Apparatus for detecting an analyte

Abstract

The apparatus comprises a detection zone comprising at least an array of magnetic field sensors, a sample introduction device configured to introduce a sample into the detection zone, and optionally a field generator optimized for generating a magnetic field and / or an electric field when the magnetizable particles do not have an aligned dipole moment, and a controller connected to receive signals from the magnetic field and / or electric field array, the controller configured to determine the amount of analyte in the sample based on signals received from the array of magnetic field sensors and / or electric field sensors, and additional features selected from one or more of the following: a set and reset module or capability for setting / resetting the magnetic sensors, a data transmission layer configured to protect signals being transmitted from one or more magnetic sensors, a plurality of magnetic field permeable zones corresponding to the area under each magnetic sensor, and a printed circuit board comprising one or more vias connecting to the magnetic field sensors.

Description

[Technical field]

[0001] The present invention relates to a device for detecting a target analyte in a sample, more particularly based on the use of nanoparticles and a sensor system for detecting the nanoparticles. The present invention also relates to a method for detecting an analyte in a sample, more particularly based on the use of nanoparticles and a sensor system. [Background technology]

[0002] There are many known devices and methods for detecting and quantifying target analytes in a sample based on the use of particles, such as magnetic particles. Such devices and systems require an indirect method of quantifying the analyte by detecting and measuring a complex bound to the analyte. Such methods typically rely on binding or recognition systems whereby a visualization aid is coated or attached to a binding molecule that binds to the analyte in the sample.

[0003] Detection and quantification of target analytes in a sample often needs to be rapid, sensitive, qualitative, and / or miniaturizable to meet the needs of in vitro diagnostics. Miniaturization of devices can result in slow and inefficient mixing in fluids due to increased viscous forces.

[0004] Point-of-care testing can reduce the turnaround time for diagnostic testing, resulting in improved workflow and thus potentially helping to improve patient care. Such systems must include sensing technology to detect biomarkers (e.g., protein or nucleic acid markers). Magnetizable particles have been used to detect analytes through manual assays for basic research to high-throughput testing.

[0005] Some portable devices use electrochemical means to detect the analyte. For example, some such devices use a potentiostat type instrument to detect the electrochemical signal produced by an enzyme-based label. Often, the label that produces the detectable electrochemical signal is further compounded with a magnetic agent (to electromagnetically manipulate the complex) and a binding agent (to bind the target analyte). Such devices can be slower in obtaining measurements.

[0006] Many existing devices that detect analytes attached to magnetizable particles require complex constructions that are not suitable or easily constructed for miniaturization in point-of-care testing applications.

[0007] The use of magnetisable particles means that an additional force can be applied to the particles, for example to separate bound particles from unbound particles.

[0008] The assessment of the analytical performance of a detection method is based on the limit of quantification (LoQ), ie the lowest biomarker concentration that can be quantified with a given required precision.

[0009] GMR has been used in sandwich-type immunoassays (such as ELISA) where molecular targets are immobilized on the sensor surface by the addition of tagged magnetic probes (see Koh and Josephson, "Magnetic Nanoparticle Sensors," Sensors, 2009:9;8130-45, and Yao and Xu, "Detection of Magnetic Nanomaterials in Molecular Imaging and Diagnostic Applications," Nanotechnol. Rev 2014:3;247-268).

[0010] Some techniques use superconducting quantum interference devices (SQUIDs) to detect and measure Néel relaxation (magnetic dipole misalignment) in magnetically labeled bacteria. In such techniques, a magnetic field is pulsed to induce alignment of the magnetic dipoles, and the subsequent dipole misalignment is detected.

[0011] It is an object of the present invention to address one or more of the above problems and / or to provide a device for detecting an analyte, a method for detecting an analyte in a sample, and / or at least to provide the public with a useful choice. Summary of the Invention

[0012] In a first aspect, a device is described comprising magnetizable particles configured to bind to an analyte, the device comprising: a detection zone comprising at least an array of magnetic field sensors; a sample introduction device configured to introduce a sample into the detection zone; Optionally, a field generator (optimized for magnetic field generation and / or electric field generation) when the magnetizable particles do not have aligned dipole moments; a controller connected to receive signals from the array of magnetic fields and / or the array of electric fields, the controller configured to determine an amount of an analyte in the sample based on signals received from the array of magnetic field sensors and / or the array of electric field sensors; i) a set and reset module or capability to set / reset the magnetic sensor; ii) a data transmission layer configured to protect signals being transmitted from one or more magnetic sensors; iii) a plurality of magnetic field permeable zones corresponding to the area under each magnetic sensor; iv) a printed circuit board with one or more vias connecting to the magnetic field sensor; or v) any combination of two or more of (i) to (iv); Equipped with.

[0013] In a further aspect, an apparatus is described for sensing a sample comprising magnetizable particles bound and unbound to an analyte, the apparatus comprising: a detection zone comprising at least an array of magnetic field sensors; a sample introduction device configured to introduce the sample into the detection zone when the bound and unbound magnetizable particles are in a fluidized state such that Brownian motion of the bound and unbound particles is induced when the sample is in the detection zone; a magnetic field generator, provided that the magnetizable particles do not have aligned dipole moments; a controller connected to receive signals from the array of magnetic field sensors and / or the array of electric field sensors representative of relative differences in the magnetic and / or electric fields of the bound and unbound magnetized particles, the controller being configured to determine a relative amount of an analyte in the sample based on the signals received from the array of magnetic field sensors; i) a set and reset module or capability to set / reset the magnetic sensor; ii) a data transmission layer configured to protect signals being transmitted from one or more magnetic sensors; iii) a plurality of magnetic field permeable zones corresponding to the area under each magnetic sensor; iv) a printed circuit board with one or more vias connecting to the magnetic field sensor; or v) any combination of two or more of (i) to (iv); Equipped with.

[0014] In a further aspect, an apparatus is described for sensing a sample comprising bound and unbound particles to an analyte, the apparatus comprising: a detection zone comprising at least an array of electric field sensors; an electric field generator that generates a current having a standard sine wave pattern; a sample introduction device configured to introduce the sample into the detection zone when the bound and unbound particles are in a fluidized state such that Brownian motion of the bound and unbound particles is induced when the sample is in the detection zone; a controller connected to receive signals from the array of electric field sensors representative of relative differences in electric fields of bound and unbound magnetized particles induced by Brownian motion of the magnetized particles, the controller being configured to determine a relative amount of an analyte in the sample based on the signals received from the array of magnetic field sensors or the array of electric field sensors; Equipped with.

[0015] In a further aspect, a method for measuring an analyte in a sample is described, the method comprising: Providing an apparatus, the apparatus comprising: a detection zone comprising at least an array of magnetic field sensors; a sample introduction device comprising magnetizable particles coated with binding molecules complementary to the target analyte; a field generator, the field generator being optimized for magnetic field generation in the presence of a magnetic field sensor, provided that the magnetizable particles do not have aligned dipole moments; a controller connected to receive signals from the array of magnetic field sensors representative of the relative difference in magnetic fields of the bound and unbound magnetized particles; i) a set and reset module or capability to set / reset the magnetic sensor; ii) a data transmission layer configured to protect signals being transmitted from one or more magnetic sensors; iii) a plurality of magnetic field permeable zones corresponding to the area under each magnetic sensor; iv) a printed circuit board with one or more vias connecting to the magnetic field sensor; or v) any combination of two or more of (i) to (iv); And, introducing a sample containing an analyte to be measured into a sample introduction device and contacting the analyte with magnetizable particles to provide both magnetizable particles bound to the analyte and magnetizable particles that are not bound to the analyte; biasing the analyte-bound and unbound magnetizable particles relative to the detection zone by a sample introduction device to position the analyte-bound and unbound magnetizable particles in the detection zone; - varying the bias sufficiently to release at least a portion of the magnetizable particles bound to the analyte and the magnetizable particles not bound to the analyte from their positions within the detection zone; determining, via the controller, a relative amount of the analyte in the sample based on signals received from the array of magnetic field sensors based on Brownian motion of magnetizable particles bound to the analyte and unbound magnetizable particles; Includes.

[0016] In a further aspect, a method for measuring an analyte in a sample is described, the method comprising: Providing an apparatus, the apparatus comprising: a detection zone comprising at least an array of electric field sensors; an electric field generator that generates a current having a standard sine wave pattern; a sample introduction device comprising particles coated with binding molecules that are complementary to the target analyte; a controller connected to receive signals from the array of electric field sensors representative of relative differences in electric fields of bound and unbound particles induced by Brownian motion of the particles; introducing a sample containing an analyte to be measured into a sample introduction device and contacting the analyte with the particles to provide both particles bound to and unbound to the analyte; biasing the analyte-bound and unbound particles relative to the detection zone by a sample introduction device to position the analyte-bound and unbound particles in the detection zone; modifying the bias sufficiently to release at least a portion of the analyte-bound and unbound particles from the vicinity of the detection zone; determining, via the controller, a relative amount of the analyte in the sample based on signals received from the array of electric field sensors based on Brownian motion of particles bound to and unbound to the analyte; Includes.

[0017] Any one or more of the following embodiments may relate to any of the above aspects.

[0018] In one configuration, the apparatus comprises: i) a set and reset module or capability to set / reset the magnetic sensor; ii) a data transmission layer configured to protect signals being transmitted from one or more magnetic sensors; iii) a plurality of magnetic field permeable zones corresponding to the area under each magnetic sensor; iv) a printed circuit board with one or more vias connecting to the magnetic field sensor; or v) Any combination of two or more of (i) to (iv).

[0019] In one configuration, the electric field generator has a frequency of 10 kHz, 100 kHz, 200 kHz, 300 kHz, 400 kHz, 500 kHz, 600 kHz, 700 kHz, 800 kHz, 900 kHz, or 1000 kHz, and a suitable range may be selected between any of these values.

[0020] In one configuration, the electric field generator has a frequency of 0.1 volts, 1 volt, 2 volts, 3 volts, 4 volts, 5 volts, 6 volts, 7 volts, 8 volts, 9 volts, or 10 volts, and a suitable range can be selected between any of these values.

[0021] In one configuration, the magnetizable particles may be magnetized prior to binding to the analyte, or prior to or during introduction of the sample to the magnetic sensing zone.

[0022] In one configuration, the array of magnetic sensors includes set and reset coils / straps that set / reset the magnetic sensors.

[0023] In one configuration, the magnetic sensor is set / reset between readings.

[0024] In one configuration, multiple magnetic sensors are connected in series to the calibration port such that one calibration signal is used to set / reset multiple magnetic sensors.

[0025] In one configuration, the magnetic sensor has a sampling rate of about 100 kHz to about 200 kHz.

[0026] In one configuration, the sensing zone is provided on a top surface of the circuit board.

[0027] In one configuration, at least one magnetic field generator or electrical generator is provided on the underside of the circuit board at a location corresponding to the sensing zone on the upper side of the circuit board.

[0028] In one configuration, the circuit board comprises multiple layers.

[0029] In one configuration, the circuit board comprises at least one top layer, a ground plane layer, and a bottom layer.

[0030] In one configuration, the circuit board includes a data transmission layer configured to shield signals being transmitted from one or more magnetic sensors from electromagnetic interference generated by other components of the circuit board.

[0031] In one configuration, the data transmission layer is located between the upper layer and the lower layer.

[0032] In one configuration, the circuit board includes a plurality of magnetic field transparent windows, each window defining a portion of the circuit board that is free of the copper layer, the transparent windows corresponding to an area of ​​the circuit board underlying each magnetic sensor.

[0033] In one configuration, the device is approximately 1 cm 2 ~about 25cm 2 The detection surface area is

[0034] In one configuration, the detection surface includes between about 6 and about 24 magnetic sensors.

[0035] In one configuration, the array of magnetic sensors is closely packed.

[0036] In one configuration, the device further comprises a housing that houses the at least one circuit board.

[0037] In one configuration, the housing further comprises an integrated display configured to render diagnostic output obtained from the circuit board.

[0038] In one configuration, the housing further comprises an integrated display and at least one circuit board configured to perform the operation of the lab-on-a-chip device.

[0039] In one configuration, the built-in display and the multiple circuit boards arranged in parallel are configured to perform the operation of a lab-on-bench device.

[0040] In one configuration, the housing that performs the operation of the lab-on-chip and lab-on-bench devices is configured to be controlled by a user interface.

[0041] In one configuration, the controller is configured to controllably bias one or more of the sample introduction device, the field generator, the array of sensors, the amplifier, and the filter.

[0042] In one configuration, the controller is configured to control the bias of the sample introduction device.

[0043] In one configuration, the sample introduction device biases the particles towards the sensor.

[0044] In one configuration, the circuit board is approximately 10 cm 2 ~about 100cm 2 The size is.

[0045] In one configuration, the sensing surface covers between about 10% and about 50% of the surface of the circuit board.

[0046] In one configuration, the device further comprises a sensor to detect the orientation of the device such that the device is operable in any orientation.

[0047] In one configuration, the sensor for detecting the orientation of the device comprises one or more of a gyroscope sensor, an inertial measurement unit, and an accelerometer.

[0048] In one configuration, the one or more magnetic sensors are analog sensors.

[0049] In one configuration, the one or more magnetic sensors comprise one or more of a magnetoresistive sensor, a Hall effect sensor, and a fluxgate sensor.

[0050] In one configuration, the apparatus further comprises a signal processing module, the signal processing module comprising: Analog-to-digital converters, an amplifier for amplifying the signal from one or more magnetic sensors; and ·Power supply It has one or more of the following:

[0051] In one configuration, the sample introduction device is removable.

[0052] In one configuration, the sample introduction device is integrated into the instrument.

[0053] In one configuration, the sensing zone comprises a plurality of wells.

[0054] In one configuration, multiple channels are arranged in a cross-hatch configuration (multiplexed design).

[0055] In one configuration, the multiple channels are arranged in a non-crosshatch configuration (parallel simplex design).

[0056] In one configuration, a number of wells are pre-loaded with the binding complex.

[0057] In one configuration, the binding complex is provided in a gel.

[0058] Reference to a range of numbers disclosed herein (e.g., 1 to 10) is intended to incorporate references to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) as well as any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7).

[0059] Numerous modifications in structure and widely differing embodiments and applications of the invention will occur to those skilled in the art to which this invention pertains without departing from the scope of the invention as defined in the appended claims. The disclosures and descriptions herein are merely illustrative and are not intended to be in any sense limiting.

[0060] Where references are made herein to external sources of information, including patents and other documents, this is generally for the purpose of providing a context for describing features of the invention. Unless specifically stated, references to such sources of information should not be construed as an admission that such sources of information are prior art or form part of the general general knowledge in the art in any jurisdiction.

[0061] The term "comprising" as used in the specification and claims means "constituting at least a part of." When interpreting each statement in this specification containing the term "comprising," features other than the one or more to which that term is attached may also be present. The related terms "comprise" and "comprises" are to be interpreted in the same manner. [Brief description of the drawings]

[0062] The present invention will now be described, by way of example only, with reference to the drawings in which: [Figure 1] FIG. 1 is a schematic representation of the components of an apparatus for detecting an analyte. [Diagram 2] FIG. 2 is a schematic representation of an apparatus for detecting an analyte. [Diagram 3] FIG. 3 is an exemplary embodiment of a microfluidic chip. [Figure 4] FIG. 4 is a functional block diagram of an apparatus for sensing a sample comprising bound and unbound particles to an analyte according to an embodiment of the present disclosure. [Diagram 5] FIG. 5 is a schematic / circuit diagram of the device showing the input and output connections as well as the various sensor modules. [Figure 6] FIG. 6 shows a schematic / circuit diagram of an embodiment of the sensing zone of the device. [Figure 7] FIG. 7 depicts a schematic / circuit diagram for an embodiment of the sensing zone of the device. [Figure 8] FIG. 8 is a schematic / circuit diagram of a signal processing module 800. [Figure 9] FIG. 9 depicts a schematic diagram of a CM module according to an embodiment. [Figure 10] FIG. 10 is a schematic diagram of the power management module of the device. [Figure 11] FIG. 11 is a schematic diagram of the display module of the device. [Figure 12] FIG. 12 is a schematic diagram of the device's orientation detection module. [Figure 13] FIG. 13 shows a schematic diagram of the set / reset circuitry of the device. [Figure 14] FIG. 14 is a 3D view of a variant of the device. [Figure 15] FIG. 15 shows a touch screen embodiment of the input user interface of the device. [Figure 16] FIG. 16 shows another embodiment of a touch screen input user interface for the device. [Figure 17] FIG. 17 is a block diagram depicting the steps of data generation and processing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] An apparatus for detecting a sample comprising bound and unbound particles for an analyte is described, the apparatus comprising a detection zone comprising an array of magnetic field sensors or an array of electric field sensors. The apparatus includes a sample introduction device configured to introduce the sample into the detection zone when the bound and unbound particles are in a fluidized state. Without being bound by theory, Brownian motion of the bound and unbound particles is induced when the sample is in the detection zone. If a magnetic sensor and / or an electric sensor is present, the particles comprise magnetizable particles, which are in a magnetized state when in the detection zone. A field generator may be present, provided that the magnetizable particles do not have an aligned dipole moment. That is, if the particles do not have an aligned dipole moment, the field generator is present, and in other cases, including the field generator is optional. The field generator is optimized for magnetic field generation if a magnetic field sensor is present, and / or optimized for electric field generator if an electric field sensor is present, the electric field generator generating a current having a standard sinusoidal pattern. The device also includes a controller connected to receive signals from the array of magnetic field sensors or from both the array of magnetic field sensors and the array of electric field sensors representing the relative difference in the magnetic or electric fields of the bound and unbound magnetized particles. The controller is configured to determine the relative amount of analyte in the sample based on the signals received from the array of magnetic field sensors or the array of electric field sensors. When a magnetic field sensor is used, the device further includes: i) a set and reset module or capability to set / reset the magnetic sensor; ii) a data transmission layer configured to protect signals being transmitted from one or more magnetic sensors; iii) a plurality of magnetic field permeable zones corresponding to the area under each magnetic sensor; iv) a printed circuit board with one or more vias connecting to the magnetic field sensor; or v) any combination of two or more of (i) to (iv); Equipped with.

