functions as constructed from the recordings of a sensor unit 70. In particular, the left distribution function 80 (left bell-shaped curve in Fig. 15) is constructed from the recordings of a first sensing element 72. The two distribution functions on the right 82, 83 (solid and dashed bell-shaped curves on the right in Fig. 15) are constructed from the recordings of a second sensing element 72. Alternatively or additionally, the distribution functions 80, 82, 83 may be determined optically, e.g. by counting the number of assaying elements 30 in a small area at either a specific timepoint or in a small time interval. Each distribution function may correspond to a specific position in or along the channel 40 and / or the substrate 42. Each distribution function 80, 82, 83 maybe determined at a specific position in or along the channel 40 and / or the substrate 42.

[0220] The distributions functions as shown in Fig. 15 may be or may correspond to histograms, and / or to density functions. A density function may be a normalized histogram. When the histogram is normalized such that the sum or the integral of the histogram over time is equal to unity, the density function may be interpreted as a probability density function. The density function may be normalized such that the integral or sum over the density function between two points in time yields the fraction of assaying elements 30 at the specific positions, e.g. at the first or the second sensing element 72, 74, in the time interval between the two points in time.

[0221] In Fig. 15, the solid distribution function 82 on the right and the dashed distribution function 83 on the right correspond to different concentrations of analyte 10 in the sample 20. On the other hand, in the example of Fig. 15, the left distribution function 80 is the same for the two different concentrations of analyte 10 in the sample 20. The distribution function 80 on the left may be a first distribution function or may correspond to a first distribution function, and any of the distribution functions 82, 83 on the right may be a second distribution function or may correspond to a second distribution function.

[0222] The change of velocity of the assaying elements 30 can be determined by determining a distance metric between the respective distribution functions at the specific positions. For instance, a distance metric maybe computed between the solid distribution function 82 on the right and the distribution function 80 on the left. The distance metric may then be compared to a reference value. The computed distance metric may be indicative of the time-of roll and / or the change of velocity for a sample 20 having a first concentration of analyte 10. Similarly, a distance metric may be computed between the dashed distribution function 83 on the right and the distribution function 80 on the left. The distance metric may then be compared to a reference value. The computed distance metric may be indicative of the time-of roll and / or the change of velocity for a sample 20 having a second concentration of analyte 20. In the specific example of Fig. 15, the difference metric may be larger for the first concentration than for the second concentration, as the solid distribution function on the right differs more from the distribution function on the left than does the dashed distribution function on the right.

[0223] Suitable distance metrics include but are not limited to f-divergences, the Kullback-Leibler divergence, the Wasserstein metric, Kolmogorov-Smirnov tests, or the like.

[0224] Alternatively or additionally, the distribution functions may be compared by fitting them to a candidate distribution function, and comparing the fitting parameters. For instance, the candidate distribution function may be a Gamma distribution, having a shape and a scale parameter. Similarly. A reference function may be fit to the candidate distribution function. By comparing the parameters as determined by the fit of each distribution as constructed from measurements of the sensor unit, the time-of- roll and / or the concentration of analyte 10 may be determined.

[0225] For instance, both the distribution function on the left of Fig. 15 and the solid distribution function on the right of Fig. 15 maybe fit to the candidate distribution function (as determined for a first analyte concentration). Then, the difference between one or more of the parameters of the candidate function maybe determined, e.g. between the shape parameters of respective gamma functions. Similarly, both the distribution function on the left of Fig. 15 and the dashed distribution function on the right of Fig. 15 (as determined for a second analyte concentration) may be fit to the candidate distribution function, and the difference between one or more of the parameters of the candidate function may be determined, e.g. between the shape parameters of respective gamma functions. The respective differences are indicative of the analyte concentration.

[0226] The candidate distribution function is not limited to the gamma distribution. Other suitable distribution functions may be chosen, such as e.g. but not limited to the lognormal distribution or the Weibull distribution. In Fig. 16, an exemplary curve of a difference of parameters of a candidate distribution function as function of the analyte concentration in a sample 20 is shown. In this particular example, the candidate distribution function is a gamma distribution and the difference corresponds to a difference of the shape parameter of respective gamma distributions as determined at two different positions in / along the channel 40 and / or the substrate 42.

[0227] In this particular example, the analyte concentration increases with decreasing difference of the parameter, here with decreasing difference of the respective shape parameters. Thus, the difference of the parameter is indicative of the analyte concentration. The analyte concentration can be determined from the difference of the parameter.

[0228] Figs. 17 and 18 show another embodiment of a device 100 and a method according to the invention. Fig. 17 shows a device 100 with a sample 20 comprising assaying elements 30 flowing through the channel 40. Figure 18 shows a comparison of analyte concentrations detected and / or determined by a method according to the invention to analyte concentrations detected and / or determined by methods of the prior art. The assaying elements 30 may be magnetic, and / or may comprise magnetic assaying elements, e.g., magnetic beads.

[0229] The device 100 comprises a channel 40 with a substrate 42, e.g., a channel 40 and a substrate 42 as described above. The device 100 may have an external force generating device 120, e.g. a magnetic field generating device (not shown in Fig. 17), which is configured to generate an external force 60, e.g. a magnetic force generated by a magnetic field (not shown in Fig. 17). Further, the device 100 comprises a sensor unit 70. The sensor unit 70 comprises two sensing elements 72, 74. The second sensing element 74 may be arranged downstream of the first sensing element 72, e.g., downstream in flow direction (for instance downstream in x-direction in Fig. 17). The embodiment as shown in Figs. 17 and 18 may have one, several or all features and / or advantages of at least one other embodiment according to the invention.

[0230] The concentration of the analyte 10 in the sample 20 may be determined by determining a depletion of the assaying elements 30, e.g., of magnetic assaying elements, along the channel 40, cf., e.g., the embodiment as shown in Figs. 17 and 18.

[0231] Determining the depletion may be carried out, e.g., by means of differential counting. For instance, it may be provided that the sensor unit 70, the first sensing element 72 and / or the second sensing element 74 may detect, may register and / or may count the assaying elements 30, e.g., magnetic assaying elements, passing through, along or over them. In some embodiments, the sensor unit 70 and / or the sensing elements 72, 74 are or comp rise / comp rises a device configured for impedance sensing and / or magnetoresistive sensing. The assaying elements 30 may be configured to be detected and / or sensed by impedance sensing and / or by magnetoresistive sensing. Alternatively or additionally, the sensor unit 70 and / or the sensing element 72, 74 may be or may comprise a coulter counter. In some embodiments, the first sensing element 72 and / or the second sensing element 74 are or comp rise / comp rises a magnetic field sensor, and / or a magnetic sensor half bridge, respectively.

[0232] The differential counting maybe determined and / or measured between two sensing elements, e.g., between the first sensing element 72 and the second sensing element 74, cf., e.g., Fig. 17. Differential counting maybe based on comparing the count of assaying elements 30 as counted by two different and / or distinct sensing elements 72, 74. It may be provided that a sensing element outputs a signal which may be used to count and / or correspond to countings of assaying elements 30. “counting by a sensor unit and / or a sensing element” may comprise that the sensor unit and / or the sensing element outputs a suitable signal, and the counting is carried out by a control unit and / or by a computer unit. Alternatively or additionally, the differential counting may be repeated at different temperatures.

[0233] However, it should be understood that a differential counting may be determined and / or measured between multiple sensor elements 72, 74, 76, and / or between two sensor elements 72, 74 separated by another sensor element 76. Multiple differential countings may be carried out. For instance, a differential counting may be carried out by comparing the number of assaying elements 30 counted by a first sensing element 72 and counted by a second sensing element 74, and a second differential counting maybe carried out by comparing the counts of a third sensing element and of a fourth sensing element. For instance, the first, the second, the third and the fourth sensing element maybe different sensing elements 72, 74, 76. However, it may be provided that, e.g., the third sensing element corresponds to or is equal to the first or the second sensing element 72, 74. Similarly, different arrangements of sensing elements 72, 74, 76 and / or differential countings are possible. A differential count may be or may correspond to a difference of the number of counted assaying elements 30 of two respective sensing elements 72, 74.