[0064] The particles may be located in the detection zone by the presence of a biasing mechanism, e.g., a magnetic field. The described devices are based on the concept of measuring detectable changes in magnetic and / or electric fields over time caused by changes in magnetizable particles, such as translational and / or rotational movement of the particle and analyte complex relative to the detection zone due to Brownian motion and / or agglomeration of the particle and analyte complex.

[0065] The particles may be functionalized with binding agents (such as antibodies) that bind to the analyte of interest. The particles used in the device may generate or be induced to generate signals that are detectable and / or measurable by a sensing module (e.g., a magnetic field sensor or an electric field sensor). For example, the particles may generate or be induced to generate magnetic fields, electric fields, light emission, fluorescence (e.g., by excitation via lasers, LEDs, microLEDs, or silicon photonics), light absorption, optical attenuated total internal reflection (e.g., induced using a light source such as a laser, LED, microLED, or silicon photon), ionic potential, vibration, acoustics, radiation that are detectable and measurable using an appropriate sensor.

[0066] The particle and analyte complexes may aggregate based on binder-bead interactions of adjacent complexes. The antibody may be designed to bind a single antigen. If the analyte uses a position on the antibody, the antibody is no longer available for interaction of that adjacent complex.

[0067] A schematic representation of an embodiment of a device 1 for detecting an analyte is shown in Figure 1. In this embodiment, the device comprises a detection surface 2, a circuit board 3 and a computation module 4. The detection surface 2 comprises a sensing zone which may include a number of magnetic and / or electrical or optical sensors 21.

[0068] When the device is turned on, the signal output of the magnetic sensor 21 may be processed through a signal processing module 7 on the circuit board 3. The signal processing module may include a number of amplifiers 22 and an analog-to-digital converter (ADC) 23. The computation module 4 includes a controller (not shown). The device 1 may also include one or more magnetic field generators (not shown).

[0069] 2 is a schematic representation of an apparatus for detecting an analyte. In particular, the apparatus may broadly comprise a detection module, a bias system, a sample introduction device, and a signal processing module comprising a signal amplifier and an analog-to-digital converter.

[0070] The device is capable of accurately, rapidly, and sensitively measuring one or more analytes in a sample. For example, an embodiment of the device (with 24 magnetic sensors, 24 amplifiers, and three 8-channel analog-to-digital converters) may be capable of generating over 450,000 high-resolution data points per second per channel, which equates to over 10 million data points per 25 seconds of continuous reading.

[0071] 4 is a functional block diagram of an apparatus for sensing a sample comprising magnetizable particles bound and unbound to an analyte, according to an embodiment. In this embodiment, the apparatus 400 may comprise a sensing module 401 configured to detect magnetic particles and output signals from a magnetic or electric field sensor on a substrate, a signal processing module 402 configured to receive and process the output of the received signal, a sample introduction device 403 configured to introduce a sample to a detection zone, a power management module 405 configured to store energy and power various components of the apparatus, a control module 406 configured to perform an on-substrate analysis on the sample by detecting the relative amount of analytes in the sample, a display module 407 configured to plot the results of the on-substrate diagnosis, and a wireless communication module 408 configured to wirelessly transmit analytical, telemetry, environmental, and diagnostic data obtained from the sample.

[0072] In an implementation, the above modules of the device may be provided in the form of an interconnected circuit board or multi-layer PCB.

[0073] The apparatus 400 may further comprise a magnetic field generator 410, an electric field generator 411, an electromagnetic field generator, and an orientation measurement module 404 configured to measure an orientation of the device.

[0074] Figure 5 shows a schematic / circuit diagram of the device showing the input and output connections as well as the various sensor modules used in the sensing process. As is evident from Figure 5, the overall design of some modules spans multiple layers of the PCB. For example, the discrete schematic level coupling of magnetic sensors, 1:1 sensor-to-sensor amplifiers / set-reset functions, analog-to-digital converters, power management modules, display modules, and various other subsystem capabilities are depicted in schematic form.

[0075] The computational module shown in Figure 5 reflects a discrete design of optional computational capabilities for fully autonomous implementations of the device. In some implementations, for example in non-fully autonomous implementations of the device, a microcontroller unit (MCU) and USB-C / Wifi / BlueTooth® connection securely streams data to a wireless / wired secondary device such as a cell phone or another computing device. This can occur between multiple device PCB "cores" within a single case (veterinary and human clinical / laboratory applications).

[0076] The device may be configured to exclude components whose functional capabilities / outputs may be accomplished by a connected device, such as, but not limited to, a cellular phone. Such capabilities may include a screen, a user interface, software, network connectivity, data processing, encryption, power magnetometers, analog-to-digital converters, accelerometers, gyroscopes, batteries, optical sensors, and speakers.

[0077] The apparatus 400 may comprise a compact form factor suitable for use as a portable point-of-care device, in addition to which the device achieves the desired accuracy, sensitivity, and speed of detecting and quantifying an analyte in a sample in order to perform the functions of the device as a portable POC device.

[0078] In some embodiments, various components of the device may be provided on one or more circuit boards, for example, the sensing surface with the magnetic sensors, the magnetic field generators, the controller, the analog-to-digital converter (ADC), the signal amplifiers, and the power supply may be provided on one or more circuit boards.

[0079] The components may be provided on separate but interconnected circuit boards, as depicted in Figure 5. For example, the sensing surface or sensing zone (including the magnetic sensor), magnetic field generator, signal generation module, signal processing module (including an analog-to-digital converter (ADC)), signal amplifier, orientation detection module, and power management module may be provided on a primary circuit board, while the controller may be provided on a secondary circuit board connected to the primary board via a connector suitable for transmitting data and maintaining integrity.

[0080] The circuit board may be a printed circuit board (PCB), for example, the circuit board may be single-sided, double-sided, multi-layer, rigid, flexible, or rigid-flexible.

[0081] A circuit board may include multiple circuit layers (copper layers), for example, a circuit board may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 circuit layers.

[0082] The circuit board may include one or more ground plane layers. Multiple ground plane layers may be used to improve signal return and reduce noise and interference to further improve the accuracy of the magnetic field sensor. The ground plane may be configured to control the oscillation frequency to eliminate or reduce interference.

[0083] The circuit board may comprise one or more data layers. By providing a dedicated data layer, the integrity of data transfer between the various components of the device may be optimized. For example, by providing a dedicated data layer, the integrity of the signals from the magnetic sensor to the amplifier, analog-to-digital converter, controller and vice versa may be maintained. By providing a dedicated data layer, the integrity of data transfer between the various components of the device may be optimized. For example, by providing a dedicated data layer, the integrity of the signals from the magnetic sensor to the amplifier, analog-to-digital converter, controller and vice versa may be maintained.

[0084] A sensing surface of the device may be provided on a top surface of a circuit board. The sensing surface defines an area housing a microfluidic chip in which one or more magnetic and / or electric sensors are provided for detecting changes in a magnetic field. The sensing surface may be provided at or near an edge of the circuit board.

[0085] One or more magnetic field generators may be provided on the bottom surface of the circuit board. The magnetic field generators may be provided on the top surface of the circuit board at locations corresponding to the locations of the sensing surfaces.

[0086] A sensing surface of the device may be provided on a lower surface of the circuit board. The sensing surface defines an area housing a microfluidic chip in which one or more magnetic and / or electric sensors are provided for detecting changes in a magnetic field. The sensing surface may be provided at or near an edge of the circuit board.

[0087] One or more magnetic field generators may be provided on the top surface of the circuit board. The magnetic field generators may be provided on the top surface of the circuit board at locations corresponding to the locations of the sensing surfaces.

[0088] The one or more magnetic field generators may be located above the circuit board, below the circuit board, adjacent to the circuit board, or parallel to the circuit board.

[0089] The circuit board may include one or more magnetic field transparent windows configured to allow transmission and / or focus the magnetic field generated by the magnetic field generators provided on the underside of the circuit board. The magnetic field transparent windows may comprise portions of the circuit board that are free of copper layers in certain areas. Each magnetic field transparent window may correspond to an area of ​​the circuit board underlying each magnetic sensor.

[0090] The circuit board is approximately 5 cm 2 , 10cm 2 , 15cm 2 , 20cm 2 , 25cm 2 , 30cm 2 , 35cm 2 , 40cm 2 , 45cm 2 , 50cm 2, 55cm 2 , 60cm 2 , 65cm 2 , 70cm 2 , 75cm 2 , 80cm 2 , 85cm 2 , 90cm 2 , 95cm 2 , or 100cm 2 and a suitable range may be selected between any of these values.

[0091] The circuit board may have footprint dimensions similar to a credit card. For example, the circuit board may have dimensions of approximately 5.5 cm x 8.5 cm x 2.5 cm. The compact dimensions of the circuit board allow the device to have relatively compact overall dimensions to improve the portability, and therefore the usability, of the device as a point-of-care diagnostic device.

[0092] The circuit board may have a sensing surface that is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the circuit board surface.

[0093] Attention is now directed to describing in detail each module of the apparatus shown in FIGS.

[0094] The sensing module (or detection unit) may comprise one or more sensors that detect and measure changes in a measurable signal over time due to translational and rotational Brownian motion of a particle as it is released from the vicinity of the sensor.

[0095] The sensor may detect and / or measure changes in a detectable signal such as magnetic, current and / or voltage (including resistance and impedance), luminescence, fluorescence, absorbance, optical attenuated total internal reflection, vibration, acoustic, ion protective, or radioactivity. In some embodiments, the sensor performs resistive pulse or electrical zone sensing.

[0096] The sensors may include magnetic field sensors, oscilloscopes, multimeters, current sensors, voltage sensors, photo sensors, optical sensors such as CMOS optical sensors used in cell phone cameras, MEMS sensors, scintillation counters, and radiation sensors. In some embodiments, the sensors include sensing elements, such as electrodes (anodes and cathodes), conductive coils, and conductive circuits.

[0097] The sensing module may comprise a sensing zone or detection surface where detection of changes in the magnetic field of the magnetizable particles over time may occur. The detection surface may comprise one or more sensors capable of rapid and sensitive detection of changes in the magnetic field, such as direction, strength and flux.

[0098] The one or more sensors may include one or more magnetic field sensors.

[0099] The magnetic sensors may be selected from spintronic sensors, atomic magnetometers (AM), nuclear magnetic resonance (NMR) systems, fluxgate sensors, Faraday induction coil sensors, diamond magnetometers, and domain wall based sensors, vibration magnetic sensors, GMR / TMR / Wheatstone bridge sensors, etc.

[0100] Volumetric-based sensors such as planar Hall effect (PHE) sensors offer simple and fast sample preparation and detection. Surface-based sensors such as giant magnetoresistance (GMR) offer lower detection limits (single particle) due to the short distance between the magnetizable particle and the sensor. Spintronic sensors can be selected from giant magnetoresistance (GMR) sensors, tunneling magnetoresistance (TMR) sensors, anisotropic magnetoresistance (AMR) sensors, and planar Hall effect (PHE) sensors.

[0101] The GMR effect was discovered in the 1980s and has traditionally been used for data recording. Spin valves offer higher sensitivity for micron-sized designs. Spin valve GMR sensors consist of artificial magnetic structures with alternating ferromagnetic and non-magnetic layers. The magnetoresistance effect is caused by the spin-orbit interaction between the conduction electrons across the different layers. The variation in magnetoresistance provides quantitative analysis by this spin-dependent sensor. GMR sensors can be used to detect DNA-DNA or protein (antibody)-DNA interactions. The dimensions of the sensor array can be tailored for the detection of individual magnetizable particles. GMR sensors can be used in combination with antiferromagnetic, ferromagnetic, ferrimagnetic, paramagnetic, and superparamagnetic particles.

[0102] The planar Hall effect is an exchange-biased Permalloy planar sensor based on the anisotropic magnetoresistance effect of ferromagnetic materials. The PHE sensor can be a spin valve PHE or a PHE bridge sensor. The PHE sensor can be capable of performing single particle sensing.

[0103] When multiple magnetic sensors are used, the multiple magnetic sensors can be configured with a set / reset function, and the set / reset for each magnetic sensor can be connected as a series circuit or connection for signaling and input-output.

[0104] The set / reset function may be incorporated into a magnetic sensor such as that provided by Bosch BMM150 geomagnetic sensor, a sensor that allows for the measurement of magnetic fields in three orthogonal axes. The use of such a sensor may simplify the design of the board, for example eliminating the need for a data transmission layer. The use of such a sensor may be implemented in a 4mm 2 ~100mm 2 The amplifier may be integrated within the sensor.

[0105] When multiple magnetic sensors are used, the multiple magnetic sensors may be configured as a series circuit or connection for the set / reset functions to eliminate hysteresis and sensor drift, i.e., the set / reset functions of each magnetic sensor of the multiple magnetic sensors are connected in series.

[0106] The accuracy and sensitivity of a magnetic sensor can be adversely affected by external forces. In particular, changes in magnetic fields and temperature can disrupt the orientation of the magnetic domains in a magnetic sensor. When the orientation of the magnetic domains is disrupted, it can become randomized, thereby reducing the accuracy and sensitivity of the sensor.

[0107] To maintain a high level of accuracy and sensitivity, the magnetic sensor may be periodically recalibrated, for example after approximately 100, 80, 60, 40, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 reading by the magnetic sensor.

[0108] The magnetic sensor may be recalibrated once per cycle of sample readings, where each cycle may consist of 10, 100, 1000, 10000, 100000 readings by the magnetic sensor.

[0109] The magnetic sensor is recalibrated after each reading.

[0110] Recalibration of the magnetic sensor can be performed using set and reset operations. Set and reset of the magnetic sensor realigns the orientation of the magnetic domains before each sampling by the sensor. By performing set and reset, the sensor can recover from any disturbance to the orientation of the magnetic domains so that the magnetic domains are in the optimal orientation for accurate and sensitive performance. By performing a "set", all the magnetic domains of the magnetic sensor in a first direction are realigned, while a "reset" realigns the magnetic domains of the magnetic sensor in a second direction opposite to the first direction. By performing a set and reset, all randomness in the magnetic domains of the magnetic sensor is removed.

[0111] The set and reset calibration can identify currents, electromagnetic bias inherent in the system, or low to high frequency interference. This unidirectional bias can then nullify the effect of any such bias in the system calculations, leading to improved sensor accuracy.

[0112] One or more of the magnetic sensors may include a set / reset coil (strap) wrapped around a sensing element (such as a magnetoresistive element) of the magnetic sensor. A calibration signal may be pulsed and sent through the set / reset coil to set and / or reset the magnetic sensor.

[0113] In an embodiment, the magnetic sensor may include an offset strap that may enable several modes of operation when a direct current is driven through the offset strap. These modes are: 1) attracting (cancelling) unwanted external magnetic fields, 2) nulling the bridge offset voltage, 3) closed loop field cancellation, and 4) auto-calibration of bridge gain. The set / reset strap may be pulsed with high current for the following advantages: 1) allowing the sensor to make highly sensitive measurements, 2) reversing the polarity of the bridge output voltage, and 3) being used periodically to improve linearity, lower cross-axis effects, and temperature effects.

[0114] The magnetic sensor circuit may be connected to a calibration port. Calibration signals may be provided through the calibration port to calibrate the magnetic sensor. The calibration signals may comprise a set calibration signal (pulse) and a reset calibration signal (pulse).

[0115] A serial configuration of the set / reset functions of the magnetic sensors allows a single calibration signal, or a single set of calibration signals, to recalibrate multiple magnetic sensors. Such a configuration can improve the speed and reliability of the sensor calibration process. For example, calibration of magnetic sensors connected in a serial configuration can occur in hundreds of thousands to millions of seconds.

[0116] Referring to Figure 13, this shows the set / reset circuit 1300 of the device. The magnetic sensor 601 is set / reset by sending a pulse of current. For example, the SR+ and SR- ports of the magnetic sensor are configured to receive a pulse of current to reset the sensor. The same amount of current can be applied simultaneously to all sensors connected in series.

[0117] The set / reset circuit may include a voltage booster circuit 1301. The voltage booster circuit 1301 may be configured to boost the voltage to set / reset all the sensors simultaneously. The set / reset port 1301 may include a set / reset port configured to source current in the sensors in series.

[0118] To achieve a high level of accuracy and sensitivity, the magnetic sensor of the device may have a high sampling rate. The magnetic sensor may sample at a sampling rate of about 10 kHz, 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz, 100 kHz, 110 kHz, 120 kHz, 130 kHz, 150 kHz, 160 kHz, 170 kHz, 180 kHz, 190 kHz, 200 kHz, 210 kHz, 220 kHz, 230 kHz, 240 kHz, or 250 kHz, with suitable ranges selected between any of these values ​​(e.g., about 10 kHz to about 250 kHz, about 10 kHz to about 200 kHz, about 10 kHz to about 150 kHz, about 10 kHz to about 100 kHz, about 100 kHz to about 250 kHz, about 100 kHz to about 200 kHz, about 100 kHz to about 150 kHz).

[0119] The ADC sampling rate of the magnetic sensor may be about 100 kHz to about 200 kHz.

[0120] The multiple magnetic sensors may have a sampling rate of about 150 kHz per channel.

[0121] The magnetic field sensor may be an on-chip magnetometer. The magnetic field sensor may have a sensitivity of at least 1 mV / V / Gauss. In some embodiments, the magnetic field sensor may detect and / or measure magnetic fields of at least about 10 milligauss, 1 milligauss, 100 microgauss, or 10 microgauss.

[0122] The magnetic field sensor may have multiple axes, for example one, two, or three axes.

[0123] The magnetic field sensor may be a Honeywell HMC1021S magnetometer. In another embodiment, the magnetic field sensor may be a Honeywell HMC1041Z magnetometer. In other embodiments, the magnetic field sensor may be selected from the group comprising a Honeywell HMC1001, HMC1002, HMC1022, HMC1051, HMC1052, HMC1053, or HMC2003 magnetometer.

[0124] The magnetic field sensor may comprise a custom-made magnetic field sensor having custom components.

[0125] In order to achieve a compact form factor with high levels of detection accuracy, sensitivity, and speed, the detection surface of the device is approximately cm 2 The device has a high density of magnetic sensors per channel. By increasing the density of magnetic sensors, a more compact microfluidic system can be used in the device. Using a more compact microfluidic system advantageously improves the speed of diagnosis by allowing the sample to travel a shorter distance in the channels of the microfluidic system. More compact microfluidics also minimizes the amount of dead volume (non-detection area) in the microfluidic system, thereby reducing the amount of sample required for diagnosis.

[0126] The detection surface is cm 2 In some embodiments, the magnetic sensors may have a sensor density of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 magnetic sensors each.

[0127] To achieve a high level of sensor density, each magnetic sensor may be configured to maximize the number of sensors that can be provided within the sensing surface with a minimal footprint. In one embodiment, the vias for the sensors are located within the perimeter of the solder pads to allow the magnetic sensors to be located closer together to achieve a high sensor density configuration.

[0128] The sensor connectors may be constructed across multiple independent planes of a multi-layer printed circuit board so that the density of planar circuit connections may be increased without conflict or interference with other connections.

[0129] A plurality of magnetic sensors may be provided on the sensing surface to simultaneously measure changes in the magnetic field. For example, the sensing surface may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 magnetic sensors.

[0130] The magnetic field sensors can be provided in a relatively small area on the device. For example, 24 magnetic field sensors can be provided in an area of ​​about 13 mm x 19 mm. Such a configuration allows for shorter sample-to-data times due to the shorter microfluidic channels used in this magnetic field sensor configuration. This configuration also allows for a smaller and more portable device.

[0131] The detection surface is approximately 1 cm 2 , 2cm 2 , 3cm 2 , 4cm 2 , 5cm 2 , 6cm 2 , 7cm 2 , 8cm 2 , 9cm 2 , 10cm 2 , 12cm 2 , 14cm 2 , 16cm 2 , 18cm 2 , or 20cm 2and a suitable range may be selected between any of these values.

[0132] The device can be configured as a mobile lab by linking multiple devices together. Linking multiple devices expands the diagnostic capabilities of the device. For example, two or more devices can be linked together to obtain a higher sensor count to further improve the speed of analyte detection and quantification across multiple samples. The devices can be connected wirelessly or via a hardwired connection.

[0133] A case may be provided that connects multiple devices together and that may provide additional functionality to the devices, for example, the case may provide additional computing power, power supplies, and communication systems.

[0134] The device may have a modular architecture, for example, sensing modules having one or more detection surfaces may be connected to the device to obtain a greater number of simultaneous readings, further reducing the sample-to-data time per analyte.

[0135] Multiple magnetic field sensors can be used to simultaneously measure changes in the magnetic field, for example 50, 60, 70, 80, 90, 100, 110, or 120 magnetic field sensors for small portable applications and in situ laboratory or clinical applications, and useful ranges can be selected between any of these values ​​(e.g., about 50 to about 120, about 50 to about 100, about 50 to about 90, about 50 to about 80, about 60 to about 120, about 60 to about 110, about 60 to about 90, about 70 to about 110, about 70 to about 90, about 80 to about 100 magnetic field sensors).

[0136] The sensing zone may include multiple electric field sensors, for example, 4, 8, 10, 14, 18, 22, 26, 30, or more electric field sensors.

[0137] 6 and 7 depict schematic / circuit diagrams for an embodiment of the sensing zone of the device. The signal from each of the magnetic sensors 601 is fed into an instrumentation amplifier 602 for amplification. The sensing zone may include a voltage regulator 603 for adjusting a reference voltage of the signal input to the instrumentation amplifier. The conditioned reference signal input to the instrumentation amplifier may provide a consistent and accurate reference point for determining voltage changes relative to the sample-to-sample voltage received from the sensor.

[0138] The detection zone may further comprise a sensor population identifier module configured to identify how many sensors are mounted on the PCB and at which of the possible sensor locations the sensors are mounted, allowing for a variety of device variants and configurations from a single PCB design.

[0139] Referring to Figure 17, an example of the data collection and processing steps of the sensing module is depicted in block diagram form. As shown, an instrumentation amplifier 1701 is configured to receive and amplify a reference signal 1702 and a signal (e.g., a voltage reading) from a magnetic sensor 1703. The amplified output signal from the instrumentation amplifier 1701 then undergoes a step of analog filtering 1704 where the raw data is filtered to remove noise. The processed analog data is then fed to an analog-to-digital converter 1705 where it is converted to the digital domain. The resulting signal from the ADC is then processed by a digital signal processing module 1706.

[0140] The sensing module 600 may include one or more instrumentation amplifiers configured to amplify the signal output from the magnetic sensor. The amplifiers may provide a large amount of gain (up to 10,000 gain) from low level signals. The amplifiers may be lower power amplifiers with overvoltage protection. One example of a suitable instrumentation amplifier is the Texas Instruments INA819.

[0141] In one implementation, the sensing module 402 may include one or more analog-to-digital converters (ADCs). The conversion or sampling resolution may be 16 bits, 24 bits, 32 bits, 64 bits, 128 bits, 256 bits, or 512 bits.

[0142] In one implementation, the ADC may be 16-bit or 24-bit and have 2, 4, 8, or 16 channels. One example of a suitable ADC is the MCP3464 8-channel 16-bit Sigma-Delta ADC by Microchip Technology.

[0143] The signal output from the multiple magnetic or electric field sensors in the detection zone is stored and processed by the signal processing module 402. The signal output of the magnetic or electric field sensors may be a voltage reading proportional to the sensed magnetic or electromagnetic field (emf). In an embodiment, the voltage reading from the magnetic field sensor may be amplified in magnitude to a higher voltage (proportional to the original voltage reading) compatible with the electronic components of data processing and collection.

[0144] The device may have an amplifier to magnetic sensor ratio that is 1:1. This arrangement may optimize the sensitivity and accuracy for each sensor. For example, a device with 24 magnetic sensors may have 24 amplifiers. The 1:1 amplifier to magnetic sensor ratio allows for a configuration where a single, isolated circuit is used for the entire analog mode of data. This configuration may eliminate the possibility of sensor crosstalk / interference when running multiple sensors simultaneously, especially when the signals are at low levels.

[0145] Referring to FIG. 8, a schematic / circuit diagram of a signal processing module 800 is shown. The signal processing module may be configured to process the amplified data output of the magnetic sensors. The amplified signal may be in raw format and may include any residual line noise or other motion / noise from the circuit board. This raw signal, which is subject to noise, is then filtered in the signal processing module through digital filtering techniques. As shown, the amplified signals from each of the sensors are fed into the filter module.

[0146] The device may switch to DC power when reading the sensor to avoid noise from the circuit board.

[0147] The digital filter may be a low pass filter, however other filtering techniques may also be applied depending on the level of noise or filtering required.

[0148] The signal processing module may further comprise a microcontroller or microprocessor, which may be a computation module (CM), reflecting the discrete design of optional computational capabilities for fully autonomous implementations of the device.

[0149] 9 depicts a schematic diagram of a CM4 module 900. As shown, the CM4 module includes GPIO interfaces 901 for a number of subsystem schematic elements, including each of the three ADC modules 803. In an embodiment, the CM4 module may further include additional and / or separate GPIO pins in the form of GPIO expanders to access and control other subsystems, such as the magnetic field generators, set / reset functions, and subsystem status.

[0150] The CM module shares a video I / O port with the PCB, resulting in an embodiment where the PCB holds a video connection (MIPI DSI or HDMI) that can be connected to the CM4 when adapted. In some embodiments, the PCB video port can allow for the connection of a capacitive touch screen. The touch screen serves as the primary user interface in these device variants. Such user interface functions include, but are not limited to, data entry, quality control information and triggers, patient information and user login credentials, workflow queue presentation and management, result report display, and the like. While the touch screen represents the primary user interface for activating, operating, and performing these tasks, the processing of such instructions and the rendering of content displayed on the screen is handled by software loaded onto the CM4. In embodiments that exclude the CM4, simpler instructions are managed by a microcontroller unit (MCU) located on the PCB. The MCU interfaces to another device (such as another PCB with CM4 or a mobile phone) either wired or wirelessly; in this mode, the MCU frees up the task of receiving information from the other device and provides it to the other device, so that the other device takes over all of the functions detailed above for the touch screen, and also performs many of the functions of CM4 in the previous embodiment (e.g., software, UI, network connectivity, sensor data storage, signal processing, report drawing, etc.). The exception is that the PCB's MCU holds direct command over the PCB hardware and also holds the collection of ADC, environmental, and telemetry data until it sends that data to the other device.

[0151] The PCB may further include a separate power module 904 for powering the module, and a ground module 905 for protection against surge voltages, short circuits, etc. The PCB module may further include a USB port 906 for receiving and transmitting data input and / or power, and LED indicators for indicating power and status for the various subsystems.

[0152] The sample introduction device may be configured to introduce the sample into the detection zone when the bound and unbound magnetizable particles are in a magnetized and fluidized state, respectively, such that upon release of the magnetized state through controlled collapse of the electric / magnetic field, Brownian motion of the bound and unbound magnetized particles once again becomes the dominant force acting on the sample in the detection zone.

[0153] With reference to FIG. 2, the sample introduction device 60 may be configured with a multiplex design. That is, the sample introduction device may be used to sample and / or measure multiple biomarkers from a single input sample at controlled intervals. For example, the sample introduction device 60 may be designed with multiple sensor-aligned wells with magnetic beads functioning to detect different angles per well. Thus, the sample introduction device 60 may be configured to perform simultaneous detection of multiple analytes in a common sample body. Additionally or alternatively, the sample introduction device may be configured to perform simultaneous multiple detection on multiple samples of a sample target.

[0154] The sample introduction device 60 may include one or more valves (not shown) that are controlled by control circuitry within the device. The one or more valves may be connected to each other.

[0155] The sample introduction device can be a microfluidic device or system.

[0156] The sample introduction device may comprise a sample well or reservoir. The sample to be analyzed may be added directly to the sample well or to the microfluidic device without further processing. The microfluidic system may comprise a fluid. The fluid may be selected from phosphate buffered saline (PBS). The phosphate buffered saline may include potassium phosphate dibasic (K2HPO4), sodium chloride (NaCl), and disodium phosphate (Na2HPO4). The PBS provides a continuous phase in which the particles are suspended.

[0157] When an electric field sensor is used to detect the Brownian motion of particles, PBS provides the property of having an impedance sufficiently different from that of the particles, thereby allowing the electric field sensor to distinguish between particles versus buffer.

[0158] Microfluidic systems can speed up analysis and reduce response times. They also offer the ability to automate sample preparation, thereby reducing the risk of contamination and human error. Furthermore, they require smaller sample volumes. Microfluidics can reduce diffusion distances by increasing the area-to-volume ratio, reduce reagent consumption through micro- and nano-fabricated channels and chambers, and / or automate all steps of the process.

[0159] Microfluidic systems enable miniaturization, which enables lab-on-a-chip applications, and may be used, for example, as part of a biosensor that includes channels for acquiring a biological sample (e.g., saliva and / or crevicular fluid and / or tears and / or sweat, etc.) and processing the fluid (e.g., mixing with one or more reagents and / or detecting interactions with biomolecules, etc.).

[0160] A microfluidic system may be implemented in the form of a microfluidic chip. A microfluidic chip comprises a set of micrometer or millimeter sized channels provided by molding or etching in a material or combination of materials, such as glass, silicon, or other types of polymers. The microfluidic channels may be interconnected to form a network of channels. The length of the channels may vary from millimeters to centimeters in length.

[0161] A microfluidic chip may include one or more ports for receiving samples and / or reagents, for example, a microfluidic chip may include a sample inlet port and a reagent port.

[0162] The microfluidic chip may comprise a plurality of detection areas that define portions of a channel where detection and quantification of an analyte or biomarker in a sample occurs. The detection areas of the microfluidic chip correspond to the locations of the magnetic sensors of the device, such that when the microfluidic chip is placed over the detection surface of the device, each detection area is vertically aligned with the corresponding magnetic sensor / sensors.

[0163] The detection area may be located anywhere along the channels, hi some embodiments, the detection area is located at a channel connection point, i.e., the detection area is located at the intersection of two or more channels.

[0164] The channel connection points may comprise reaction / detection wells, which may have dimensions larger than the channels.

[0165] Microfluidics may require some degree of sample preparation. Sample preparation may include cell lysis, washing, centrifugation, separation, filtration, and elution. In some embodiments, sample preparation is performed off-chip. Alternatively, sample preparation is performed on-chip.

[0166] Microfluidic chips can be provided in a "ready to use" format. For example, the microfluidic chip can be preloaded with all the necessary components for analyte detection and quantification and cell separation (e.g., binding agent complexes and reagents). That is, the "ready to use" format requires only the addition of sample to the microfluidic device.

[0167] The reaction / detection wells may be pre-loaded with a binding agent complex that binds one or more target analytes. The binding agent complex may be provided within a gel matrix in the reaction / detection wells. For example, each reaction / detection well may contain a hydrogel, an agarose gel, or an agar that contains the binding agent complex. The binding agent complex is described in more detail later in this specification.

[0168] Binding agent complexes and / or reagents may be added to the reaction / detection wells prior to use.

[0169] The microfluidic system may include rigid or flexible materials and may include electronics that may be integrated into the microfluidic chip. The electronics may include wireless communication electronics.

[0170] The microfluidic system may be a flow-through or fixed system. For example, the microfluidic system may include magnetic field sensors or other sensors that are fixed relative to the microfluidic system.

[0171] Microfluidic systems may operate passively, for example, under passive diffusion, i.e., they do not require actively generated flow to be effective.

[0172] A microfluidic system may include a network of reservoirs that may be connected by microfluidic channels that may be configured for active or passive metering, which may allow sample fluid to be drawn into the microfluidic channels and passed into a sample chamber.

[0173] The channels may be arranged in a cross-hatch configuration, which is a multiplexed design.

[0174] Alternatively, the channels may be arranged in a non-crosshatch configuration, which is a parallel simplex design.

[0175] A microfluidic system may include microfluidic channels configured to allow access to different sample and / or detection regions on the device at different times. For example, a microfluidic device integrated into or on an aligner may be configured to provide timing via temporal sampling of fluids. For example, a microfluidic system may be designed to allow sampling in a chronological order with controlled timing. In some variations, the timing of fluids in the microchannels may be actively timed by opening of the channel, for example, via opening of valves (e.g., electromechanical valves, solenoid valves, pressure valves). Examples of valves that control fluids in a microfluidic network include piezoelectric, electrokinetic, and chemical approaches.

[0176] The channels of the microfluidic chip may include wicking structures that may improve the velocity of fluid transported by capillary action. The wicking structures may include porous media, such as paper-based materials.

[0177] A microfluidic chip may comprise multiple microfluidic channels arranged in series, fluid may be drawn into the microfluidic channels at a measured rate, and the timing of sample access to the channels may be staggered.

[0178] Microfluidics may perform signal multiplexing. That is, microfluidics may be used to sample and / or measure multiple biomarkers at controlled intervals. For example, microfluidics may be used to provide access to one or more sample chambers. Microfluidics may include one or more valves controlled by control circuitry within the device. The one or more valves may be connected to each other. Thus, microfluidics may be configured to perform simultaneous detection of multiple analytes in a common sample body. Additionally or alternatively, microfluidics may be configured to perform simultaneous multiple detection of multiple samples of a sample target.

[0179] The microfluidic channels are approximately 0.001 mm 2 ~0.01mm 2 , 0.01mm 2 ~0.1mm 2 , 0.1mm 2 ~0.25mm 2 , 0.25mm 2 ~0.5mm 2 , 0.1mm 2 ~1mm 2 , 0.5mm 2 ~1mm 2 , 1mm 2 ~2mm 2 , or 2 mm 2 ~10mm 2 and a useful range may be selected between any of these values.

[0180] In some embodiments, the microfluidics accommodate a predetermined sample volume in the range of about 0.1 μL to 1 μL, 1 μL to 5 μL, 5 μL to 10 μL, 10 μL to 20 μL, or 20 μL to 50 μL, or more, and a useful range may be selected between any of these values.

[0181] An example of a sample introduction device / microfluidic chip is shown in Figure 3. The microfluidic chip may include multiple channels arranged to direct the sample from a sample insertion area to a detection area and functionalized particles for analyte detection.

[0182] The channels have cross-sectional dimensions as described above, more preferably about 0.01 mm 2 (0.1 mm x 0.1 mm). The channels may have variable lengths. For example, the channels may be 1 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 120 mm, 140 mm, 160 mm, 180 mm, 200 mm, 250 mm, or 300 mm long, with useful ranges between any of these values ​​(e.g., about 1 mm to 10 mm, 1 mm to 20 mm, , 1 mm to 50 mm, 1 mm to 100 mm, 1 mm to 200 mm, 1 mm to 300 mm, 10 mm to 20 mm, 10 mm to 40 mm, 10 mm to 60 mm, 10 mm to 80 mm, 10 mm to 100 mm, 50 mm to 100 mm, 50 mm to 150 mm, 50 mm to 200 mm, 50 mm to 250 mm, 50 mm to 300 mm, 100 mm to 200 mm, or 100 mm to 300 mm in length.