[0234] A depletion of assaying elements 30 may comprise an immobilization of at least some of the assaying elements 30. For instance, when an assaying element 30 with an analyte 10 bonded thereto is stuck and / or immobilized on the substrate 42, e.g. by one or more surface binder elements 44, the sensing elements 72, 74 may count a different number of assaying elements 30. Since the number and / or the ratio of assaying elements 30 that are immobilized and / or stuck on the substrate 42 may depend on the analyte concentration, the higher the differential count, the higher the analyte concentration. Thus, the analyte concentration may depend on, may be correlated with and / or may be a function of the differential counting, and / or the differential counting may be employed to determine an analyte concentration.

[0235] In Fig. 18 the method according to the invention is compared to conventional assays. Whereas conventional assays may detect and / or may determine analyte concentrations over 2 to 3 orders of magnitude, the method and the device according to the invention may detect and / or may determine analyte concentrations over 3 to 4 orders of magnitude, or even more. Thus, the method and the device according to the invention allow for a significantly improved detection and / or determination of the analyte concentration over a wide range, and provide assays with higher range of detection. It may be provided that the ratio and / or number of stuck and / or immobilized assaying elements 30 of Fig. 30b, Fig. 31a and / or Fig. 31b is determined with differential counting.

[0236] Alternatively or additionally, optical methods may be used to determine the concentration of the analyte and / or to carry out the differential counting. The sensor unit 70 may be or may comprise one or more optical devices 78 (not shown in Fig. 17), e.g., one or more cameras. The sensor unit 70, the optical device 78 and / or the camera may take snapshots at specific times, and / or may capture photos or videos. The sensor unit 70, the optical device 78 and / or the camera may temporally and / or spatially capture the movement and / or the position of the assaying elements 30. The sensor unit 70, the optical device 78 and / or the camera may take snapshots at specific times, and / or may capture photos or videos in a field of view 79.

[0237] In some embodiments, more than one optical device 78 and / or more than one camera is provided, such as to have a larger total field of view 79 at sufficient resolution. It may be provided that different optical devices 78 and / or cameras have different fields of view 79, respectively. In some embodiments, at least two, multiple or all different fields of view 79 may at least partially overlap. Alternatively or additionally, it may be provided that at least two, multiple or all fields of view 79 do not overlap. A field of view (not shown in Fig. 17) may encompass the substrate 42, fully or at least part of it. The field of view may be along the flow direction, e.g., in x-direction of Fig. 17. Different and / or overlapping fields of view 79 may form a composite field of view and / or a total field of view. It may be provided that a first optical device 78 and / or camera is arranged upstream of the substrate 42 and / or such that its field of view 79 may cover a region upstream of the substrate 42. A second optical device 78 and / or camera may be arranged downstream of the substrate 42 and / or such that its field of view 79 may cover a region downstream of the substrate 42. The optical device 78 and / or the camera may detect one or more of color, size, luminescence and granularity of the assaying elements 30. Thereby, in some embodiments, multiplexing is enabled.

[0238] For instance, the movement and / or the position of the assaying elements 30, e.g., magnetic assaying elements, in the channel 40 and / or on the substrate 42, e.g., the moving or rolling along / on the substrate 42, may be captured. Then, the velocity and / or the position of the assaying elements 30 may, e.g., be determined by comparing different positions of the assaying elements 30 at different times. Immobilized and / or stuck assaying elements 30 may be identified and / or may be determined, e.g., by comparing the positions and / or the velocities at different times. The differential count may comprise, may correspond to and / or may be equal to the number of immobilized and / or stuck assaying elements 30.

[0239] Figs. 19 to 21 show another embodiment of a device too and a method according to the invention. For instance, determining a concentration of the analyte 10 and / or a depletion of assaying elements 30, e.g., of magnetic assaying elements, may be carried out by detection of jolting of assaying elements 30. Fig. 20 shows detected jolts, which occur when an assaying element 30 is stuck and / or immobilized, e.g., on the substrate 42. Fig. 21 shows a comparison of analyte concentrations detected and / or determined by a method according to the invention to analyte concentrations detected and / or determined by methods of the prior art. The embodiment as shown in Figs. 19 to 21 may have one, several or all features and / or advantages of at least one other embodiment according to the invention.

[0240] Fig. 19 shows a device too with a sample 20 comprising assaying elements 30 flowing through the channel 40. The device too comprises a channel 40 with a substrate 42, e.g., a channel 40 and / or a substrate 42 as described above. The device too may comprise an external force generating device 120, e.g., a magnetic field generating device (not shown in Fig. 19), which is configured to generate an external force 60 (not shown in Fig. 19), e.g., a magnetic force generated by a magnetic field. Further, the device too comprises a sensor unit 70. The sensor unit 70 comprises one or more optical devices 78. The optical device 78 may be or may comprise one or more cameras. The sensor unit 70, the optical device 78 and / or the camera may take snapshots at specific times, and / or may capture photos or videos. The sensor unit 70, the optical device 78 and / or the camera may temporally and / or spatially capture the movement and / or the position of the assaying elements 30.

[0241] In some embodiments, more than one optical device 78 and / or more than one camera is provided, such as to have a larger total field of view 79 at sufficient resolution. It may be provided that different optical devices 78 and / or cameras have different fields of view 79, respectively. In some embodiments, at least two, multiple or all different fields of view 79 at least partially overlap. Alternatively or additionally, it may be provided that at least two, multiple or all fields of view 79 do not overlap. Different and / or overlapping fields of view 79 may form a composite field of view and / or a total field of view.

[0242] A field of view 79 may encompass the substrate 42, fully or at least part of it. The field of view 79 may be along the flow direction, e.g., in x-direction of Fig. 19. It may be provided that a first optical device 78 and / or camera is arranged upstream of the substrate 42 and / or such that its field of view 79 may cover a region upstream of the substrate 42. A second optical device 78 and / or camera may be arranged downstream of the substrate 42 and / or such that its field of view 79 may cover a region downstream of the substrate 42. The optical device 78 and / or camera may detect one or more of color, size, luminescence and granularity of the assaying elements 30. Thereby, in some embodiments, multiplexing is enabled.

[0243] In some embodiments, determining a concentration of the analyte 10 is carried out by detection of jolting of assaying elements 30, e.g., of magnetic assaying elements 30, which may occur when bonding to at least one of the surface binder elements 44, cf. e.g. in the embodiment as shown in Figs. 19 to 21. The detection of jolting may be carried out by optical means, such as e.g. by employing the optical device 78 in Fig. 19.

[0244] When an analyte bonded assaying element 30 is stuck and / or immobilized on the substrate 42, the binding interaction may impart a force onto the assaying element 30 moving or rolling along / on the substrate 42, such that the assaying element 30 jolts. This jolting maybe detected by the sensor unit 70, e.g., optically and / or by the optical device 78, cf., e.g., Fig. 19. In Fig. 20, the signal spikes correspond to a binding of an analyte bonded assaying element 30 to at least one surface binder element 44, and / or to a jolt of an assaying element 30.

[0245] Detecting the jolting of assaying elements 30 may correspond to, may relate to and / or may correlate with differential counting. Thus, the concentration of the analyte 10 may be determined by detecting the jolting, and / or by counting the number of jolts. The analyte concentration may depend on, maybe correlated with and / or maybe a function of the jolting, and / or the detection of jolting maybe employed to determine an analyte concentration.