[0183] The above dimensions of the channel facilitate passive capillary flow.

[0184] In use, a sample is introduced into the microfluidic device via the sample insertion area, which may include an inlet port.

[0185] A filter membrane may be present in the insert area 4 to separate and allow passage of desired components of the sample. For example, plasma from blood can pass into the microfluidic chip, but cells cannot. The presence of a filter membrane depends on the nature of the sample and whether the sample contains components that are desired not to pass into the microfluidic chip.

[0186] The plasma-cell separation may be due to or related to the device configuration.

[0187] Once introduced into the insertion area, the sample then contacts the microfluidic channel and flows through the remainder of the channel circuit.

[0188] The microfluidic system may be implemented as a lab-on-chip. The lab-on-chip may include one or more magnetic sensors 3 in proximity to the channel 2. For example, the microfluidic device 1 may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 magnetic sensors arranged around the periphery of the microfluidic device 1.

[0189] The lab-on-a-chip may comprise two or more magnets, such as permanent magnets or electromagnets, positioned in close proximity to the channel that may be actuated to draw magnetizable particles through the liquid in channel 2 to enhance mixing. Mixing may be performed for, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes, and a suitable range may be selected between any of these values. The timing of mixing may depend on the assay requirements, such as sample volume, viscosity, composition, and detection range of the target analyte.

[0190] To effect mixing, magnets (e.g., electromagnets) can be placed at substantially opposite ends of the channel or microfluidic device. For example, the magnets can be controlled or switched such that they push / pull the magnetizable particles towards one end of the well / channel or microfluidic device 1 and then the effect is reversed, pulling the magnetizable particles towards the other end of the well / channel or microfluidic device. This cycle can be repeated multiple times until a desired level of mixing is achieved.

[0191] As will be understood by those skilled in the art, Brownian motion or Brownian diffusion may mean that particles may move in any direction, including toward a magnetic field sensor or an electric field sensor. The magnetic signal detected by the magnetic field sensor is based on the net movement of bound and unbound magnetizable particles. The electric signal detected by the electric field sensor is based on the change in impedance as the particles move through a continuous phase (e.g., PBS).

[0192] When the bound and unbound particles are located near the magnetic or electric field sensor 40, the bound and unbound particles may be located at or near the wall of the sample well or reservoir until they are released. When the particles are released from the vicinity of the magnetic or electric field sensor 40, they may move translationally or rotationally. Assuming that the particles are close to the surface of the sample well or reservoir immediately prior to release from the bias system, the bound and unbound magnetizable particles may typically initially tend to move with a degree of freedom of movement of about 180° relative to the surface of the sample well or reservoir.

[0193] The apparatus may include a bias system configured to control the position of the particles to be within a proximity of a detection zone of a detection module (such as a sensor that detects and measures the particles). The bias system may exert a force on bound and unbound particles in the sample such that the particles are positioned at a starting position for detection / measurement by the detection module. When the force exerted by the bias system is relaxed or removed, the particles are released to undergo Brownian motion for detection by the detection module.

[0194] The bias system may include one or more bias units.

[0195] The starting position for detection / measurement by the sensing module may be the position where the particle is closest to the detection unit.

[0196] Particles may or may not be tethered. Tethered particles are tethered to larger secondary particles (macromolecules). Non-tethered particles may diffuse freely throughout the sample, while tethered particles have limited diffusivity and may diffuse freely in the sample within the tether connection. Tethered particles are described in more detail later in this specification.

[0197] In embodiments where the particles are not tethered (i.e., are free to diffuse within the sample), closest proximity to the detection unit may be a location at the face of the sample / reaction well adjacent to the detection unit. For example, the biasing system may exert a force to move bound and unbound particles that are free to diffuse within the sample / reaction well toward the face of the microfluidic chip closest to the detection system.

[0198] In embodiments in which the particle is tethered, closest proximity to the detection unit may be the closest position to the detection unit permitted by the tether connection.

[0199] The bias system may comprise an active or a passive system.

[0200] An active bias system uses energy from a power supply to generate forces that are used to position particles within the sensing zone of a detection system. For example, an active bias system may convert power from a battery to generate magnetic fields, electric fields, acoustic waves, electromagnetic waves, differential pressure to position particles at a starting position for detection / measurement. Active bias systems may include magnetic field generators, electric field generators, acoustic tweezers, centrifuge systems, and active pumps.

[0201] A passive bias system may passively position particles in a sensing zone of a detection system without requiring an external energy input. Passive positioning may be achieved using one or a combination of features (e.g., in a microfluidic device) to position particles. For example, a passive bias system may include a trapping element that traps particles flowing in a microchannel of a microfluidic device to position the particles at a starting position for detection / measurement. A passive bias system may include other passive mechanisms, such as a capillary pump.

[0202] Other bias systems may include the use of soluble or dissolvable materials to position or immobilize particles, as well as emulsion and liquid phase approaches to position particles.

[0203] The various biasing systems are described in detail in the following paragraphs.

[0204] The bias system may comprise one or more magnetic field generators that generate a magnetic field optimized to magnetize the magnetizable particles and / or position the magnetizable particles in the microfluidic chip. The magnetic field generators may comprise magnets.

[0205] The magnetic field generator may generate a magnetic field in a direction perpendicular to the sensor, for example, the magnetic field generator may generate a magnetic field from above and / or below the magnetic field sensor such that the magnetic field is perpendicular to the body of the magnetic field sensor.

[0206] The magnetic field generator may generate a magnetic field in a direction parallel to the sensor, for example, the magnetic field generator may generate a magnetic field from the side of the magnetic field sensor such that the magnetic field is parallel to the body of the magnetic field sensor.

[0207] The device may comprise a combination of magnetic field generators that generate magnetic fields in directions perpendicular and parallel to the sensor.

[0208] The magnet may comprise an electromagnet that may exert a field of strength of about 0.5 Gauss, 1 Gauss, 5 Gauss, 10 Gauss, 15 Gauss, 20 Gauss, 25 Gauss, 30 Gauss, 35 Gauss, 40 Gauss, 45 Gauss, or 50 Gauss, with a suitable range being selected between any of these values.

[0209] The magnets can be controlled or switched to position the magnetizable particles in proximity to the magnetic sensor, within the detection area of ​​the microfluidic chip.

[0210] The magnet may exert a magnetic field strength of about 0.01 Gauss, 0.05 Gauss, 0.1 Gauss, 0.2 Gauss, 0.3 Gauss, 0.4 Gauss, 0.5 Gauss, 0.6 Gauss, 0.7 Gauss, 0.8 Gauss, 0.9 Gauss, 1 Gauss, 5 Gauss, 10 Gauss, 50 Gauss, or 100 Gauss, and a suitable range may be selected between any of these values.

[0211] In some embodiments, the magnetizable particles have a particle size of about 1 nm to about 100 nm, with suitable ranges being selected between any of these values. The controller may bias the particles through the generation of an external force, which acts to increase any inter-particle, particle-solvent, or binding forces.

[0212] In some embodiments, the magnetizable particles have a particle size of about 0.5 μm to 5 μm, with suitable ranges being selected between any of these values. The controller may bias the particles through the generation of an external force, which acts to completely counter any inter-particle, particle-solvent, or binding forces.

[0213] The magnetic field generator may be configured to generate a magnetic field from below and / or above the sensing surface.

[0214] The bias system may comprise one or more electromagnetic field (EMF) generators that generate an electric field optimized to position particles within a sensing zone of the detection system. The electric field generator generates an electric field in the sample to move particles within the sample. The EMF generator may comprise a power supply unit, a rotating armature AC generator such as a stator of any form, or a rotating field AC generator such as a rotor, or a multi-layer generator.

[0215] The power supply unit may be a DC power supply unit.

[0216] The electric field generator may output a voltage of about 0.1 volts, 1 volt, 2 volts, 3 volts, 4 volts, 5 volts, 6 volts, 7 volts, 8 volts, or 9 volts, and a suitable range may be selected between any of these values.

[0217] The electric field generator may output a wattage of 100 Watts, 120 Watts, 140 Watts, 160 Watts, 180 Watts, 200 Watts, 220 Watts, 240 Watts, 260 Watts, 280 Watts, 300 Watts, 320 Watts, or 340 Watts, 360 Watts, 380 Watts, 400 Watts, 420 Watts, 440 Watts, 460 Watts, 480 Watts, 500 Watts, and a suitable range may be selected between any of these values.

[0218] The electric field generator may include sensing elements, such as electrodes (anodes and cathodes), conductive coils, and conductive circuits. For example, a cathode and an anode may be provided in the sample well.

[0219] The electrodes may be operated at alternating current (AC) frequencies of 10 kHz, 100 kHz, 1000 kHz, 10000 kHz.

[0220] The electric field generator may be configured to generate an electric field beside, above, or around the sensing surface.

[0221] The device may include one or more electric field generators that generate an electric field to facilitate dielectrophoresis (DEP).

[0222] The electric field generator may comprise one or more electrode pairs.

[0223] The electrodes may be operated with direct current (DC) or alternating current (AC) at a voltage of 1 volt, 2 volts, 3 volts, 4 volts, 5 volts, 6 volts, 7 volts, 8 volts, 9 volts, or 10 volts.

[0224] The electrodes may be operated at alternating current (AC) frequencies of 10 kHz, 100 kHz, 1000 kHz, 10000 kHz.

[0225] The electrodes can be controlled or switched to position the particles within the detection area of ​​the microfluidic chip and in proximity to the detection surface.

[0226] The electric field generator may be configured to generate an electric field beside, above, or around the sensing surface.

[0227] In some embodiments, the bias system may be implemented using dielectrophoresis. Dielectrophoresis-based bias systems use a non-uniform electric field via electrodes to control particle movement. The frequency of such a non-uniform electric field may be set to controllably position particles within the fluid of specific sizes and shapes.

[0228] The bias system may be based on acoustics, cavitation, vibration, or acoustohydrodynamics.

[0229] The bias system may comprise one or more acoustic or electrical tweezers that generate acoustic waves to position particles within a detection zone of a detection system. Acoustic tweezers use the force of sound waves or acoustic radiation to move particles within a sample. For example, standing surface acoustic waves (SSAW) through the application of interdigital transducers (IDTs) (which may be orthogonally positioned) focus particles within a detection zone of a sample introduction device.

[0230] A sample introduction device, such as a microfluidic device, can be designed with specific features (such as microchannel shape and dimensions) that optimize the effectiveness of the SSAW generated by the IDT. For example, the sample introduction device can include a pressure node.

[0231] The bias system may be implemented using the piezoelectric effect: a piezoelectric film, membrane, or reflector may be used to position particles within the sensing zone at a starting position for detection / measurement by the sensing module.

[0232] The sample introduction device may incorporate acoustic vortex designs and features that enhance particle positioning within the detection zone. The vortices may be generated through a combination of actuation, flow, holographic transducer, and microfluidic lens features to control the vortex forces to a very fine degree of operation.

[0233] The biasing system may comprise a centrifugal force system that uses centripetal force to position the particles within the detection zone of the detection system. In this embodiment, a sample introduction device (such as a sample receptive chip or a microfluidic chip) may be subjected to centrifugal force at a suitable speed and for a suitable amount of time to position the particles within the detection zone.

[0234] When a centrifugal force system is used, the sample introduction device may include a sample vessel having one or more channels with a circular or semicircular cross section. The sample vessel channels may have a radius of about 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, and a suitable range may be selected between any of these values.

[0235] The sample introduction device containing the sample was rotated at approximately 100 rpm (min -1 ), 150 rpm (min -1 ), 200 rpm (min -1 ), 250 rpm (min -1 ), 300 rpm (min -1 ), 350 rpm (min -1), 400 rpm (min -1 ), 450 rpm (min -1 ), 500 rpm (min -1 ), 550 rpm (min -1 ), 600 rpm (min -1 ), 650 rpm (min -1 ), 700 rpm (min -1 ), 750 rpm (min -1 ), 800 rpm (min -1 ), 850 rpm (min -1 ), 900rpm(min -1 ), 950 rpm (min -1 ), or 1000 rpm (min -1 ) and a suitable range may be selected between any of these values.

[0236] The sample introduction device containing the sample may be centrifuged for a predetermined time of about 0.5 minutes, 0.75 minutes, 1 minute, 1.25 minutes, 1.5 minutes, 1.75 minutes, 2 minutes, 2.25 minutes, 2.5 minutes, 2.75 minutes, 3 minutes, 3.25 minutes, 3.5 minutes, 3.75 minutes, 4 minutes, 4.25 minutes, 4.5 minutes, 4.75 minutes, 5 minutes, 5.25 minutes, 5.5 minutes, or 6 minutes, with a suitable range selected between any of these values.

[0237] For example, the sample introduction device containing the sample was rotated at 520 rpm (min -1 ) can be subjected to centrifugal force.

[0238] After centrifugation for a predetermined amount of time, the sample introduction device may slow down to a stop for a period of time. For example, the centrifuge may slow down for a period of 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds.

[0239] Once fully stopped, the sample vessel remains stationary throughout the remainder of the detection and measurement. The sensor is positioned proximate to the circular channel (at the outer circumference) to perform the detection and measurement.

[0240] The bias system may include laminar flow patterning and micromixers with laminar flow. This may be implemented as pinch flow fractionation (PFF) using microfluidic features, microbubblers, and other complementary design elements or inclusions. Additional microfluidic design features may be utilized to interrupt the laminar flow or otherwise trigger the release of particles to diffusion forces (including Brownian motion).

[0241] The biasing system may comprise an active pump or suction system, which may be implemented in conjunction with a capture element provided in the sample introduction device.

[0242] In some embodiments, a capture element may be provided in a sample well or microchannel of a microfluidic device to capture particles. The capture element may be located in a location in the sample introduction device that corresponds to a detection zone of a detection module. The capture element may include a permeable or semi-permeable material that allows sample fluid to pass through while retaining particles. For example, the capture element may include a gel, such as an agarose gel.

[0243] In some embodiments, the agarose gel may comprise 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3% agarose gel, and a suitable range may be selected between any of these values.

[0244] In some embodiments, the capture element may include an angled ramp.

[0245] Pressure or suction created by an active pump or suction system forces the particles to become trapped in a capture element located in close proximity to the sensing module. When the pressure or suction is relaxed or removed, the particles are free to undergo Brownian diffusion that is detected and measured by the sensing module.

[0246] An active pump or suction system combined with the sample introduction device can be configured to create a hydrodynamic effect such that freely moving particles become trapped in the recirculating flow to position the particles in proximity to the sensor.

[0247] The active pump may be operated in cycles of active flow and passive flow. In each cycle, the active pump may be operated for a predetermined time to establish active flow and deactivated to allow passive flow for a predetermined period of time. For example, the active pump may be operated for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds and deactivated for a period of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 seconds, with suitable ranges being selected between any of these values.

[0248] The active pump may run for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles before the sensing module acquires data.

[0249] The biasing system may include a passive pump configured to passively position the particles within a sensing zone of the detection system without requiring an external energy input. A passive pump may be any microfluidic design feature that enhances and / or controls capillary effects without requiring an active pump.

[0250] A passive pump, such as a capillary pump, can be implemented using microfluidic design features that enhance the capillary effect within the microfluidic chip, so that sample fluid can be passively drawn through a capture element (as described for an active pump) to position the beads in proximity to the sensor.

[0251] The passive pump can be adjusted for a set amount of time taking into account the control fluid dynamics of the microfluidic design, so that after the set amount of time, the capillary effect disappears by the conducted fluid entering a larger chamber within the microfluidic design (or other design example).

[0252] The particles may be incorporated or immobilized in a soluble or dissolvable material.The particles may be incorporated or immobilized in a location within the sample introduction device that corresponds to a detection zone of the detection module.

[0253] The particles may be incorporated or immobilized such that a surface of the particle remains available for binding the target analyte in the sample. Such functionalized particles may be loaded into the sample introduction device in a dry state utilizing any one of the applicable adhesive compounds known to dissolve in liquids. Upon introduction of the sample (such as, for example, plasma), the soluble or dissolvable material dissolves to release particles that undergo Brownian motion that is detectable and measurable by the sensing module.

[0254] The soluble or dissolvable material may have biodegradable and biocompatible properties.

[0255] Soluble or dissolvable materials may include soluble chemicals, reagent films, and adhesives including, but not limited to, sodium alginate, calcium alginate, gelatin, agar, agarose, latex adhesives, hydrogels, cellulose membranes, polyvinyl alcohol, and the like.

[0256] The bias system may be based on emulsion and liquid phase approaches, such as Pickering emulsions. According to such approaches, particles may be transported using an emulsion that is controllable to return to a liquid phase via changes in pH, temperature, and / or ionic strength. The particles may be transported in the emulsion in proximity to a sensing module and then released by returning from the emulsion phase to a liquid phase by a controlled change in one or more of the known triggers for such a phase change. Once the emulsion returns to a liquid phase, the beads are subject to effects such as Brownian motion and the sensor may detect the beads.

[0257] Optionally, one or more of the above bias systems may be used in combination to achieve an improved bias effect, for example, a magnetic generator may be used in combination with an active pump and suction system to achieve an improved effect.

[0258] The orientation detection module may comprise a sensor that detects the orientation of the device. The sensor that detects the orientation may comprise a gyroscope-based sensor, an inertial measurement unit, and / or an accelerometer. The sensor allows the device to operate in any orientation. The operation of the device or the performance of the method effectively does not depend on gravity. That is, the device can perform the method regardless of how the device is oriented. For example, the device may be operable in an inverted configuration with the magnetic field sensor facing up the sample reservoir or microfluidic device.

[0259] 12, there is shown a device orientation detection module 1200. In an embodiment, the orientation detection module comprises an accelerometer 1201 configured to detect the orientation of the device.

[0260] The power management module may include an on-board power supply controller to control / power the device. The power supply may be an AC input and / or a DC input.

[0261] The power control module allows the device to select the power source to minimize signal noise and maximize performance. For example, whenever available, AC power (provided by the USB-C input) is required when the magnetic sensor is reading / sensing, and during these times, the DC power battery is temporarily utilized.

[0262] The AC input may include receiving power externally from a USB Type-C based connection provided on the device.

[0263] The on-board DC input power supply may include a rechargeable lithium-ion battery. In some embodiments, the power supply is a 3.7V, 1200mAh lithium-ion battery.

[0264] In an embodiment, the power management module may include a power rectifier and / or a boost regulator to regulate the voltage (from 3.3V to 5V).

[0265] In an embodiment, the power management module may include a regulator to maintain the power at 3.3V.

[0266] The power management module may comprise a switching unit for switching from AC to DC mode when external power is not available.

[0267] The power management module may further comprise a battery status indicator for determining and indicating the power level in the battery when external power is not available.

[0268] The power management module may provide a battery charge percentage display in the user interface.

[0269] The power management module may provide a quality control indication at the start of each test attempt to determine whether sufficient power remains in the battery to complete each test.

[0270] Referring to Figure 10, the power management module of the device is shown. The power management module 1000 comprises a power management unit 1005 configured to determine whether there is 5V input power received via the USB connector. If such power from the USB connector is detected, the battery charger chip 1001 is configured to charge the internal battery. If no input power is detected, power is received from the internal battery.

[0271] A booster circuit 1002 in this module is configured to boost the voltage of the battery. In an embodiment, a battery monitor circuit 1003 is configured to determine the power level of the battery. The power management module also includes a voltage regulator 1004 for powering certain low-voltage components of the device. For example, the components operate at an input range of 3.3V.

[0272] The control module may comprise a controller that may be connected to the device to receive signals from the array of magnetic field sensors 40 or the array of electric field sensors 50, the signals representing the relative net changes in the magnetic or electric fields of the bound and unbound magnetized particles induced by Brownian motion or diffusion of the magnetized particles.

[0273] The controller may be configured to determine the relative amount of the analyte in the sample based on signals received from the array of magnetic field sensors or the array of electric field sensors.

[0274] The wireless communication module may comprise a wireless and / or cellular communication module. The wireless communication module may be configured for wi-fi and / or Bluetooth low energy wireless communication. The cellular communication module may be configured for 3G, 4G, and / or 5G cellular communication.

[0275] The communications module may facilitate communication of the device with one or more external networks or devices, including other PCB cores within the same core (e.g., in multi-core design embodiments). In some embodiments, the device may be wirelessly connected to a computer or mobile communications device. In some embodiments, the device may be connected to an Internet of Things (IoT) network.

[0276] In an embodiment, the communications module is configured to wirelessly transmit telemetry, environmental, and diagnostic data acquired on the sample to another network device.

[0277] The device may include a built-in display. A display module 1100 is shown in Fig. 11 with an input module 1101 configured to send and receive signals and information commands from the CM module. The display module may include an ESD protection circuit 1102 and provide signals from the ESD protection circuit to the built-in display 1103.

[0278] Here, the device's internal quality control steps are presented below. When the device activates (PCB switch / remote switch / timer-based power on / accelerometer sensor / remote command from network core or device), the device may start a series of internal quality controls (QC). The QC control may include cycling test parameters across all subsystems, reading ambient conditions such as device temperature, ambient magnetic field above the sensor, set & reset of all sensors, utilizing the sensor in various set-reset modes to determine which sensor location is mounted on the PCB, device system health status, components, error conditions (e.g., high G events since last power on, which may indicate potential structural damage), before measuring and then recording any system-generated interference or bias of potential algorithm offsets.

[0279] The following QC checks signal generation can occur in the following steps: Input / software / firmware (local or remote) commands the action. In some embodiments this may include input data from a connectivity core (PCB), a mobile phone, or from a Near Field Communication tag with embedded data. The NFC tag may come from a single-use NFC tag included in a disposable diagnostic chipset and provide information for the system to use in terms of analysis, biomarkers, sensor location, typical sensitivity range for the result, batch number, expiry date, associated variety, associated fluid type (blood, tears, saliva, etc.), electromagnet requirement, assay type, analyte binding kinetics, and wait time, read cycle, frequency, duration, mathematical confidence interval, pass-extend-failover test values. This process may occur in the background while the login / customer / patient details are selected. · Software / UI / indicator may instruct user to insert microfluidic / sample. The software can be configured to incorporate any input instructions in the form of UI / indicators and initiate an associated sequence of operations on the PCB and attached peripherals (battery / USB C / indicator LEDs / screens / coils, etc.). Depending on the assay (embodiment), the electromagnetic field generator (electromagnet) can be powered and follow a predefined sequence of on / intensity curve / off / potential polarity switches and potential repetitions. These can control the magnetic particles for optimized performance and fast binding kinetics of analytes to functionalized magnetic beads. In some embodiments, the electromagnets and electromagnetic generating fields can be controlled and optimized for short reaction times using power control circuits, e.g., H-bridge circuits. In some embodiments, further quality control testing can be done by using existing sensors to determine environmental changes that are synchronized to sample introduction into the device, so that liquid movement, location, velocity, and viscosity can be determined. After a minimum dwell time of >10 μs (to ensure the device is not reading Neel relaxation), the sensor can be set / reset quickly (to ensure absolute time series alignment or magnetic set-reset) without pause after set-reset, and after a few millionths of a second, the analog magnetic field sensor is read at up to a total of 450,000 readings per second (across the 24 sensor array). This occurs via the following approach: within each field sensor, the analog dynamic magnetic field environment is continuously sensed and converted to a voltage and fed to a sensor-dedicated 10,000x amplifier. All sensor-dedicated amplifier circuits are equidistant / near equidistant in length (to ensure data parity and timing). The amplifier then feeds it (the amplified voltage signal) to one of three analog-to-digital converters that tally up the data at 16-bit resolution. Depending on the required / desired read time and number of read cycles, there are tens of millions of data points per chip for processing in less than a minute.

[0280] Either between each reading or some other number of readings, the sensor can be set / reset to maintain maximum consistency and data integrity. For this same reason, the data circuitry is protected by ground plane circuit layers above and below the data circuitry to minimize any interference and maintain maximum signal relevance. The ADC progressively streams / transmits data to either the MCU or CM for processing, storage, forwarding transmission, e.g., a connected device or a mobile phone. Active data analysis is performed such that a feedback loop is created where data acquisition can be actively extended or concluded depending on the clarity, quality, consistency, clarity, etc. of the data being read and processed against device parameters (including parameters from telemetry, environmental viscometer, near-field input, QC checks, temperature, etc.).

[0281] In an embodiment, the device comprises a housing that houses at least one circuit board. The housing may further comprise a built-in display configured to depict status and / or diagnostic output obtained from the circuit board.

[0282] In an embodiment, the housing with the built-in display and at least one circuit board is configured to operate as a lab-on-a-chip device. In another embodiment, the housing with the built-in display and multiple circuit boards arranged in parallel is configured to operate as a lab-on-bench device.

[0283] In an embodiment, the housing that performs the operation of the lab-on-chip and lab-on-bench devices is configured to be controlled by a user interface.

[0284] This housing / case can have minimal openings, provide a uniform surface that is easily sanitized, and keep the sample on the outside of the device (any portion entering the device is completely encapsulated in plastic in the sample introduction device and close to the sensor face). The instrument can be configured for operation in either bench-top mode (screen facing up at a small angle) or wall-mounted mode (screen facing outward at a small angle). The entry point for the sample introduction device can be adjusted / oriented between these two implementations.

[0285] In a veterinary clinical room, a wall-mounted embodiment may solve the problem arising from animals' tendency to knock over anything on a bench or desk, where fluids often come into contact with items in these same locations.

[0286] Referring to FIG. 14, a CAD design of a variation of the device is shown. In this embodiment, the housing is shown with a rectangular opening with curved upper corners that houses a 7 inch capacitive touch screen. In some embodiments, this screen represents the primary user interface through on-board software on the device or a tethered device. A number of variations on the device embodiments are provided below.

[0287] The device may have a single core with a single computing module. The device may have a case, a screen, a battery, and optionally passive or active cooling. In this embodiment, the sample entry point may be located on the left or right side or at the front center. The device may autonomously manage one UI, network and diagnostic functions of the device, as well as QC processes. It will be understood that smaller versions may be implemented for more mobile applications, such as mobile veterinarians, home testing by patients and owners with results returned to the clinic / veterinarian, and emergency implementations for critical presentations at hospital and veterinary reception.

[0288] The device may have a larger capacity with a core with two compute modules, optionally including a case, a screen, a battery, optionally passive or active cooling, and a separate dedicated compute unit. In this embodiment, the separate dedicated compute unit may handle power output for the core, data I / O for the core, UI for the screen, and also network connection / workflow queues and communications for implementing the management software. The core (without the compute module) may operate as a slave to the central unit and simultaneously take in power and data through a usb-c connection. In other embodiments using a cat5 / 6 connection cable or similar, the core may stream its raw results to the compute unit for calculation, report rendering, and network / screen presentation modes. These implementations may have either a front-facing, left-right positioned chipset opening, or a left-side and right-side case opening. The larger capacity implementations may be configured to be suitable for smaller veterinary campus labs and shared / multiple animal clinical rooms (as well as human analogs such as small GP clinics).

[0289] Variations of the above devices are expected to fit within similar housing dimensions to each other and to FIG. 14, and are configured for diagnostic testing using peripheral blood pricks or systemic blood samples.

[0290] 15 shows an embodiment of a touch screen user interface for a device used in a veterinary environment (in a lab-on-a-chip or lab-on-bench environment). In this embodiment, a user (veterinarian or laboratory clinician) can enter data about the sample being processed. In this example, the user can select whether the sample belongs to a dog or a cat, and add any additional notes on the test (e.g., about patient information about the animal). A unique test display is then presented for the sample being run, which can be retrieved later during analysis of the results.

[0291] In some embodiments, the device may include network and workflow integration for implementing management software and applications, which may allow integration of remote ordering of tests and results within clinic (human or veterinary) software systems and platforms.

[0292] In non-networked embodiments / configurations, the controller is configured to render graphical images on the screen and a data file relating to the results of the diagnosis. This may include environmental and various telemetry indicators relating to the device during operation of the device. This information may then be sent (along with the input reference number, the patient's name and details entered just prior to the start of the test) to a designated email or cloud storage source.

[0293] FIG. 16 shows an example of an exemplary user interface of the apparatus depicting the diagnostic results of a processing sample device.

[0294] The device may be configured to operate as a personal health assistant. In embodiments, the device may be connected to any one of the personal assistant devices such as Amazon Echo®, Google Nest®, Apple Watch®, or any smart device using a virtual assistant such as Microsoft Cortana®, Amazon Alexa®, or Apple Siri®.

[0295] The apparatus may be embedded or connectable to a personal assistant device. Such an embodiment allows for sharing of one or more components between the apparatus and the personal assistant device. For example, the embedded personal assistant device may utilize the processing power, memory, network connectivity, cloud storage, power supply of the apparatus, and vice versa.

[0296] The integration of instruments and personal health devices will enable improved integration of contextual health data and services such as telehealth appointments and platforms, real-time telehealth prescribing of diagnostic panels, online medication fulfillment, fitness and wellness data and programs linked to diagnostic results, telehealth expert advice, voice control and remote authentication of devices, and HIPAA approved medical record apps.

[0297] The integration of appliances and personal health devices enables a holistic approach to healthcare by providing the contextual benefit of health or medical data while providing the home diagnostics necessary for a complete suite of remote healthcare or preventive healthcare services.

[0298] The devices may be backward compatible with older devices. Such embodiments allow devices to be connected to a larger portion of the population, expanding access to remote populations and expanding healthcare options to populated areas, especially during periods of restricted social mobility.

[0299] The virtual assistant may be built into the device.

[0300] The device can be configured to provide alerts, reminders, and set goals, and to schedule appointments with medical professionals to discuss the results of the diagnosis.

[0301] A method for detecting an analyte in a sample is described, comprising the steps of: contacting a sample comprising a target analyte with particles that produce or can be induced to produce a detectable signal, the particles being coated with binding molecules complementary to the target analyte to produce bound and unbound binding agent complexes; Applying a bias field to position particles with both bound and unbound binding agent complexes near a detection module (the "capture" step); modifying the bias field sufficiently to release at least a portion of the particles having both bound and unbound binding agent complexes from the vicinity of the sensing module (the "releasing" step); and Measuring a change in a detectable signal detected from the particle as a result of a net movement of the particle relative to the sensing module, the movement being either translational and / or rotational.

[0302] The described method is based on the concept of bringing particles and analyte complexes that generate or are induced to generate a detectable signal in close proximity to a sensing module (i.e., either a magnetic field sensor or an electric field sensor). The bias field strength is modulated to allow the particles and analyte complexes to diffuse (i.e., by translational and / or rotational movement) away from the magnetic field sensor or electric field sensor. The sensing module then measures the change in the detectable signal generated by the particles over time by Brownian motion or diffusion, which allows the quantification of the amount of particle-analyte complexes and then the determination of the amount of analyte in the sample. That is, bound and unbound binding agent complexes are distinguished based on their diffusion properties, which are determined from the net flux value read by the change in the sensing module over time. The particles (i.e., both bound and unbound complexes) are physically moved relative to the sensing module so that bound and unbound complexes can be distinguished (assuming that they move to different extents due to different diffusion properties).

[0303] Broadly speaking, there may be three stages in the method of analyzing a sample. The first stage may be a pre-sample baseline sensing stage. This stage is performed to obtain a baseline reading in the absence of a sample. The baseline reading provides a base comparison for subsequent sample readings. The pre-sample baseline sensing stage may be performed for 1, 2, 3, 4, or 5 seconds, with suitable ranges selected between any of these values ​​(e.g., about 1 to about 5 seconds, about 1 to about 4 seconds, about 2 to about 5 seconds, about 2 to about 3 seconds, or about 3 to about 5 seconds).

[0304] The second step can be to load the sample into the device. This step can include mixing of the sample and analyte-binding agent complexing (i.e., where the functionalized particles bind to the analyte). This step can be performed for about 3, 4, 5, 6, 7, or 8 minutes, with suitable ranges selected between any of these values ​​(e.g., about 3 to about 8 minutes, about 3 to about 7 minutes, about 3 to about 5 minutes, about 4 to about 8 minutes, about 4 to about 6 minutes, or about 5 to about 8 minutes).

[0305] The third step can be a sample read step, i.e., the particles are positioned near a sensing module, the bias field is changed to release at least a portion of the bound and unbound binding agent complexes, and the sensing module measures the resulting change in signal detected from the particles related to the net movement of the particles relative to the magnetic sensor. This step can be performed for about 1, 2, 3, 4, 5, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 seconds, or 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 seconds, with preferred ranges being any of these values. (e.g., about 10 seconds to about 20 seconds, about 10 seconds to about 18 seconds, about 10 seconds to about 15 seconds, about 11 seconds to about 20 seconds, about 11 seconds to about 19 seconds, about 11 seconds to about 16 seconds, about 11 seconds to about 15 seconds, about 12 seconds to about 20 seconds, about 12 seconds to about 18 seconds, about 12 seconds to about 15 seconds, about 13 seconds to about 20 seconds, about 13 seconds to about 19 seconds, about 13 seconds to about 17 seconds, or about 13 seconds to about 15 seconds).

[0306] Particles may be attached to other objects, such as larger secondary particles or molecules. Magnetizable particles may also be attached to surfaces. Attachment to other objects or surfaces allows magnetizable beads to be located in specific locations while retaining the ability to undergo Brownian diffusion (within the limits of the attachment or tether connection) that is detectable and measurable by the device.

[0307] Advantageously, by being tethered, particles can retain the ability to undergo Brownian diffusion while being positioned at specific locations in a larger shared volume, such that multiple types of magnetizable particles (types with analyte recognition or other properties) can all be in their discrete locations (e.g., aligned with a specific magnetic sensor) while still being within a shared volume, thereby enabling multiplexed detection of different target analytes in a volume.

[0308] Such multiplexed detection is made possible by tethering non-magnetizable beads or surfaces of microchannels, as the non-magnetizable beads can act as anchors to keep the tethered particles in place through a combination of size, surface chemistry, and interactions with the local environment.

[0309] For example, a magnetizable particle may be molecularly tethered to a larger non-magnetizable particle, such as a latex bead, such that the magnetizable particle is positioned in a particular area by the larger non-magnetizable bead, but can still diffuse freely within the confines of the tether connection. In another example, a magnetizable particle may be molecularly tethered to a surface, such as a surface of a microfluidic device that corresponds to a detection zone of a detection module.

[0310] The non-magnetizable particles may comprise any suitable non-magnetizable particles, including, but not limited to, latex beads, polystyrene beads, or other types of polymer beads.

[0311] In some embodiments, a non-magnetizable particle such as a latex bead with surface chemistry (such as amine and carboxy groups) can have a molecular tether (e.g., polyethylene glycol - PEG) attached to the particle such that one end of the molecular tether is attached to the latex bead (with chemistry compatible with the latex bead surface) and the other end is attached to the magnetizable bead (with chemistry compatible with the magnetic bead surface, e.g., biotin on the tether attached to streptavidin on the magnetic bead surface), thus forming a tether connection between the two beads.