[0246] In Fig. 21 the method according to the invention is compared to conventional assays. Whereas conventional assays may detect and / or may determine analyte concentrations over 2 to 3 orders of magnitude, the method and device according to the invention may detect and / or may determine analyte concentrations over 3 to 4 orders of magnitude, or even more. Thus, the method and the device according to the invention allow for a significantly improved detection and / or determination of the analyte concentration over a wide range, and provide assays with higher range of detection. It may be provided that the ratio and / or number of stuck and / or immobilized assaying elements 30 of Fig. 30b, Fig. 31a and / or Fig. 31b is determined by detecting jolting of assaying elements 30.

[0247] Figure 22 shows a device too according to an embodiment of the invention, where a laminated flow 114 is employed. The laminated flow may be or may comprise a sheath flow. The device too comprises a channel 40 with a substrate 42, e.g., a channel 40 and a substrate 42 as described above. The substrate 42 comprises surface binder elements 44. The device too may comprise an external force generating device 120, e.g., a magnetic field generating device 120. The external force generating device 120 may be configured to generate an external force 60 and / or a magnetic field.

[0248] The laminated flow 114 may separate the sample flow 112 (which may correspond to the flow 110 of Figs. 1 to 21) from the substrate 42 in the channel 40. In some embodiments, the laminated flow 114 separates the sample flow 112 from the substrate 42 in the channel 40 along the full length of the channel 40, or at least partially. In some embodiments, the laminated flow 114 separates the sample flow 112 from the substrate 42 in the channel 40 at least over / above the substrate 42. In some embodiments, more than one laminated flow 114 is provided (not shown in Fig. 22). If more than one laminated flow 114 is provided, the laminated flows may be arranged and / or may flow over / above each other. The laminated flows may substantially flow in respective planes parallel to the surface of the substrate 42, where the planes may be arranged above each other with respect to a direction perpendicular to the surface of the substrate 42.

[0249] The external force 60, e.g., a magnetic force generated by a magnetic field, may be configured such that the assaying elements 30, e.g., magnetic assaying elements, are moved from the sample flow 112 into and / or through the laminated flow 114 towards the substrate 42. The external force 60 may pull or may attract the assaying elements 30 from the sample flow 112 into and / or through the laminated flow 114 towards the substrate 42. A height of the laminated flow 114, e.g., in z-direction in Fig. 22, maybe larger than the diameter of the assaying elements 30, or the diameter of the largest of the assaying elements 30.

[0250] By using a laminated flow 114, cellular or debris background from or in the sample 20 may be prevented from interfering with the moving or rolling of the assaying elements 30. The laminated flow 114 may reduce or may minimize interference from hematocrit. Figs. 23 to 25 show embodiments of devices too and methods according to the invention which are configured and / or used for multiplexing. The method and device according to the invention maybe suitable for carrying out heterogeneous assays. It may be provided that the sample 20 comprises or contains more than one, and / or multiple different kinds or types of analyte 10, at least one or multiple thereof, or respective concentrations, are to be detected and / or analyzed. For instance, in Figs. 23, 24 and / or 25, different exemplary embodiments which may determine the concentration of different types and / or kinds of analyte 10 are shown.

[0251] The respective devices too of Figs. 23, 24 and 25 comprise a respective channel 40 with a substrate 42, and a respective sensor unit 70 (not shown in Figs. 23 to 25). The devices too may comprise a respective external force generating device 120 (not shown in Figs. 23 to 25), e.g., a magnetic field generating device to generate a magnetic field. The embodiments as shown in Figs. 23, 24 and / or 25 may have one, more or all features and / or advantages of at least one other embodiment according to the invention. The assaying elements 30 may be or comprise magnetic assaying elements.

[0252] The substrate 42 may have different types and / or kinds of surface binder elements 44, 45, 46 for different types and / or kinds of analyte 10 to be detected and / or analyzed, cf., e.g., Figs. 23, 24 and 25. For instance, the substrate 42 may comprise first surface binder elements 44, second surface binder elements 45 and / or third surface binder elements 46. The first surface binder elements 44 may be configured to bind to a first kind and / or type of analyte 10, the second surface binder elements 45 may be configured to bind to a second kind and / or type of analyte 10, and / or the third surface binder elements 46 may be configured to bind to a third kind and / or type of analyte 10. The different kinds and / or types of surface binder elements 44, 45, 46 may be, e.g., arranged one behind the other, e.g., alternating, and / or in a substantially random arrangement. In some embodiments, the different kinds and / or types of surface binder elements 44, 45, 46 are arranged in homogeneous groups on portions of the substrate 42, cf., e.g., Fig. 25. It may be provided that the homogeneous groups are separated by a sensing element 76, cf., e.g., in the embodiment of Fig. 25.

[0253] The assaying elements 30 may have different sizes or diameters, cf., e.g., Fig. 23. It may be provided that all assaying elements 30 having substantially the same size or diameter have the same kind and / or type of capture binder elements 35, 36, 37 and / or may be configured to bind to the same type and / or kind of analyte 10. However, assaying elements 30 of different size or diameter may have different kinds and / or types of capture binder elements 35, 36, 37 and / or may be configured to bind to different types and / or kinds of analyte 10. The assaying elements 30 maybe grouped into specific and / or distinct groups, according to their size or diameter. It may be provided that all assaying elements 30 of the same group have the same kind and / or type of capture binder elements 35, 36, 37. It maybe provided that all assaying elements 30 of the same group have substantially the same size or diameter, are substantially identical or have substantially the same features.

[0254] For instance, assaying elements 30 having a first size or diameter may have first capture binder elements 35, assaying elements 30 having a second size or diameter may have second capture binder elements 36, and / or assaying elements 30 having a third size or diameter may have third capture binder elements 37. The first capture binder elements 35, the second capture binder elements 36, and / or the third capture binder elements 37 may be different. Thus, the assaying elements 30 of first size or diameter may be configured to bind to a first type and / or kind of analyte 10, the assaying elements 30 of second size or diameter may be configured to bind to a second type and / or kind of analyte 10, and the assaying elements 30 of third size or diameter maybe configured to bind to a third type and / or kind of analyte 10.

[0255] Alternatively or additionally, the assaying elements 30 may have different magnetic moments, cf., e.g., Fig. 24. For instance, some of the assaying elements 30 may be or may comprise a different material than some other of the assaying elements 30, such that their respective magnetic moments may differ.

[0256] The assaying elements 30 may be grouped into specific and / or distinct groups, according to their magnetic moment. It may be provided that all assaying elements 30 of the same group have the same kind and / or type of capture binder elements 35, 36, 37. It maybe provided that all assaying elements 30 of the same group have substantially the same magnetic moment, are substantially identical or have substantially the same features.

[0257] It may be provided that all assaying elements 30 having substantially the same magnetic moment have the same kind and / or type of capture binder elements 35, 36, 37 and / or are configured to bind to the same type and / or kind of analyte 10. However, assaying elements 30 of different magnetic moment may have different kinds and / or types of capture binder elements 35, 36, 37, and / or maybe configured to bind to different types and / or kinds of analyte 10. For instance, assaying elements 30 having a first magnetic moment may have first capture binder elements 35, assaying elements 30 having a second magnetic moment may have second capture binder elements 36, and / or assaying elements 30 having a third magnetic moment may have third capture binder elements 37. The first capture binder elements 35, the second capture binder elements 36, and / or the third capture binder elements 37 may be different. Thus, the assaying elements 30 of first magnetic moment maybe configured to bind to a first type and / or kind of analyte 10, the assaying elements 30 of second magnetic moment may be configured to bind to a second type and / or kind of analyte 10, and the assaying elements 30 of third magnetic moment may be configured to bind to a third type and / or kind of analyte 10.