[0312] The molecular tether can be about 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm long. As described above, the amount of analyte in the sample is determined based on a change in the signal detected by the sensing module. The sensing module detects the change based on the net movement of the particle. When the particle is released from the vicinity of the sensing module, the particle with both bound and unbound binding agent complexes moves away from the sensing module. This movement is random based on Brownian diffusion.

[0313] Typically, the sensing module is located near or adjacent (non-sample side) to the surface of the sample well or sample reservoir. When the bound and unbound particles are located near the magnetic field sensor, they may be located at or near the wall of the sample well or sample reservoir until they are released. When the particles are released from the vicinity of the sensing module, they may move translationally and / or rotationally. Assuming that the particles are close to the surface of the sample well or sample reservoir, the bound and unbound particles may usually move with an initial degree of freedom of movement of about 180° relative to the surface of the sample well or sample reservoir. Brownian diffusion means that the particles may move in any direction, including towards the magnetic field sensor. The magnetic signal detected by the magnetic field sensor is based on the net movement of the bound and unbound particles.

[0314] Advantages of the present invention may include rapid detection (see, eg, Example 2) and highly sensitive detection methods (see, eg, Examples 1 and 3).

[0315] Considering collisions between analytes and particles that are free in solution, the diffusion collision step can be separated into (1) a process of diffusion transport through the fluid volume and (2) a process of alignment near the surface. If the volume transport generates the first collisions between the particles and the analytes of interest, the subsequent alignment process near the surface deals with the alignment rate of the binding sites of the reactants. The volume transport is essentially a translational process, while the alignment is determined by both the translational and rotational mobilities of the reactants.

[0316] When free components react in solution, ordering processes (i.e., rotational diffusion) are a significant limitation due to the highly inherent ordering constraints, but volume transport (i.e., translational diffusion) is not a limitation. When one of the components is attached to a surface, volume transport can become a limitation.

[0317] The magnetic properties of nano- and micro-sized particles differ from those of the corresponding bulk magnetic materials. Magnetizable particles are usually classified as paramagnetic, ferromagnetic, ferrimagnetic, antiferromagnetic, or superparamagnetic based on the magnetic behavior of the magnetizable particles in the presence or absence of an applied magnetic field.

[0318] The diagnostic material does not exhibit a dipole moment in the absence of a magnetic field, and in the presence of a magnetic field, the diagnostic material aligns with respect to the direction of the magnetic field.

[0319] Paramagnetic particles exhibit random dipole moments in the absence of a magnetic field, and in the presence of a magnetic field, the paramagnetic particles align with the direction of the magnetic field.

[0320] In a perpendicular magnetic field, superparamagnetic particles, exhibiting aligned magnetic moments, can repel one another, increasing the equilibrium spacing and reducing correlated particle migration.

[0321] A parallel magnetic field can result in attractive forces between equilibrium superparamagnetic particles, resulting in a higher degree of correlated particle movement.

[0322] Ferromagnetic materials exhibit aligned dipole moments.

[0323] Ferromagnetic and antiferromagnetic materials exhibit alternatingly aligned dipole moments.

[0324] In one embodiment, the magnetizable particles are paramagnetic particles. Such particles become magnetic when subjected to a magnetic field. When the magnetic field is removed, the particles begin to lose their magnetic properties.

[0325] In an alternative embodiment, the magnetizable particles are ferromagnetic particles, i.e. they always exhibit magnetic properties whether or not they are subjected to a magnetic field.

[0326] Commercially available magnetizable particles include Dynaparticles M-270, Dynaparticles M-280, Dynaparticles MyOne T1, and Dynaparticles MyOne C1 from Thermo Fisher Scientific, μMACS MicroParticles, SPHERO® Superparamagnetic Particles, SPHERO® Paramagnetic Particles from Miltenyi Biotec, and SPHERO® Ferromagnetic Particles from Spherotech.

[0327] In one embodiment, the magnetizable particles used are Spherotech SVFM-20-5 (2.0 micrometers to 2.9 micrometers).

[0328] The magnetizable particles may be streptavidin-coated ferromagnetic particles, which may be functionalized with a biotinylated "detection" antibody.

[0329] The magnetizable particles may be formed by ferrites, which themselves are formed from iron oxides (such as magnetite and maghemite). Various methods are known for synthesizing iron oxide and metal substituted ferrite magnetizable particles, such as co-precipitation, pyrolysis, and hydrothermal. The co-precipitation process uses stoichiometric amounts of ferrous and ferric salts in an alkaline solution in conjunction with a water-soluble surface coating material such as polyethylene glycol (PEG), where the coating provides colloidal stability and biocompatibility. The size and characteristics of the magnetizable particles can be influenced by the concentration of the reducing agent, pH, ionic strength, temperature, iron salt source, or Fe. 2+ vs. Fe 3+ can be controlled by adjusting the ratio of

[0330] The size and shape of the magnetizable particles can be tuned by varying the reaction conditions, such as the type of organic solvent, heating rate, surfactant, and reaction time. This method leads to a narrow size distribution of magnetizable particles in the size range of 10 nm to 100 nm. 2+ can be replaced by other metals to increase the saturation magnetization.

[0331] The inventors have also found that larger particles can be effective. For example, the particles can have a size of about 0.5 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, or 5 μm, and suitable ranges can be selected between any of these values.

[0332] The magnetizable particles may be coated with a hydrophobic coating during the synthesis process, in which case the method of producing the magnetizable particles may include an additional step of ligand exchange so that the magnetizable particles can be dispersed in water for further use.

[0333] Magnetizable particles can be produced by polyol hydrothermal reduction, which produces water-dispersed magnetizable particles in the size range from tens to hundreds of nanometers. The size and surface functionalization of the iron oxide magnetizable particles can be optimized by adjusting the solvent system, reducing agent, and type of surfactant used. This process can be used to synthesize FePt magnetizable particles.

[0334] Magnetizable particles can be produced by the inverse water-in-oil micelle method, which forms a microemulsion of aqueous nanodroplets of iron precursor stabilized by surfactants in an oil phase, with magnetic nanoparticles obtained by precipitation. Iron oxide nanocrystals can be organized into magnetizable particles with diameters greater than 100 nm by mixing the microemulsion and a silica sol-gel, which can be obtained via co-precipitation.

[0335] Metallic magnetizable particles can be either monometallic (e.g., Fe, Co, or Ni) or bimetallic (e.g., FePt and FeCo). Alloy magnetizable particles can be synthesized by physical methods including vacuum deposition and gas phase evaporation. These methods produce high saturation magnetization (~207 emu / g(A m 2 %. / kg) and 3+ and Co 2+ can be synthesized via reduction of a salt of

[0336] The magnetizable particles may include a single metal or metal oxide core. The magnetizable particles may include multiple cores, multiple layers of magnetic and non-magnetic materials. The magnetizable particles may include a silica or polymer core coated with a magnetic shell. The non-magnetic core particles may include silica or other polymers.

[0337] In some embodiments, the magnetizable particles may include alternating magnetic orientation layers separated by insulating layers.

[0338] The magnetizable particles may include a dielectric silica core coated with a magnetic shell. The magnetic shell may be formed from Co, FePt, or Fe3O4. The shell may also include a stabilizer such as a silica shell or a polyelectrolyte layer. The magnetizable particles may be mesoporous magnetizable particles.

[0339] Coatings on magnetizable particles can determine the interactions between the magnetizable particles and biomolecules (such as analytes) and their biocompatibility. Coatings can be used to determine surface charges, which, together with the coating, can modify the hydrodynamic size of the magnetic particles. The hydrodynamic size of the magnetizable particles can modify the function of the magnetic particles.

[0340] The magnetizable particles may be coated with specific coatings that provide electrostatic and steric repulsive forces, which may help to stabilize the magnetizable particles, preventing their aggregation or co-precipitation.

[0341] The magnetizable particles may include a coating formed from an inorganic material. Such magnetizable particles may be formed with a core-shell structure. For example, the magnetizable particles may be coated with biocompatible silica or gold (e.g., alloy magnetic nanoparticles, FeCo and CoPt, coated with silica). The shell may provide a platform for modifying the magnetizable particles with ligands (e.g., thiols). Other inorganic coating materials may include titanates or silver. For example, silver-coated iron oxide magnetizable particles may be synthesized and integrated in a carbon paste.

[0342] The shell may be formed from silica. The advantage of coating with silica is that the silica-coated magnetizable particles can be covalently bonded with multifunctional molecules and surface reactive groups. The silica shell may be produced, for example, by the Stober or Phillips process using the sol-gel principle or a combination thereof. The core of the magnetizable particles may be coated with tetraethoxysilane (TEOS), for example, by hydrolysis of TEOS under basic conditions, which condenses and polymerizes the TEOS into a silica shell at the surface of the magnetic core. Cobalt magnetizable particles may be coated using a modified Stober process, mixing 3-aminopropyltrimethoxysilane and TEOS.

[0343] The Phillips process forms a silica shell of sodium silicate on the magnetic core. A second layer of silica may be deposited by the Stober process. The reverse microemulsion process may be used to coat with silica. The process may be used with a surfactant. The surfactant may be selected from Igeoal CO-520 to provide a silica shell thickness of about 5 nm to about 20 nm. Preferably, the reagent for producing the silica shell is selected from an amino-terminated silane or an alkene-terminated silane. Preferably, the amino-terminated silane is (3-aminopropyl)trimethoxysilane (APTMS). Preferably, the alkene-terminated silane is 3-methacryloxypropyltrimethoxysilane.

[0344] The magnetizable particles may be coated with gold. The gold-coated iron oxide nanoparticles may be synthesized by any one of chemical methods and reverse microemulsion. The gold-coated magnetizable particles may be synthesized by directly coating gold onto the magnetizable particle core. Alternatively, the gold-coated magnetizable particles may be synthesized by using silica as an intermediate layer of the gold coating. Preferably, reduction is the method used to deposit the gold shell onto the magnetizable particles.

[0345] Metal oxide or silica coated magnetic cores can be first functionalized with 3-aminopropyltrimethoxysilane prior to electrostatic deposition of about 2 nm to about 3 nm gold nanocrystal seeds (from chloroauric acid) to the surface followed by addition of a reducing agent to form a gold shell. Preferably, the reducing agent is a mild reducing agent selected from sodium citrate or tetrakis(hydroxymethyl)phosphonium chloride. In some embodiments, the gold shell is formed from the reduction of gold(III) acetate (Au(OOCCH3)3). In some embodiments, the gold shell is formed on metal magnetic cores (e.g., nickel and iron) by reverse micelles.

[0346] The magnetizable particles can be functionalized with organic ligands. This can be done in situ (i.e., the functional ligands are provided in the magnetizable particles during a synthesis step) or post-synthesis. The magnetizable particles can be functionalized with terminal hydroxyl (-OH), amino (-NH2), and carboxyl (-COOH) groups. This can be achieved by modifying the surfactants (e.g., dextran, chitosan, or poly(acrylic acid)) used in the hydrothermal synthesis.

[0347] Post-synthesis functionalization of magnetizable particles may allow customized ligand functionalization on any magnetizable particle surface. Post-synthesis functionalization may be performed by ligand addition and ligand exchange. Ligand addition involves the adsorption of amphiphilic molecules (containing both hydrophobic segments and hydrophilic components) to form a bilayer structure. Ligand exchange replaces the original surfactant (or ligand) with a new functional ligand. Preferably, the new ligand contains a functional group that is capable of binding at the magnetizable particle surface via either strong chemical bonds or electrostatic attraction. In some embodiments, the magnetizable particles also contain functional groups for stabilization in water and / or biofunctionalization.

[0348] The magnetizable particles may be coated with ligands that improve ionic stability. The functional groups may be selected from carboxylates, phosphates, and catechols (e.g., dopamine). The ligands may be siloxane groups for coating hydroxyl-rich surfaces (e.g., metal oxide magnetic particles or silica-coated magnetic particles). The ligands can be small silane ligands linking magnetizable particles and various functional ligands (e.g., amines, carboxylates, thiols, and epoxides). The silane ligands can be selected from N-(trimethoxysilylpropyl)ethylenediaminetriacetic acid and (triethoxysilylpropyl)succinic anhydride to provide carboxylate terminated magnetic particles. The functional groups can be selected from phosphonic acid and catechol (to provide hydrophilic end groups). The functional groups can be selected from amino terminated phosphonic acid. The functional groups can be selected from 3-(trihydroxysilyl)propylmethylphosphonate for dispersion in aqueous solutions. The ligands can be selected from dihydroxyhydrocinnamic acid, citric acid, or thiomalic acid for magnetizable particles for dispersion in water.

[0349] In some embodiments, the magnetizable particles are functionalized with a polymer ligand, which may be selected from natural polymers (e.g., starch, dextran, or chitosan), PEG, polyacrylic acid (PAA), poly(methacrylic acid) (PMAA), poly(N,N-methylene-bisacrylamide) (PMBBAm), and poly(N,N-methylenebisacrylamide-co-glycidyl methacrylate) (PMG).

[0350] The functional groups on the magnetizable particle surface act as linkers for binding with complementary biomolecules. The biomolecules can be small biomolecules. The small biomolecules can be selected from vitamins, peptides, and aptamers. The biomolecules can be larger biomolecules. The larger biomolecules can be selected from DNA, RNA, and proteins.

[0351] With regard to nucleic acid attachment, the nucleic acid may be attached by non-chemical methods (e.g., electrostatic interactions) or chemical methods (e.g., covalent bonds). The nucleic acid chain may be modified with a functional group. The functional group may be selected from thiols or amines, or any combination thereof.

[0352] Binding of larger biomolecules can rely on the biomolecule's intrinsic binding interactions with a wide range of subtractive and synthetic analogs, such as inherent receptor-substrate recognition (i.e., antigen-antibody and biotin-avidin interactions).

[0353] Specific pairs of proteins can be used to immobilize species on magnetic particles. Physical interactions include electrostatic interactions, hydrophilic-hydrophobic interactions, and affinity interactions.

[0354] In some embodiments, the biomolecule has an opposite charge to that of the magnetic polymer coating (e.g., polyethylenimine or polyethyleneimine), for example, a positively charged magnetizable particle will bind to a negatively charged DNA.

[0355] Magnetizable particles may utilize biotin-avidin interactions: biotin molecules and tetrameric streptavidin have site-specific attractive forces that reduce non-specific binding and control the orientation of interacting biomolecules, e.g., exposure of the Fab region of an antibody to an antigen.

[0356] The magnetizable particles may be attached to biomolecules using covalent bonds, which may be selected from homobifunctional / heterobifunctional crosslinkers (amino groups), carbodiimide bonds (carboxy groups), maleimide bonds (amino groups), direct reactions (epoxide groups), maleimide bonds (thiol groups), Schiff base condensation (aldehyde groups), and click reactions (alkene / azide groups).

[0357] The magnetizable particles may have an average particle size of about 5 nm, 10 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm, with suitable ranges between any of these values ​​(e.g., about 5 nm to about 500 nm, about 5 nm to about 400 nm, about 5 nm to about 250 nm, about 5 nm to about 100 nm, about 5 nm to about 50 nm, about 10 nm to about 500 nm, etc.). nm, about 10 nm to about 450 nm, about 10 nm to about 300 nm, about 10 nm to about 150 nm, about 10 nm to about 50 nm, about 50 nm to about 500 nm, about 50 nm to about 350 nm, about 50 nm to about 250 nm, about 50 nm to about 150 nm, about 100 nm to about 500 nm, about 100 nm to about 300 nm, about 150 nm to about 500 nm, about 150 nm to about 450, or about 200 nm to about 500 nm.

[0358] The magnetizable particles may have an average particle size of about 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm, and a suitable range may be selected between any of these values ​​(e.g., about 500 nm to about 1000 nm, about 500 nm to about 850 nm, about 500 nm to about 700 nm, about 550 nm to about 1000 nm, about 550 nm to about 800 nm, about 600 nm to about 1000 nm, about 600 nm to about 900 nm, about 650 nm to about 1000 nm, about 650 nm to about 950 nm, about 650 nm to about 800 nm, or about 700 nm to about 1000 nm).

[0359] The magnetizable particles may have an average particle size of about 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm, and a suitable range may be selected between any of these values ​​(e.g., about 1000 nm to about 5000 nm, about 1000 nm to about 4000 nm, about 1500 nm to about 5000 nm, about 1500 nm to about 4500 nm, about 1500 nm to about 3500 nm, about 2000 nm to about 5000 nm, about 2000 nm to about 4000 nm, about 2500 nm to about 5000 nm, about 2500 nm to about 3500 nm, about 3000 nm to about 5000 nm).

[0360] The variation in particle size of the magnetizable beads may be less than 25%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%, and a suitable range may be selected between any of these values.

[0361] A method for detecting an analyte in a sample is described that includes: contacting a sample comprising a target analyte with particles, the particles being coated with a binding molecule complementary to the target analyte to produce bound and unbound binding agent complexes; Positioning particles with both bound and unbound binding agent complexes near a magnetic or electric field sensor; modifying the magnetic or electric field sufficiently to release at least a portion of the particles having both bound and unbound binding agent complexes from the vicinity of the magnetic or electric field sensor; and Measuring the change in magnetic or electrical signal detected from the net movement (i.e. translational or rotational) of the particle relative to the magnetic or electrical sensor, respectively.

[0362] As shown in FIG. 1, a setup according to an embodiment of the method may broadly include a microfluidic device or sample well, a sensor, a magnet, a signal amplifier, an analog-to-digital converter, and a computer.

[0363] The target analyte may be any substance or molecule that is complementary to the binding molecules provided on the magnetizable particles and that can be bound by said binding particles, for example, the target analyte may be selected from the group including proteins, peptides, nucleic acids, lipids or carbohydrates, biochemical and biological agents, viruses, bacteria, etc.