[0258] It may be provided that some of the assaying elements 30, or groups of the assaying elements 30, have the same size, but differ in magnetic moment. It may be provided that some of the assaying elements 30, or groups of the assaying elements 30, have the same magnetic moment, but differ in size. Alternatively, some of the assaying elements 30, or groups of the assaying elements 30, may differ both in size and in magnetic moment.

[0259] When the assaying elements 30 of specific size and / or of specific magnetic moment have specific capture binder elements 35, 36, 37 such as to bind to a specific analyte 10, the velocity decrease of the assaying elements 30 of different size and / or of different magnetic moment may be employed to determine the concentration of the different specific analytes 10. Thus, multiplexing and / or differentiation between different analytes 10 is possible.

[0260] Assaying elements 30 of different size or diameter may be distinguished by the sensor unit 70 and / or by the sensing elements 72, 74, 76. Similarly, assaying elements 30 of different magnetic moment maybe distinguished by the sensor unit 70 and / or by the sensing elements 72, 74, 76. Alternatively and / or additionally, an optical device 78 maybe used, e.g. to determine the size and / or the velocity of the assaying elements 30, and thus the associated analyte concentration.

[0261] Alternatively or additionally, at least two assaying elements may have different acoustic properties, different electrical properties. It may be provided that the assaying elements may be distinguished or grouped according to their different acoustic properties, different electrical properties, and / or different optical properties.

[0262] The optical property may comprise a color or a marker, such that e.g. an assaying element may be colored or may be a marked assaying element. The acoustic property may comprise a compressibility and / or a density, such as e.g. a compressibility and / or density of an assaying element. The electric property may comprise an electrical impedance and / or an electrical charge, such as e.g. an electrical impedance and / or electrical charge of an assaying element.

[0263] In some embodiments, the assaying elements maybe distinguished, grouped and / or identified according to their acoustic properties, electrical properties, and / or optical properties. For instance, the device too may be configured to detect the acoustic properties, electrical properties, and / or optical properties. In some embodiments, the sensor unit 70 is configured to detect the acoustic properties, electrical properties, and / or optical properties. In some embodiments, the optical device 78 may detect differently colored or marked assaying elements.

[0264] When the assaying elements 30 having a specific acoustic property, electrical property, and / or optical property have specific capture binder elements 35, 36, 37 such as to bind to a specific analyte 10, the velocity decrease of the assaying elements 30 having different acoustic properties, electrical properties, and / or optical properties may be employed to determine the concentration of the different specific analytes 10. Thus, multiplexing and / or differentiation between different analytes 10 is possible.

[0265] Thus, referring, e.g., to Figs. 23 and 24, the concentration of the respective different types and / or kinds of analyte 10 may be determined by determining a decrease of the velocity of the assaying elements 30 along the channel 40, and / or by determining a depletion of the assaying elements 30 as described above. For this, there may be provided at least two sensing elements 72, 74 (not shown in Figs. 22, 23 and 24), e.g., the first sensing element 72 being arranged upstream of the surface binder elements 44, 45, 46 and the second sensing element 74 being arranged downstream of the surface binder elements 44, 45, 46. Since the sensor unit 70 and / or the sensing elements 72, 74 may distinguish the different kinds of assaying elements 30 (e.g. as defined by their different size and / or different magnetic moment) a decrease of the velocity of the assaying elements 30 and / or a depletion of each assaying elements 30 of different size and / or of different magnetic moment may be detected, e.g. for each group of assaying elements 30 of different size and / or of different magnetic moment may be detected. Since assaying elements 30 of different size and / or of different magnetic moment are associated with different types and / or kinds of analyte 10, the concentration of analytes 10 may be determined as outlined above. Alternatively or additionally, an optical device 78 may be used, e.g. to determine the velocity, the number and / or the size of the assaying elements 30 and thus the associated analyte concentration.

[0266] Similarly, a velocity decrease of depletion of assaying elements 30 having different acoustic properties, electrical properties, and / or optical properties maybe determined.

[0267] Alternatively or additionally, referring to Fig. 25, it may be provided that the assaying elements 30, all of the assaying elements 30 or at least some of the assaying elements 30, have multiple different types and / or kinds of capture binder elements 35, 36, 37, or groups of different kinds and / or types of capture binder elements 35, 36, 37. It may be provided that the assaying elements 30, all of the assaying elements 30 or at least some of the assaying elements 30, do neither differ in size or diameter nor in magnetic moment (or do not have different acoustic properties, electrical properties, and / or optical properties), but have multiple different types and / or kinds of capture binder elements 35, 36, 37, and / or groups of different kinds and / or types of capture binder elements 35, 36, 37. For instance, an assaying element 30 may have one or more capture binder elements 35 of first type and / or kind, one or more capture binder elements 36 of second type and / or kind, and / or one or more capture binder elements 37 of third type and / or kind. The first capture binder elements 35, the second capture binder elements 36, and / or the third capture binder elements 37 may be different. The first capture binder elements 35 may be configured to bind to a first type and / or kind of analyte 10, the second capture binder elements 36 may be configured to bind to a second type and / or kind of analyte 10, and the third capture binder elements 37 may be configured to bind to a third type and / or kind of analyte 10. Correspondingly, the assaying elements 30 may capture analytes 10 of first type and / or kind, analytes 10 of second type and / or kind, and / or analytes 10 of third type and / or kind.

[0268] In the embodiment of Fig. 25, multiplexing and / or differentiating between different analytes 10 are / is carried out by providing homogeneous groups of surface binder elements 44, 45, 46. The surface binder elements 44, 45, 46 of one specific homogeneous group may be configured to bind to one specific analyte 10. Then, the decrease of the velocity of the assaying elements 30 and / or of the analyte bonded assaying elements 30, along / on the substrate 42 in the regions of the specific homogeneous groups may be employed to determine the concentration of the corresponding specific analyte 10. Thus, multiplexing and / or differentiation between different analytes 10 are / is possible.

[0269] The decrease of the velocity of the assaying elements 30 along the channel, and / or the depletion of the assaying elements 30 may be determined by two sensing elements 76 separated by a homogeneous group of surface binder elements 44, 45, 46, respectively, as, e.g., seen in Fig. 25.

[0270] For instance, when a first type of analyte 10 may bind to first surface binder elements 44, assaying elements 30 bound to the first analyte 10 may be slowed down and / or may be immobilized on the substrate 42 in the region of the homogeneous group of first surface binder elements 44. Thus, for determining the analyte concentration of a first type and / or kind of analyte 10, the decrease of the velocity of the assaying elements 30 along the channel 40, and / or the depletion of the assaying elements 30 may be determined as outlined above by the first sensing element 76 arranged upstream of and adjacent to the homogeneous group of first surface binder elements 44 and by the second sensing element 76 arranged downstream of and adjacent to the homogeneous group of first surface binder elements 44. In other words, the change and / or decrease of the velocity of the assaying elements 30 along the channel 40, and / or the depletion of the assaying elements 30 maybe determined by the sensing elements 76 sandwiching the homogeneous group of first surface binder elements 44.

[0271] Similarly, when a second type of analyte 10 may bind to second surface binder elements 45, assaying elements 30 bound to the second analyte 10 may be slowed down and / or may be immobilized on the substrate 42 in the region of the homogeneous group of second surface binder elements 45. Thus, for determining the analyte concentration of a second type and / or kind of analyte 10, the decrease of the velocity of the assaying elements 30 along the channel 40, and / or the depletion of the assaying elements 30 maybe determined as outlined above by the first sensing element 76 arranged upstream of and adjacent to the homogeneous group of second surface binder elements 45 and by the second sensing element 76 arranged downstream of and adjacent to the homogeneous group of second surface binder elements 45. In other words, the decrease of the velocity of the assaying elements 30 along the channel 40, and / or the depletion of the assaying elements 30 may be determined by the sensing elements 76 sandwiching the homogeneous group of second surface binder elements 45.