[0364] The target analyte may be a protein or a fragment thereof selected from the group including an antibody, an enzyme, a signal transduction molecule, or a hormone.

[0365] The target analyte may be a nucleic acid selected from the group including DNA, RNA, cDNA, mRNA, or rRNA.

[0366] The method may detect multiple target analytes in a single sample, for example, the method may detect 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 40 or more, or 50 or more target analytes in a single sample.

[0367] The sample to be analyzed can be any sample that can contain one or more target analytes For example, the sample can be a clinical, veterinary, environmental, food, forensic, or other suitable biological sample.

[0368] The clinical sample may be selected from a body fluid, for example the body fluid may be selected from blood, sweat, saliva, urine, sputum, semen, mucus, tears, cerebrospinal fluid, amniotic fluid, gastric fluid, gingival crevicular fluid or interstitial fluid.

[0369] The environmental sample may be selected from the group including water, soil, or aerosol.

[0370] An advantage of the present invention can be that the sample preparation is not laborious or difficult to prepare: it utilizes established biochemistry for the functionalization and attachment of molecules on either the microfluidic or magnetizable particle surfaces.

[0371] The sample to be analyzed can be added directly to the sample well or microfluidic device without further processing.

[0372] The sample may undergo one or more sample processing steps. It will be understood that suitable sample processing steps may depend on the type and / or nature of the sample to be analyzed. In some embodiments, the sample processing step may be selected from the group including dilution, filtration, or extraction (e.g., liquid-liquid, solid-phase). This may also be achieved through the use of microfluidic features and designs, or centripetal forces. For example, a whole blood sample may be filtered using a cellulose-based filter or other filter to separate the plasma to be analyzed.

[0373] The first step of the method may involve mixing the sample to be analyzed with a preparation comprising freely diffusible magnetizable particles coated with a binding molecule (binding agent complex) that is complementary to the target analyte in the sample well or sample reservoir. Where appropriate, the term "binding agent complex" may be used interchangeably to refer to magnetizable particles coated with a binding molecule.

[0374] In some embodiments, the magnetizable particles may have limited diffusivity. This may occur when the magnetizable particles are crosslinked or derivatized with a polymer. The polymer may be a hydrogel or a PEG linker. This may occur when the device is used in a multiplex assay for the detection of multiple targets or samples in one sample.

[0375] The method can improve the rate at which the binding molecule binds the target analyte by providing a binding agent complex that can move and diffuse freely in the solution. When the sample and the preparation of the binding agent complex are combined, the binding agent complex can diffuse freely and the binding molecule can interact with the target analyte throughout the sample volume. Because both the binding agent complex and the target analyte are freely diffusible and suspended in the sample volume, the average physical distance between the target analyte and the binding agent complex can be small. Thus, the rate of binding can be improved and the binding equilibrium can be reached significantly faster.

[0376] In detection assays such as ELISA, binding molecules such as antibodies are immobilized on macro-scale objects, e.g., the surface of a test well. In such methods, the physical distance between the target analyte and the antibody can vary significantly depending on the location of the analyte within the sample volume. For example, a target analyte near the top of the sample volume may be very far from the immobilized antibody and is unlikely to be captured and bound. Thus, the rate of binding may be limited by the rate at which the target analyte diffuses within the sample volume toward the immobilized antibody.

[0377] The sample and binding agent complex may be allowed to bind for a suitable amount of time for the binding molecules to reach binding equilibrium. In some embodiments, a suitable amount of time for allowing binding to reach equilibrium may be about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 45 seconds, 60 seconds, 90 seconds, 120 seconds, 180 seconds, 240 seconds, 300 seconds, or 360 seconds, with useful ranges between any of these values ​​(e.g., about 1 second to 30 seconds, 1 second to 60 seconds, The time can be selected from the following: 1 second to 120 seconds, 10 seconds to 30 seconds, 10 seconds to 60 seconds, 10 seconds to 90 seconds, 30 seconds to 60 seconds, 30 seconds to 90 seconds, 30 seconds to 120 seconds, 60 seconds to 90 seconds, 60 seconds to 120 seconds, 60 seconds to 180 seconds, 90 seconds to 120 seconds, 90 seconds to 180 seconds, 90 seconds to 240 seconds, 180 seconds to 240 seconds, 180 seconds to 300 seconds, 180 seconds to 360 seconds).

[0378] A magnetic field generator may be used to induce magnetohydrodynamic mixing of the sample to improve the rate at which binding equilibrium is reached. In such embodiments, a magnetic field generator is used to induce movement of binding agent complexes within the sample volume.

[0379] A signal that allows quantification of the analyte in the sample is generated by measuring the net change in the magnetic field as the bound and unbound analyte complexes move relative to the magnetic field sensor.

[0380] A further step of the method may include applying a magnetic field to the sample to position the binding agent complexes near a magnetic field sensor. A magnetic field generator as described in paragraph 0376 may be used to generate a magnetic field to manipulate the bound and unbound binding agent complexes into positions that allow the magnetic field sensor to effectively measure changes in the magnetic field generated by the magnetizable particles.

[0381] In some embodiments, the binding agent complex may be positioned near the magnetic field sensor using microfluidics, electrophoresis, optical tweezers, acoustics, piezoelectrics, pumps and / or suction, passive capillary pumps, or other suitable means, hi other embodiments, the binding agent complex may be positioned by centrifugation.

[0382] In some embodiments, the magnetic field can be generated in a direction that moves the magnetizable particles in the sample volume toward the magnetic field sensor. The magnetic field sensor can be provided at any location relative to the test well or the microfluidic device. For example, if the magnetic field sensor is located below the test well or sample reservoir, the magnetic field moves the magnetizable particles toward the bottom of the test well or sample reservoir. In another example, if the magnetic field sensor is located above the test well or sample reservoir, the magnetic field moves the magnetizable particles toward the top of the test well or sample reservoir.

[0383] For centrifugation, the sensor may be oriented on a vertical axis with the sensor's sensing axis pointing horizontally inwards or outwards.

[0384] The magnetic field generated can be static or dynamic.

[0385] The strength of the generated magnetic field can be modulated.

[0386] Without being bound by theory, the modulation of this magnetic field (i.e., the bias field) has the primary function of aligning the magnetizable particles to the sensor during detection to achieve the highest detection sensitivity. For ferromagnetic particles, assuming that the ferromagnetic particles themselves have a permanent magnetic field, the bias field is switched off resulting in misalignment of the magnetic particles. For paramagnetic (or superparamagnetic) particles, the magnetic field of the particles needs to be induced by an external field, so the bias field provides the additional function of inducing such a field.

[0387] Because different particles (whether by chemical composition or physical size) may require different bias field strengths and configurations, the bias field may be modulated to support different magnetizable particles.

[0388] The magnetic field may be generated and positioned to maximize its effect on the magnetizable particles but minimize its effect on the magnetic field sensor. The magnetic field generator may be generated and / or positioned in close proximity to the magnetic field sensor. In some embodiments, the magnetic field generator is located above, below, or beside the magnetic field sensor. In some embodiments, the magnetic field generator may be located in the same vertical or horizontal plane as the magnetic field sensor.

[0389] The magnetic field generator may not be activated or the magnetic field may not be present entirely.

[0390] A further step of the method may include modifying the magnetic field sufficiently to release at least a portion of the binding agent complexes from the vicinity of the magnetic field sensor when the bound and unbound binding agent complexes are located near the magnetic field sensor.

[0391] The magnetic field may be gradually reduced.

[0392] The magnetic field can be instantly removed.

[0393] The shape of the magnetic field can be varied.

[0394] When the magnetic field applied to the sample is reduced and / or removed, the bound and unbound binding agent complexes may be released from the magnetic field and free to diffuse (translationally) away from the vicinity of the magnetic field sensor. When the magnetic field applied to the sample is reduced and / or removed, the binding agent complexes may also rotate (rotationally move) relative to the magnetic field sensor.

[0395] According to this method, bound and unbound binder conjugates can be distinguished based on changes in molecular diffusion properties according to Graham's law of molecular diffusion, which states that the diffusion rate is inversely proportional to the square root of its molecular weight. The diffusion rate can be calculated using the following formula:

[0396]

number

[0397] Where: R A = diffusion rate for molecule A, R B = diffusion rate for molecule B M A = molecular weight of molecule A, and M B = molecular weight of molecule B.

[0398] Since the binding agent conjugate bound to the target analyte has a larger molecular weight compared to the unbound binding agent conjugate, the unbound binding agent conjugate will have a higher diffusion rate according to Graham's law. Therefore, the bound and unbound binding agent conjugates can be distinguished based on their kinetic profiles.

[0399] A further step of the method may include measuring the change in the magnetic signal detected from the magnetizable particles as they move (via translational and / or rotational movement) relative to the magnetic field sensor. The magnetic field sensor as detailed in the previous paragraph measures the change in magnetic field strength generated by the magnetizable particles over time. The method uses the magnetic field change over time, which requires only one binding molecule for the binding of the target analyte.

[0400] In some embodiments, the magnetic field change over time can be determined by measuring the magnetoresistance effect and the signal decrease over time.

[0401] The magnetic field signal generated by a magnetizable particle with respect to a magnetic field sensor follows the equation for a magnetic dipole field.

[0402]

number

[0403] where B is the field, r is the vector from the position of the dipole to the position where the field is being measured, and r is the absolute value of r, the distance from the dipole.

[0404]

number

[0405] is a unit vector parallel to r, m is the (vector) dipole moment, and μ0 is the permeability of free space.

[0406] Based on the equation for a magnetic dipole field, the detected signal decreases with the cube of the distance from the magnetic field sensor. In conjunction with the diffusion kinetics described above, this phenomenon can be used for signal generation as described in the following paragraphs.

[0407] Due to the higher diffusion rate of the unbound binding agent complex, the unbound binding agent complex may migrate further away from the sensor at a faster rate when compared to the binding agent complex bound to the target analyte. The difference in diffusion rate generates a magnetic field decay signal over time. The decay rate depends on the molecular weight of the bound and unbound binding agent complexes, where the unbound binding agent complex has a faster decay rate compared to the bound binding agent complex.

[0408] The decay rate can be modeled with a decay curve, which can be used to distinguish between bound and unbound binding agent conjugates, for example, a fast decay curve can indicate an unbound binding agent conjugate and a weak decay curve can indicate a bound binding agent conjugate.

[0409] The method may include the following steps multiple times to generate a signal curve over time, distinguish between bound and unbound binding agent conjugates, and quantify the target analyte. Applying a magnetic field to position magnetizable particles in the vicinity of a magnetic field sensor. · Altering the magnetic field sufficiently to release at least a portion of the magnetizable particles from the vicinity of the magnetic field sensor. Measuring the change in the magnetic signal detected from the magnetizable particles as they move away from the magnetic sensor.

[0410] The method may include a reference calibration step by measuring the total magnetic field strength generated by the bound or unbound binding agent complex.

[0411] The magnetic field signal generated by the magnetizable particles may be due to intrinsic properties of the magnetizable particles, or the magnetic field signal may be induced by an external magnetic field.

[0412] The magnetic field sensor is positioned to maximize detection of the magnetizable particles by the magnetic field sensor but minimize detection of the magnetic field generators.

[0413] The magnetic field or signal from a magnetizable particle may be inherent to the atomic structure of the magnetizable particle, or may be induced by an external magnetic field.

[0414] Data acquisition by the sensor may be synchronized with the microfluidic device. This may allow the detected data from the sensor to be characterized as between sample data or environmental or ambient data. For example, detection of a signal by a magnetic sensor in the absence of sample injection into the microfluidic device characterizes the data as environmental or ambient data. Characterizing the data as environmental or ambient data may help establish background and may help prepare calibration data.

[0415] When a magnetic sensor detects a signal resulting from injection of a sample into a microfluidic device that is consistent with positioning magnetizable particles in proximity to the magnetic sensor, such data may be characterized as sample data.

[0416] Utilizing such a sensing system may provide an embodiment in which sample displacement and detection time may be ascertained and microfluidic quality control measurements may be performed.

[0417] Data acquisition from the sensor can be continuous, i.e., the magnetic sensor continuously transmits a signal and characterizes the data as sample data or background data based on synchronization of data collection with the injection of a sample into the microfluidic device.

[0418] Sensor data may be acquired over a period of time to measure changes in the magnetic signal from the magnetizable particles. An action or event may be inferred from the change in the sensed magnetic signal. The action or event may include movement of the magnetizable particles due to fluid flow, external magnetic forces, or diffusion.

[0419] The method may include processing raw data output from the magnetic field sensor to quantify the amount of target analyte in the sample. Processing of the raw data may be performed using a combination of the hardware and software implementations described in detail in the preceding paragraphs.

[0420] The evaluation of the analytical performance of a detection method is often performed by measuring a dose-response curve from which the limit of detection (LoD) can be derived. The LoD is the lowest amount of a substance such as a biomarker that can be detected for a selected confidence level. The selected assay (biomarker, biomaterial, sample matrix, incubation time, etc.) can have a strong influence on the LoD. The limit of quantification (LoQ), which is the lowest biomarker concentration that can be quantified with a given required accuracy, is also used. If the dose-response curve has good sensitivity, i.e. the signal changes significantly as a function of the target concentration, the LoQ is close to the LoD.

[0421] The methods may be provided with a LoQ of about 0.05 pg / mL, 0.1 pg / mL, 0.2 pg / mL, 0.3 pg / mL, 0.4 pg / mL, 0.5 pg / mL, 0.6 pg / mL, 0.7 pg / mL, 0.8 pg / mL, 0.9 pg / mL, 1.0 pg / mL, 1.5 pg / mL, or 2.0 pg / mL, and suitable ranges may be selected between any of these values.

[0422] The methods may be provided for an LoD of about 0.1 pg / mL, 1.1 pg / mL, 1.2 pg / mL, 1.3 pg / mL, 1.4 pg / mL, 1.5 pg / mL, 1.6 pg / mL, 1.7 pg / mL, 1.8 pg / mL, 1.9 pg / mL, or 2.0 pg / mL, and suitable ranges may be selected between any of these values.

[0423] The present invention describes methods, reagents and systems for detecting and quantifying an analyte in a sample.

[0424] It will be appreciated that the method may be broadly used in any application requiring detection and / or quantification of a target analyte. In particular, the method may include: (i) Expedited decision-making; (ii) high sensitivity determination; (iii) quantitative determination; Or any combination of (i) to (iii). It can be used in applications requiring

[0425] For example, suitable applications may include clinical, veterinary, environmental, food safety, or forensic applications.

[0426] In some embodiments, clinical applications may include diagnostic detection of biomarkers in samples that may be indicative of a clinical condition. In one example, the method may be used for rapid, sensitive, and quantitative diagnostic detection of specific antibodies in blood samples that may indicate possible infection by a pathogen. In a further example, the method may be used for diagnostic detection of specific protein biomarkers that are overexpressed in cancer. Diagnostic detection may be performed in samples across different species.

[0427] The clinical condition may be selected from infections, e.g., infections caused by bacteria, fungi, viruses (e.g., hepatitis, SARS-CoV-19, and HIV) (e.g., biomarkers such as antibodies for hepatitis, SARS-CoV-19, and HIV), parasites (e.g., microbial parasites [e.g., malaria], nematodes, insect parasites).

[0428] The clinical condition may be selected from diseases such as cardiac disease (biomarkers such as BNP), cancer (e.g. solid organ cancer, blood cancer, other cancers), (e.g. biomarkers such as Ca-125 and other tumor markers), neurological disease (e.g. multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease) (e.g. biomarkers such as CNS immunoglobulins), respiratory disease (e.g. biomarkers such as serum ACE), liver disease (e.g. liver function tests and biomarkers such as albumin), kidney disease (e.g. biomarkers such as creatinine and protein).

[0429] The clinical condition may be selected from organ damage or failure, for example, brain damage (e.g., a biomarker such as glial fibrillary acidic protein or GFAP), kidney damage (e.g., a biomarker such as serum creatine), heart damage (e.g., a biomarker such as creatine kinase-muscle), lung damage (e.g., a biomarker such as intercellular adhesion molecule-1 or ICAM1), or liver damage (e.g., a biomarker such as alkaline phosphatase).

[0430] The clinical condition may be selected from endocrine disorders, such as diabetes (e.g., biomarkers such as insulin, high HbA1C, thyroid dysfunction, thyroid hormones, pituitary disorders (e.g., biomarkers such as ACTH, prolactin, gonadotropins, thyroid stimulating hormone, growth hormone, antidiuretic hormone), parathyroid disorders (e.g., biomarkers such as parathyroid hormone), adrenal disorders (e.g., biomarkers such as cortisol, aldosterone, adrenaline, DHEAS), sex hormone imbalance (e.g., biomarkers such as androgens and estrogens), carcinoid tumors (e.g., biomarkers such as 5-HIAA, VIPoma, serum VIP), high bone turnover (e.g., biomarkers such as P1NP).

[0431] The clinical condition may be selected from lipid disorders (eg, biomarkers such as cholesterol and triglycerides).

[0432] The clinical condition may be selected from nutritional disorders (eg, vitamin deficiencies, malabsorption syndromes, malnutrition, vitamin metabolism disorders), (eg, biomarkers such as vitamin levels, iron levels, mineral levels, etc.).

[0433] The clinical condition may be selected from inflammation or an inflammatory disorder (eg, biomarkers such as ESR, CRP, and other acute phase proteins).

[0434] The clinical condition may be selected from an autoimmune disease (eg, a biomarker such as a particular antibody marker).

[0435] The clinical condition may be selected from an allergic disease (eg, a biomarker such as tryptase).

[0436] The clinical condition may be selected from physical trauma such as electric shock (eg, a biomarker such as creatine kinase).