[0272] Similarly, when a third type of analyte 10 may bind to third surface binder elements 46, assaying elements 30 bound to the third analyte 10 may be slowed down and / or may be immobilized on the substrate 42 in the region of the homogeneous group of third surface binder elements 46. Thus, for determining the analyte concentration of a third type and / or kind of analyte 10, the decrease of the velocity of the assaying elements 30 along the channel 40, and / or the depletion of the assaying elements 30 maybe determined as outlined above by the first sensing element 76 arranged upstream of and adjacent to the homogeneous group of third surface binder elements 46 and by the second sensing element 76 arranged downstream of and adjacent to the homogeneous group of third surface binder elements 46. In other words, the decrease of the velocity of the assaying elements 30 along the channel 40, and / or the depletion of the assaying elements 30 may be determined by the sensing elements 76 sandwiching the homogeneous group of third surface binder elements 46.

[0273] Alternatively or additionally, one or more optical devices 78 may be used, e.g., to determine the size, the velocity and / or the number of the assaying elements 30, e.g., in the regions of the respective homogeneous groups of surface binder elements 44, 45, 46, and thus the associated analyte concentration. While with reference to Figs. 23, 24 and 25 three different types and / or kinds of analyte 10, of assaying elements 30, of capture binder elements 35, 36, 37 and / or of surface binder elements

[0274] 44, 45, 46 are described, the number of analytes 10, assaying elements 30, capture binder elements 35, 36, 37 and / or surface binder elements 44, 45, 46 is not limited thereto. Similarly, the arrangement of surface binder elements 44, 45, 46 may be different and / or not limited thereto. It may be provided that different assaying elements 30 as described and or characterized above are mixed and / or used.

[0275] In some embodiments, at least one, multiple or all capture binder elements 35, 36, 37 is or comprises a tetraspanin, e.g., CD81, CD63 and / or CD9. In some embodiments, at least one, multiple or all surface binder elements 44, 45, 46 is or comprises a tetraspanin, e.g., CD81, CD63 and / or CD9.

[0276] It may be provided that the capture binder elements 35, 36, 37 differ, but the surface binder elements 44, 45, 46 may be the same, i.e. may be of same kind and / or type. In some embodiments, the surface binder elements 44, 45, 46 may be ubiquitous for all analytes 10 of interest.

[0277] In some embodiments, it may be provided that there is only one type and / or kind of capture binder elements 35, 36, 37, e.g. some type or kind of antibody for all capture binder elements 35? 36, 37, and only one type and / or kind of surface binder elements 44, 45, 46, e.g. some type or kind of antibody for all surface binder elements 44, 45, 46. The type and / or kind of the capture binder elements 35, 36, 37 may be the same as that of the surface binder elements 44,

[0278] 45, 46. Alternatively, the type and / or kind of the capture binder elements 35, 36, 37 may be different from that of the surface binder elements 44, 45, 46. In some embodiments, it may be provided that the capture binder elements 35, 36, 37 of at least one, multiple or all assaying elements 30 are of the same type and / or kind as at least some, multiple or all of the surface binder elements 44, 45, 46.

[0279] It may be provided that the capture binder elements 35, 36, 37 of all assaying elements 30 are the same, i.e. are the same kind and / or type, but the surface binder elements 44, 45, 46 differ. In some embodiments, the capture binder elements 35, 36, 37 of at least one, multiple or all assaying elements 30 match at least one, multiple or all of the surface binder elements 44, 45,

[0280] 46, such that the respective capture binder elements 35, 36, 37 and the surface binder elements 44, 45, 46 form matched antibody pairs. It may be provided that the embodiments as described with reference to Figs. 23, 24 and 25 are combined. For instance, the surface binder elements 44, 45, 46 may be arranged in homogeneous groups, and the size and / or the magnetic moments (or acoustic properties, electrical properties, and / or optical properties) of the assaying elements 30 may differ.

[0281] When the assaying elements 30 are spherical or substantially sphere-shaped, they may move or roll along / on the substrate 42 such that substantially the same part of their surface repeatedly contacts the substrate 42 when moving or rolling. For instance, the assaying elements 30 may move or roll substantially along a circumferential disc on their surface. Thus, the analyte 10 bound to the capture binder elements 35, 36, 37 arranged outside said circumferential disc may not come into contact with the substrate 42 and / or with the surface binder elements 44, 45, 46, at all, or at least not as often as the analyte 10 bound to the capture binder elements 35, 36, 37 arranged on / within said circumferential disc.

[0282] Thus, in some embodiments, it may be provided that the assaying elements 30 moving or rolling on the substrate 42 are manipulated, and / or that an orientation or a moving or rolling direction of the assaying elements is varied. Figs. 26, 27 and 28 show some corresponding exemplary embodiments according to the invention.

[0283] The embodiments as shown in Figs. 26, 27 and 28 comprise at least one, several and / or all features and / or advantages of a device too and / or of a method as described herein, for instance but not limited to those described above and / or shown in Figs. 1 to 25. Particularly, the embodiment as shown in Figs. 26, 27 and 28 comprise or include one or more of a channel 40, an external force generating device 120, e.g., a magnetic field generating device, an analyte 10 and / or a sample 20, e.g., as seen in Fig. 1 or 2, which are not shown in Figs. 26, 27 and / or 28. The assaying elements 30 may be magnetic, and / or may comprise magnetic assaying elements.

[0284] In some embodiments, the channel 40 has a pattern 48, as may be seen, e.g., in Figs. 26, 27 and 28. The pattern 48 may be configured to change, to manipulate or to vary the movement, the direction and / or the orientation of the assaying elements 30, e.g., when flowing within the channel 40 and / or when moving or rolling along / on the substrate 42. The pattern 48 may change, may manipulate or may vary the movement, the direction and / or the orientation of the assaying elements 30 perpendicular or at least at an angle greater than o degrees, preferably 10 degrees or less, more preferably 6 degrees or less, relative to the flow direction, e.g. in x-direction in Figs. 26, 27, and 28. In some embodiments, said angle is maximally 150, more preferably maximally io°. The pattern 48 may deflect and / or may divert the assaying elements 30 flowing within the channel 40 and / or moving or rolling along / on the substrate 42. The pattern may exert a force onto an assaying element 30 resulting in a change of movement, direction or orientation. The pattern 48 may be or may comprise a mechanical and / or a magnetic structure suitable for manipulating and / or varying the movement, the direction and / or the orientation of the assaying elements 30 relative to the flow direction.

[0285] The pattern 48 may be a mechanical pattern 48. The mechanical pattern 48 may be and / or may comprise notches, recesses, ridges or the like. Alternatively or additionally, the pattern 48 may by a magnetic pattern 48. The magnetic pattern 48 may exert a magnetic force, e.g., as indicated by reference numeral 130 in Fig. 28, onto the assaying elements 30 such as to manipulate or vary the movement, the direction or the orientation of the assaying elements 30. The magnetic pattern 48 may modify or may modulate a magnetic field, e.g., a magnetic field provided by a magnetic field generating device.

[0286] In some embodiments, the pattern 48 is arranged in the channel 40, e.g., in and / or on an inner sidewall of the channel 40, but not overlapping with the substrate 42, cf. e.g. the exemplary embodiment as seen in Fig. 26. In some embodiments, the pattern 48 is arranged in the channel 40, e.g., in and / or on an inner sidewall of the channel 40, but not in and / or on the substrate 42, cf., e.g., Fig. 26. In some embodiments, the pattern 48 is arranged upstream and / or downstream of the substrate 42. In some embodiments, the pattern 48 only overlaps with the substrate 42, and / or is only on and / or in the substrate 42, cf., e.g., Fig. 27. Alternatively or additionally, the pattern 48 may at least partially overlap the substrate 42, and / or maybe arranged on and / or in the substrate 42, cf., e.g., Figs. 27 or 28.

[0287] The pattern 48 may be or may comprise chevrons. The pattern 48 may be or may comprise a chevron pattern. The pattern 48 maybe chevron-like or may have a chevron form.

[0288] Alternatively or additionally, the orientation and / or direction of movement of the assaying elements may be changed and / or varied by the external force. In some embodiments, the external force may be controlled accordingly, and / or may change or differ over time and / or locally.

[0289] Alternatively or additionally, the shape of an assaying element 30 may deviate from a sphere, or may be not perfectly spherical (not shown in the figures). Then, the movement of such an assaying element 30 when moving rolling may vary (during the time-of-roll), e.g., perpendicular to the flow direction such as in y-direction in Figs. 26, 27, and / or 28. In some embodiments the channel 40 is tapered (cf. the exemplary embodiment as shown in Fig. 29), at least partially or completely. For instance, the channel 40 may be tapered in a direction perpendicular to the flow direction, e.g., perpendicular to the x-direction in Fig. 29. In some embodiments, the channel 40 is tapered in y-direction and / or in z-direction. When the channel 40 is tapered, the cross-section may vary. When the cross-section increases, the flow velocity may decrease. The channel 40 maybe tapered such that its cross-section increases in flow direction, e.g., in x-direction in Fig. 29. In some embodiments, the assaying elements 30 are guided and / or directed to the sensing unit 70 by the tapering of the channel 40. The channel 40 may be tapered such that the assaying elements 30 may be guided and / or directed to the sensor unit 70, and / or to a sensing element 72, 74, 76.

[0290] When the channel 40 is tapered, the driving force on the assaying elements 30 by the flow 110 may decrease, and the velocity of an analyte bonded assaying element 30 may be further decreased. Alternatively or additionally, the likelihood of an analyte bonded assaying element 30 to be immobilized and / or stuck maybe increased.

[0291] In a preferred embodiment, the device too comprises a second external force generating device (not shown in the figures) which is configured to generate a second external force. The second external force generating device may be arranged downstream of the sensor unit 70, and / or downstream of the most downstream sensing element 74 or downstream of the most downstream optical device 78. In some embodiments, the second external force generating device is or comprises a second magnetic field generating device (not shown in the figures) and the second external force is a magnetic force. The second magnetic field generating device may be or may comprise a permanent magnet. The second external force generating device may be configured to generate the second external force varying in amplitude and / or spatial direction. The second external force may vary over time. It may be provided that the second external force generating device is a sorting unit for sorting assaying elements 30. For instance, the second external force may be used to sort assaying elements 30, e.g., sorting the assaying elements 30 downstream of the sensor unit 70. By sorting, the assaying elements 30 may be sorted into different groups. In some embodiments, different kinds / or types of analyte are bound to assaying elements 30 of different groups. Assaying elements 30 of different groups, and / or analyte bound to the assaying elements 30 of the different groups, may be used for further analysis or diagnostics.

[0292] The second external force may be a second magnetic force, but is not necessarily limited thereto. The second external force may be different from the external force, e.g. may differ in amplitude, power and / or frequency. It may be provided that the second external force is controlled, and / or may vary, e.g. overtime and / or locally.

[0293] Alternatively or additionally, the orientation and / or direction of movement of the assaying elements may be changed and / or varied by the second external force. In some embodiments, the second external force may be controlled accordingly, and / or may change or differ over time and / or locally.

[0294] Figs 30 and 31 show exemplary measurements as carried out according to the disclosure. In Fig. 30a, the analyte 10 maybe or may comprise e.g. Her2neu+ EVs, the concentration of which in the sample has been assayed according to the disclosure. In Fig. 30a, the mean velocity difference as well as the mean delay of assaying elements 30 has been measured with a method and / or a device according to the disclosure. In some embodiments, the capture binder elements 35, 36, 37 and / or the surface binder elements 42 are or comprise anti-Her2neu antibodies. The sample maybe or may comprise a buffer, e.g., a suitable chemical. In Fig. 30a, the mean velocity difference corresponds to a change of velocity of the assaying elements 30. The mean delay corresponds to a corresponding time of roll, as, e.g., determined by the sensor unit 70. As can be seen, the mean velocity difference and the mean delay increases monotonically with the concentration of the analyte 10. Thus, the concentration can be determined from the mean velocity difference and / or the mean delay.

[0295] In Fig. 30b, the analyte 10 may be or may comprises IL-6, the concentration of which in the sample has been assayed according to the disclosure. In Fig. 30b, the ratio of stuck and / or immobilized assaying elements 30 (relative immobilized particles) is shown as function of the concentration of the analyte, here, e.g., IL-6, in the sample. The ratio of stuck and / or immobilized assaying elements 30 can be determined, e.g., by differential counting, and / or by detection of jolting of the assaying elements 30. The stuck and / or immobilized assaying elements 30 have been determined according to the disclosure. In some embodiments, the capture binder elements 35, 36, 37 and / or the surface binder elements 42 are or comprise anti- IL6 antibodies. The sample may be or may comprise a buffer, such as e.g. a suitable chemical. As can be seen, the ratio of stuck and / or immobilized assaying elements 30 increases monotonically with the concentration of the analyte 10. Thus, the concentration can be determined from the mean velocity difference and / or the mean delay. Moreover, the concentration can be determined in a range of nearly four decades and / or order of magnitude. For example, in Fig. 30b, the range of detection spans concentrations between around 1 ng / mL and 10000 ng / mL. In Fig. 31a and fig. 31b, flow cytometry has been employed to determine the ratio of stuck and / or immobilized assaying elements 30 (corresponding to the normalized signal). In Fig. 31a and 31b, optical flow cytometry and magnetic flow cytometry are compared. Fig. 31a shows the concentration of analyte 10 in a buffer, and Fig. 31b the concentration of the analyte 10 in a buffer (for the optical flow cytometry) and in a blood sample (for the magnetic flow cytometry). The ratio of stuck and / or immobilized assaying elements 30 has been determined according to a method and / or device of the disclosure. The ratio of stuck and / or immobilized assaying elements 30 can be determined, e.g., by differential counting, and / or by detection of jolting of the assaying elements 30. In Fig. 31a and 31b, the ratio of stuck and / or immobilized assaying elements 30 (relative immobilized particles) is shown as function of the concentration of the analyte, here biotin, in the sample. In some embodiments, the capture binder elements 35? 36, 37 and / or the surface binder elements 42 are or comprise avidin. As can be seen, the measured ratio of stuck and / or immobilized assaying elements 30 as determined by the two respective measurements (i.e. by optical flow cytometry and magnetic flow cytometry) are in quite close agreement. In particular, the ratio of stuck and / or immobilized assaying elements 30 can be determined accurately also for samples which are opaque (and where optical methods may fail), e.g., in blood. Thus, the magnetic flow cytometry is suitable, e.g., to determine the ratio (or number) of stuck and / or immobilized assaying elements 30 in opaque samples, and thus also the concentration of analyte 10. As can be seen, the ratio of stuck and / or immobilized assaying elements 30 increases monotonically with the concentration of the analyte 10. Thus, the concentration can be determined from the mean velocity difference and / or the mean delay. Moreover, the concentration can be determined in a range of nearly four decades and / or order of magnitude. For example, in Fig. 31a and 31b, the range of detection spans concentrations between around o mg / mL and 100 ng / mL.

[0296] In Fig. 31, an exemplary velocity decrease as function of the analyte concentration in a sample 20 is shown. In this example, the assaying elements 30 have capture binder elements 35, the substrate 42 has surface binder elements 44, and both the capture binder elements 35 and the surface binder elements 44 are configured to bind to the analyte 10 in the sample 20. The analyte may comprise one or more of CD9+ / 63+ / 81+.

[0297] In this example, the velocity decrease increases monotonically. In particular, the velocity decrease increases with increasing analyte concentration, such that the determined velocity decrease is indicative of the analyte concentration. In other words, the concentration of analyte 10 in the sample 20 can be determined from the velocity decrease. The disclosure is not limited to the specific exemplary embodiments as shown and / or discussed. Only those features as given by the independent claims may be required and / or necessary for some embodiments. It may be provided that an embodiment has only some features of some other embodiment. The features as disclosed in the claims, the specification and the figures maybe relevant for the realization of the invention in any combination thereof.

[0298] List of reference numerals analyte sample assaying element

[0299] (first) capture binder element second capture binder element third capture binder element element test analyte channel substrate

[0300] (first) surface binder element second surface binder element third surface binder element surface test analyte pattern external force sensor unit first sensing element second sensing element sensing element optical device first distribution function second distribution function second distribution function device flow sample flow laminated flow external force generating device force

Claims

Claims: i. A method for detecting a concentration of an analyte (to) in a sample (20), the method comprising: providing a device (too) having a channel (40), the channel (40) having a substrate (42), the substrate (42) having surface binder elements (44, 45, 46) configured to bind to the analyte (10) in the sample and / or the substrate (42) having a surface test analyte (47); mixing assaying elements (30), preferably magnetic assaying elements (30), into the sample (20), the assaying elements (30) having capture binder elements (35, 36, 37) configured to bind to the analyte (10) in the sample and / or the assaying elements (30) having an element test analyte (38) configured to bind to the surface binder elements; flowing the sample (20) through the channel (40); moving the assaying elements (30) to the substrate (42) with an external force (60), preferably with a magnetic force; moving the assaying elements (30) along / on the substrate (42) and changing the velocity of at least one assaying element (30) by an interaction, wherein the interaction comprises one or more of:(i) an interaction, preferably a binding interaction, between at least one surface binder element (44, 45, 46) of the substrate (42), the analyte (10) and at least one capture binder element (35, 36, 37) of said at least one assaying element (30);(ii) an interaction, preferably a binding interaction, between the surface test analyte (47) of the substrate (42) and at least one of the capture binder elements (35, 36, 37) of said at least one assaying element (30); and / or(iii) an interaction, preferably a binding interaction, between the element test analyte (38) of said at least one assaying element (30) and at least one of the surface binder elements (44, 45, 46) of the substrate (42); and determining a concentration of the analyte (10) using a sensor unit (70) by determining a change of velocity of the assaying elements (30) along the channel (40) and / or by determining a depletion of the assaying elements (30) along the channel (40), preferably by differential counting of the assaying elements (30), and / ordetermining binding kinetics, preferably binding constants, between the analyte (to) and the capture binder elements (35, 36, 37) and / or between the analyte (10) and the surface binder elements (44, 45, 46), wherein the binding kinetics are determined using the sensor unit (70) by determining a change of velocity of the assaying elements (30) along the channel (40).

2. The method of claim 1, wherein moving the assaying elements (30) along / on the substrate (42) comprises rolling the assaying elements (30) along / on the substrate (42).

3. The method of any of the preceding claims, wherein the surface test analyte is of the same kind and / or type as the analyte, and / or wherein the element test analyte is of the same kind and / or type as the analyte.

4. The method of any of the preceding claims, wherein determining the concentration of the analyte (10) comprises determining a time-of-roll between a first sensing element (72, 76) and a second sensing element (74, 76).

5. The method of claim 4, wherein the first sensing element (72, 76) is arranged upstream of the substrate (42), and wherein the second sensing element (74, 76) is arranged downstream of the substrate (42) and / or downstream of the first sensing element (72, 76),6. The method of claim 4 or 5, wherein the first sensing element (72, 76) is or comprises a magnetic field sensor, and / or wherein the second sensing element (74, 76) is or comprises a magnetic field sensor.

7. The method of any of the preceding claims, wherein determining the change of velocity comprises determining a first distribution function (80) of the assaying elements (30) at a first position in the channel (40) and a second distribution function (82, 83) of the assaying elements at a second position in the channel (40), wherein the first distribution function (80) is a distribution function of the time difference between the timepoint of a respective assaying element (30) crossing the first position and a first reference timepoint, and the second distribution function (82, 83) is a distribution function of the time difference between the timepoint of a respective assaying element crossing the second position and a second reference timepoint.

8. The method of claim 7 with further reference to any of claims 4 to 6, wherein the first position is a position of the first sensing element (72, 76) and / or wherein the second position is a position of the second sensing element (74, 76).

9. The method of any of the preceding claims 7 or 8, wherein the first distribution function (80) comprises a density function or a histogram and wherein the second distribution function (82, 83) comprises a density function or a histogram, wherein the change of velocity is determined by a time difference between a peak, preferably a maximum value, of the second distribution function (82, 83) and a peak, preferably a maximum value, of the first distribution function (80).

10. The method of any of the preceding claims 7 or 8, wherein the first distribution function (80) comprises a cumulative function and wherein the second distribution function (82, 83) comprises a cumulative function, wherein the change of velocity is determined by a time difference between an inflection point of the second distribution function (82, 83) and an inflection point of the first distribution function (80).

11. The method of any of the preceding claims 7 or 8, wherein the change of velocity is determined by a distance metric of the second distribution function (82, 83) and the first distribution function (80), and / or by a change of shape of the second distribution (82, 83) function compared to the first distribution function (80).

12. The method of claim 11, wherein the change of shape is determined by fitting the first distribution function (80) and the second distribution function (82, 83) to the same candidate distribution function, and comparing the respective fitting parameters.

13. The method of claim 12, wherein the candidate distribution function is a normal distribution function, a gamma distribution function, a lognormal distribution function or a Weibull distribution function.

14. The method of any of the preceding claims 7 to 13, wherein the first reference timepoint is equal to the second reference timepoint.

15. The method of any of the preceding claims 11 to 13, wherein the first reference timepoint is equal to the timepoint of a first crossing of the first position by an assaying element (30), and / or wherein the second reference timepoint is equal to the timepoint of a first crossing of the second position by an assaying element (30).

16. The method of any of the preceding claims, wherein determining the concentration of the analyte (10) comprises an optical detection, wherein the optical detection preferably comprises capturing a movement and / or a position of the assaying elements (30) spatially and / or temporally resolved in a field of view (79).

17. The method of any of the preceding claims, wherein changing the velocity comprises immobilizing said at least one of the assaying elements (30).

18. The method of any of the preceding claims, wherein the external force (60) and a flow velocity of the sample (20) are adjusted such that at least one of the assaying elements (30) rolls along / on the substrate (42).

19. The method of any of the preceding claims, wherein the external force (60) is adjusted such that the assaying elements (30) are pushed away from the substrate (42) and / or sorted, preferably after determining the concentration of the analyte (10).

20. The method of any of the preceding claims, wherein in the channel (40) a laminated flow (114) is flown between the sample (20, 112) and the substrate (42) such as to separate the sample (20, 112) from the substrate (42), and wherein the assaying elements (30) are moved by the external force (60) from the sample (20, 112) through the laminated flow (114) to the substrate (42).

21. The method of any of the preceding claims, wherein at least one of the assaying elements (30) has at least two different capture binder elements (35, 36, 37), wherein the different capture binder elements (35, 36, 37) are configured to bind to different analytes (10) and / or wherein the different capture binder elements (35, 36, 37) are configured to bind to the same analyte (10).

22. The method of any of the preceding claims, wherein the capture binder elements (35, 36, 37) of at least one or all assaying elements (30) are of the same type and / or kind as at least one or all surface binder elements (44, 45, 46).

23. The method of any of the preceding claims, wherein at least two of the assaying elements (30) have different sizes, different magnetic moments, different acoustic properties, different electrical properties, and / or different optical properties.

24. The method of any of the preceding claims, wherein the assaying elements (30) comprise at least two groups of assaying elements (30) and the assaying elements (30) of each group have the same type and / or kind of capture binder elements (35, 36, 37).

25. The method of any of the preceding claims, wherein the substrate (42) has at least two different surface binder elements (44, 45, 46), wherein the different surface binder elements (44, 45, 46) are configured to bind to different analytes (10), wherein preferably the different surface binder elements (44, 45, 46) are grouped into homogeneous groups, preferably separated by sensing elements (76).

26. The method of any of the preceding claims, wherein an orientation and / or a rolling direction of the assaying elements (30) are / is varied when the assaying elements (30) are moving along / on the substrate (42) or when the assaying elements (30) are moving downstream of the substrate (42) or downstream of the sensor unit (70).

27. The method of any of the preceding claims, wherein a second external force acts on the assaying elements (30) when the assaying elements (30) are moving downstream of the substrate (42) and / or downstream of the sensor unit (70), the second external force preferably sorting the assaying elements (30).

28. A device (too) for detecting a concentration of an analyte (10) in a sample (20), the device (too) comprising: a channel (40), wherein the channel (40) has a substrate (42), the substrate (42) having surface binder elements (44, 45, 46) and / or the substrate (40) having a surface test analyte (47), the channel (40) being configured such that a sample (20) comprising an analyte (10) and assaying elements (30), the assaying elements (30) having capture binder elements (35, 36, 37) and / or having element test analyte (38), can flow therethrough; wherein the device (too) is configured such that, when the sample (20) that comprises the analyte (10) and assaying elements (30) flows through the channel, an external force (60) moves the assaying elements (30) to the substrate (42) and the assaying elements (30) move along / on the substrate (42), wherein the device is further configured such that when the assaying elements (30) move along / on the substrate (42), the velocity of at least one assaying element is changed by an interaction, the interaction comprising one or more of:(i) an interaction, preferably a binding interaction, between at least one surface binder element (44, 45, 46) of the substrate (42), the analyte (10)and at least one capture binder element (35, 36, 37) of said at least one assaying element (30);(ii) an interaction, preferably a binding interaction, between the surface test analyte of the substrate and at least one of the capture binder elements (35, 36, 37) of said at least one assaying element (30); and / or(iii) an interaction, preferably a binding interaction, between the test analyte of said at least one assaying element and at least one of the surface binder elements (44, 45, 46); and the device further comprising a sensor unit (70) configured to determine a change of velocity of the assaying elements (30) along the channel (40), and / or to determine a depletion of the assaying elements (30) along the channel (40), preferably by differential counting of the assaying elements (30), and / or to determine binding kinetics, preferably binding constants, between the analyte (10) and the capture binder elements (35, 36, 37) and / or between the analyte (10) and the surface binder elements (44, 45, 46).

29. The device (too) of claim 28, the device (too) comprising an external force generating device (120) configured to generate the external force (60).

30. The device (too) of claim 29, wherein the external force generating device (120) is or comprises a magnetic field generating device and the external force (60) is a magnetic force, wherein preferably said magnetic field generating device is or comprises a permanent magnet.

31. The device (too) of any of the preceding claims claim 28 to 30, wherein the sensor unit (70) comprises a first sensing element (72, 76) and a second sensing element (74, 76), wherein the sensor unit (70) is configured to determine the concentration of the analyte (10) by determining a time-of-roll between the first sensing element (72, 76) and the second sensing element (74, 76),32. The device (too) of claim 31, wherein the first sensing element (72, 76) is arranged upstream of the substrate (42), wherein preferably the second sensing element (74, 76) is arranged downstream of the first sensing element (72, 76) and / or the substrate (42), wherein preferably the first sensing (72, 76) element and / or the second sensing element (74, 76) comprise / comprises or are / is a magnetic field sensor, respectively.

33. The device (too) of any of the preceding claims claim 28 to 32, wherein the sensor unit (70) is configured to determine the change of velocity by determining a first distributionfunction (80) of the assaying elements (30) at a first position in the channel (40) and a second distribution function (82, 83) of the assaying elements (30) at a second position in the channel (40), wherein the first distribution function (80) is a distribution function of the time difference between the timepoint of a respective assaying element (30) crossing the first position and a first reference timepoint, and the second distribution function (82, 83) is a distribution function of the time difference between the timepoint of a respective assaying element (30) crossing the second position and a second reference timepoint.

34. The device (too) of claim 33 with further reference to claim 32, wherein the first position is a position of the first sensing element (72, 76) and / or wherein the second position is a position of the second sensing element (74, 76).

35. The device (too) of any of the preceding claims claim 33 or 34, wherein the first distribution function (80) comprises a density function or a histogram and wherein the second distribution function (82, 83) comprises a density function or a histogram, wherein the change of velocity is determined by a time difference between a peak, preferably a maximum value, of the second distribution function (82, 83) and a peak, preferably a maximum value, of the first distribution function (80).

36. The device (too) of any of the preceding claims 33 or 34, wherein the first distribution function (80) comprises a cumulative function and wherein the second distribution function (82, 83) comprises a cumulative function, wherein the change of velocity is determined by a time difference between an inflection point of the second distribution function (82, 83) and an inflection point of the first distribution function (80).

37. The device (too) of any of the preceding claims 33 or 34, wherein the change of velocity is determined by a distance metric of the second distribution function (82, 83) and the first distribution function (80), and / or by a change of shape of the second distribution function (82, 83) compared to the first distribution function (80).

38. The device (too) of any of the preceding claims 28 to 37, wherein the sensor unit (70) is or comprises an optical device (78), wherein the optical device (78) is preferably configured to determine the concentration of the analyte (10) by capturing a movement and / or a position of the assaying elements (30) spatially and / or temporally resolved in at least one field of view (79).39- The device (100) of any of the preceding claims 28 to 38, wherein the substrate (42) has at least two different surface binder elements (44, 45, 46), wherein the different surface binder elements (44, 45, 46) are configured to bind to different analytes (10), wherein preferably the different surface binder elements (44, 45, 46) are grouped into homogeneous groups, preferably separated by sensing elements (76).

40. The device (too) of any of the preceding claims claim 28 to 39, wherein the device is configured to vary an orientation and / or a rolling direction of the assaying elements (30) when and / or before the assaying elements (30) are rolling along / on the substrate (42) or downstream of the substrate (42) or downstream of the sensor unit (70).

41. The device (too) of claim 40, wherein the channel (40) and / or the substrate (42) have / has a mechanical and / or magnetic pattern (48), preferably a mechanical and / or magnetic chevron pattern.

42. The device (too) of any of the preceding claims claim 28 to 41, wherein the channel (40) is tapered.

43. The device (too) of any of the preceding claims 28 to 42, wherein the device (too) comprises a second external force generating device which is configured to generate a second external force, wherein preferably the second external force generating device is arranged at a position along the substrate (42) or downstream of the sensor unit (70).

44. The device (too) of claim 43, wherein the second external force generating device is or comprises a second magnetic field generating device and the second external force is a magnetic force, wherein preferably said second magnetic field generating device is or comprises a permanent magnet, and / or wherein preferably the second external force generating device is configured to generate the second external force varying in amplitude and / or spatial direction.

45. The device (too) of claim 43 or 44, wherein the second external force generating device is configured to sort the assaying elements (30).

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