[0437] The clinical condition can be selected from an immunodeficiency disorder (eg, common variable immunodeficiency) (eg, biomarkers such as complement, white blood cells, and immunoglobulins).

[0438] The clinical condition may be selected from a coagulation disorder (eg, thrombophilia) (eg, biomarkers, eg, biomarkers such as clotting factors and other markers).

[0439] The clinical condition may be selected from genetic or acquired enzyme disorders, deficiencies or excesses, and other congenital or acquired metabolic disorders (e.g., Bartter's syndrome, congenital adrenal hyperplasia) (e.g., biomarkers such as electrolytes, enzyme levels, metabolic products of enzymes, etc.).

[0440] The clinical condition may be selected from electrolyte disturbances (eg, biomarkers such as electrolytes) such as hyperkalemia and hypernatremia.

[0441] The clinical condition may be selected from adverse drug effects or toxicity (e.g., biomarkers such as drug levels and levels of drug metabolites).

[0442] The clinical condition may be selected from exposure to chemical or biological weapons, or adverse effects or poisoning from other environmental chemical and biological agents.

[0443] Veterinary specific clinical conditions may be selected from renal failure, FIV / AIDS (cats), cancer, and any biomarker for organ function / failure.

[0444] In some embodiments, the clinical condition may be a condition in a veterinary subject, such as a feline, canine, bovine, ovine, equine, porcine, or murine.

[0445] In some embodiments, environmental applications may include detection of contaminants in environmental samples. The environmental contaminants may be selected from contaminants such as lead, particulate matter, microplastics, and hormones.

[0446] For example, the method may be used to monitor and quantify heavy metals in water samples.

[0447] In some embodiments, food safety applications may include detection of pathogens in food samples, for example, the method may be used to rapidly and sensitively detect contamination with bacterial pathogens in pasteurized milk.

[0448] Working Example The aim of this study was to test the sensitivity and range of detection of a device using ferromagnetic particles, which generate their own magnetic field without needing to be magnetized by an external magnetic field.

[0449] The main components of the device and the sensor data parameters are summarized below. Magnetic sensor: Honeywell HMC 2003 magnetometer Amplifier: Honeywell HMC2003 built-in amplifier ·electromagnet: 5V DC with 10N force Sensor data acquisition: Approximately 0.007 seconds per reading 2,500 reads per sample Total read time approximately 17.5 seconds

[0450] The device consists of an electromagnet at the top and a microfluidic chip in the middle placed on top of a magnetic sensor at the bottom.

[0451] The magnetizable particles used are Spherotech SVFM-20-5 (2.0 micrometers - 2.9 micrometers) streptavidin-coated ferromagnetic particles. The magnetizable particles are functionalized with the biotinylated "detection" anti-human albumin antibody from the DY1455 ELISA kit.

[0452] The experimental protocol is summarized below. Concentration of human albumin recombinant protein to be tested (DY1455 ELISA kit): Sample 1-0 pg / mL (control) Sample 2 - 0.1pg / mL Sample 3-1pg / mL Sample 4-10pg / mL Sample 5-100pg / mL Sample 6-1,000pg / mL For each protein concentration tested: 5 microliters of magnetizable particles (Spherotech ferromagnetic beads 1% w / v) 2 nanograms of anti-albumin antibody (biotinylated "detection" antibody from the DY1455 ELISA kit) All components are mixed and detected in a test volume of 50 microliters

[0453] After being introduced into the microfluidic chip, the magnetizable particles were positioned over the sensor using an electromagnet. The electromagnet was activated to bring the magnetizable particles into close proximity to the magnetic sensor. The electromagnet was controlled to collapse the bias field, and the magnetic field sensor measured the change in magnetic field strength produced by the magnetizable particles over time as the magnetizable particles diffused away from the magnetic sensor. The device determines the amount of analyte in the sample by measuring the net movement of the magnetizable particles relative to the magnetic field sensor.

[0454] Magnetic sensor data was acquired for each concentration of human albumin.

[0455] For each concentration of human albumin samples tested below, the average sensor readings over 2,500 sample readings, expressed in volts (V), are shown in Table 1.

[0456] [Table 1]

[0457] The results demonstrate the sensitivity and range of the device to detect an analyte (human albumin) using functionalized ferromagnetic particles over at least five orders of magnitude from 0.1 pg / mL to 1,000 pg / mL.

[0458] The purpose of this test is to demonstrate the optimization of the upper dynamic range of detection of human albumin in Example 1 by using increased amounts of detection antibody.

[0459] The same apparatus as described in Example 1 is used in Example 1a.

[0460] The experimental protocol is modified by using 20 nanograms of anti-albumin antibody instead of the 2 nanograms in Example 1. A higher concentration of 10,000 pg / mL was also tested.

[0461] For each concentration of human albumin tested, the average sensor readings over 2,500 sample readings, expressed in volts (V), are shown in Table 2.

[0462] [Table 2]

[0463] The purpose of this test is to demonstrate the flexibility of the device and method to detect analytes in opposite physical orientations.

[0464] The experimental protocol used is as described in Example 1, except that the highest concentration of human albumin tested was 100 pg / ml.

[0465] The components of the device used in this test are as described in Example 1, except that the device consists of a magnetic sensor at the top, the microfluidic chip is inverted (upside down) and located below the magnetic sensor, and the electromagnet is located at the bottom.

[0466] For each concentration of human albumin samples tested below, the average sensor readings over 2,500 sample readings, expressed in volts (V), are shown in Table 3.

[0467] [Table 3]

[0468] The purpose of this test is to demonstrate the detection of analytes using a device that employs suction and microfluidic features to position particles near a sensor without the use of electromagnets.

[0469] The main components of the device and the sensor data parameters are summarized below. Magnetic sensor: Honeywell HMC 2003 magnetometer Amplifier: Honeywell HMC2003 built-in amplifier Sensor data acquisition: Approximately 0.004 seconds per reading 5,000 readings per sample Total read time approximately 20 seconds

[0470] The magnetizable particles used are Spherotech SVFM-20-5 (2.0 micrometers - 2.9 micrometers) streptavidin-coated ferromagnetic particles. The magnetizable particles are functionalized with the biotinylated "detection" anti-human albumin antibody from the DY1455 ELISA kit.

[0471] The microfluidic chip is constructed with a 1.5% low melting point agarose trap. A pump is used to generate a small suction-induced flow of particles in the microfluidic channel. Magnetizable particles in the suction-induced flow are captured by the agarose trap, bringing the particles into close proximity to the magnetic sensor, while allowing the sample fluid to flow through the agarose trap. Suction was set at 2 microliters per second, every 5 seconds, with 1 second of activation followed by 4 seconds of no aspiration (passive flow).

[0472] The experimental protocol is summarized below. Concentration of human albumin recombinant protein to be tested (DY1455 ELISA kit): Sample 1-0 pg / mL (control) Sample 2 - 0.1pg / mL Sample 3-1pg / mL Sample 4-10pg / mL Sample 5-100pg / mL Sample 6-1,000pg / mL Sample 7-10,000pg / mL For each protein concentration sample tested: 5 microliters of magnetizable particles (Spherotech ferromagnetic beads 1% w / v) 2 nanograms of anti-albumin antibody (biotinylated "detection" antibody from the DY1455 ELISA kit) All components are mixed and detected in a test volume of 50 microliters.

[0473] Once the sample is introduced into the microfluidic chip, the pump is operated for three cycles (i.e., 1 second of active flow followed by 4 seconds of passive flow). At the end of the third cycle of pump operation, the magnetic sensor acquires data for approximately 20 seconds.

[0474] For each concentration of human albumin tested, the average sensor readings over 1,250 sample readings (of approximately 5 seconds), expressed in volts (V), are shown in Table 4.

[0475] [Table 4]

[0476] The purpose of this test is to demonstrate detection of an analyte using a device that employs centrifugation to position particles near a sensor.

[0477] The main components of the device and the sensor data parameters are summarized below. Magnetic sensor: Honeywell HMC 2003 magnetometer Amplifier: Honeywell HMC2003 built-in amplifier Sensor data acquisition: Approximately 0.004 seconds per reading 8,750 reads per sample Total read time approximately 35 seconds

[0478] The magnetizable particles used are Spherotech SVFM-20-5 (2.0 micrometers - 2.9 micrometers) streptavidin-coated ferromagnetic particles. The magnetizable particles are functionalized with the biotinylated "detection" anti-human albumin antibody from the DY1455 ELISA kit.

[0479] The experimental protocol is summarized below. Concentration of human albumin recombinant protein to be tested (DY1455 ELISA kit): Sample 1-0 pg / mL (control) Sample 2 - 0.1pg / mL Sample 3-1pg / mL Sample 4-10pg / mL Sample 5-100pg / mL Sample 6-1,000pg / mL Sample 7-10,000pg / mL For each protein concentration tested: 20 microliters of magnetizable particles (1% w / v 2 μm ferromagnetic beads from Spherotech) 8 nanograms of anti-albumin antibody (biotinylated "detection" antibody from the DY1455 ELISA kit) All components are mixed and detected in a test volume of 200 microliters

[0480] A sample vessel with a circular channel with a radius of 42 mm was used to accommodate the sample.

[0481] The sample vessel containing the sample was rotated at 520 rpm (min -1 ) and decelerates to a stop over approximately 10 seconds. After the sample vessel decelerates to a stop, it is maintained in a stationary position. A magnetic sensor was positioned adjacent to the circular channel (at the outer circumference).

[0482] For each concentration of human albumin sample tested, the average sensor readings over 2,500 sample readings (of approximately 10 seconds), expressed in volts (V), are shown in Table 5. The sensor values ​​in Table 5 are set to reflect the negative step results, with higher concentrations recording lower values.

[0483] [Table 5]

[0484] The purpose of this test is to demonstrate detection of an analyte using a device that employs a passive bias system to position particles near a sensor without the use of magnets or electromagnets.

[0485] The main components of the device and the sensor data parameters are summarized below. Magnetic sensor: Honeywell HMC 2003 magnetometer Amplifier: Honeywell HMC2003 built-in amplifier Sensor data acquisition: Approximately 0.004 seconds per reading 2,500 reads per sample Total read time of about 10 seconds

[0486] The magnetizable particles used are Spherotech SVFM-20-5 (2.0 micrometers - 2.9 micrometers) streptavidin-coated ferromagnetic particles. The magnetizable particles are functionalized with the biotinylated "detection" anti-human albumin antibody from the DY1455 ELISA kit.

[0487] The experimental protocol is summarized below. Concentration of human albumin recombinant protein to be tested (DY1455 ELISA kit): Sample 1-0 pg / mL (control) Sample 2-1pg / mL Sample 3-10pg / mL Sample 4-100pg / mL Sample 5-1,000pg / mL Sample 6-10,000pg / mL For each protein concentration tested: 1 microliter of magnetizable particles (Spherotech ferromagnetic beads 1% w / v) 0.4 nanograms of anti-albumin antibody (biotinylated "detection" antibody from the DY1455 ELISA kit) All components are mixed and detected in a test volume of 50 microliters.

[0488] Magnetizable particles functionalized with anti-human albumin antibodies are added to the detection area of ​​the microfluidic chip, and the sample is added to the sample port of the microfluidic chip.

[0489] The microfluidic chip consists of a permeable plug containing 1.5% low melting point agarose. The agarose plug is positioned within the microfluidic chip to capture particles of 2 micrometer size in an area corresponding to a magnetic sensor. A capillary pump (a passive microfluidic structure) located downstream from the agarose pump is used to establish passive suction that is sufficient to draw liquid through the microfluidic chip. In conjunction with a 5 minute suction-induced flow downstream from the plug, the agarose plug collects and captures magnetizable particles in close proximity to the sensor.

[0490] For each concentration of human albumin sample tested, the average sensor readings over 2,500 sample readings (of approximately 10 seconds), expressed in volts (V), are shown in Table 6.

[0491] [Table 6]

[0492] The purpose of this test is to demonstrate detection of an analyte using a device that employs a passive system to position particles near a sensor without the use of magnets or electromagnets.

[0493] The test described in Example 6 was modified by randomizing the order in which the samples were measured to ensure that the readings were accurate for the samples.

[0494] The equipment used is as described in Example 6 with the sensor data parameters summarized below. Sensor data acquisition: Approximately 0.006 seconds per reading 1,250 reads per sample Total read time of about 7 seconds

[0495] The experimental protocol and microfluidic chip design are as described in Example 6.

[0496] For each concentration of human albumin sample tested, the average sensor readings over 2,500 sample readings (approximately 5 seconds) expressed in volts (V) are shown in Table 7.

[0497] [Table 7]

[0498] The purpose of this test is to demonstrate detection of an analyte using a device that employs a passive system to position particles near a sensor without the use of magnets or electromagnets.

[0499] The test described in Example 6 was modified by running the samples in order from lowest to highest concentration to ensure that the readings were accurate for the samples. Otherwise, the equipment and experimental protocol were as described in Example 6.

[0500] For each concentration of human albumin tested, the average sensor readings over 2,500 sample readings (approximately 5 seconds) expressed in volts (V) are shown in Table 8.

[0501] [Table 8]

[0502] The purpose of this test is to demonstrate the detection of an analyte using electrical sensing.

[0503] The electrical sensing platforms are summarized below. Copper electrodes - 0.1mm diameter configured for 0.3mm gap separation. The anode is connected in series with a 1 ohm resistor The entire platform is driven by a signal generator: Signal Wave Pattern-AC 2 volts peak-to-peak 1 megahertz frequency Keithley Instruments 3390 Arbitrary Waveform Generator Voltage detection detected by oscilloscope: Agilent Technologies' InfiniiVision DSO5034A Voltage detection of the sample by probing the cathodic and anodic copper electrodes Sample current detection by probing the cathode and anode of a 1 ohm resistor Sensor data acquisition: 10 microseconds per read 1,000 readings per sample 10ms total read time

[0504] The magnetizable particles used are Spherotech SVFM-20-5 (2.0 micrometers - 2.9 micrometers) streptavidin-coated ferromagnetic particles. The magnetizable particles are functionalized with the biotinylated "detection" anti-human albumin antibody from the DY1455 ELISA kit.

[0505] The experimental protocol is summarized below. Concentration of human albumin recombinant protein to be tested (DY1455 ELISA kit): Sample 1-0.1pg / mL Sample 2-1pg / mL Sample 3-10pg / mL Sample 4 - 1,000pg / mL Sample 5-10,000pg / mL For each protein concentration tested: 1 microliter of magnetizable particles (Spherotech ferromagnetic beads 1% w / v) 0.4 nanograms of anti-albumin antibody (biotinylated "detection" antibody from the DY1455 ELISA kit) All components are mixed and detected in a test volume of 10 microliters.

[0506] The sample was pipetted into a closed rectangular channel of a sample introduction device, and copper electrodes were affixed to the side walls of the channel such that there was a 0.3 mm gap between the electrodes. An electromagnet was positioned directly above the channel.

[0507] After the sample is loaded into the channel of the sample introduction device, the signal generator is switched on for the above-mentioned conditions. The electromagnet is switched on for 2 seconds and then switched off. The oscilloscope records for the above-mentioned conditions (both voltage and current).

[0508] The sensor data is processed according to the following steps: 1. Impedance is derived by taking the voltage reading and dividing it by the current reading for each time step. 2. The change in impedance for each time step is derived by taking the difference between the time step and the previous time step. 3. The difference impedance data is then filtered for any absolute value greater than 100 ohms. 4. The filtered impedance data for each sample is then summed.

[0509] For each concentration of human albumin sample tested, the sensor readings, expressed as total impedance (ohms), are shown in Table 9.

[0510] [Table 9]

[0511] Although the embodiments have been described with reference to a number of exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

[0512] Many modifications will be apparent to those skilled in the art without departing from the scope of the invention as described herein with reference to the accompanying drawings.

Claims

1. 1. An apparatus for sensing a sample comprising bound and unbound particles to an analyte, the apparatus comprising: a detection zone comprising at least an array of magnetic field sensors and / or an array of electric field sensors; a sample introduction device configured to introduce the sample into the detection zone; a field generator, the field generator being optimized for magnetic field generation if a magnetic field sensor is present and / or for electric field generation if an electric field sensor is present, provided that the magnetizable particles do not have aligned dipole moments, the electric field generator generating a current having a standard sinusoidal pattern; a field generator, in the presence of a magnetic field sensor, wherein the particles comprise magnetizable particles, the magnetizable particles being in a magnetized state when in the sensing zone; a controller connected to receive signals from the array of magnetic fields and / or the array of electric fields, the controller configured to determine an amount of analyte in the sample based on the signals received from the array of magnetic field sensors and / or the array of electric field sensors; Equipped with In the presence of a magnetic sensor, the apparatus further comprises: i) a set and reset module or capability to set / reset the magnetic sensor; ii) a data transmission layer configured to protect the signals being transmitted from one or more magnetic sensors; iii) a plurality of magnetic field permeable zones corresponding to the area under each magnetic sensor; iv) a printed circuit board with one or more vias connecting to the magnetic field sensor; or v) An apparatus comprising any combination of two or more of (i)-(iv).

2. The device of claim 1 , wherein the magnetizable particles can be magnetized prior to binding to the analyte or prior to or during introduction of the sample to a magnetic sensing zone.

3. 3. The apparatus of claim 1, wherein the array of magnetic sensors comprises set and reset coils / straps for setting / resetting the magnetic sensors.

4. 3. The device of claim 1 or 2, wherein the set and reset module or capability is incorporated into the magnetic sensor.

5. 3. The device of claim 1, wherein the magnetic sensor is set / reset between readings.

6. 3. The apparatus of claim 1, wherein a plurality of magnetic sensors are connected in series to the calibration port such that one calibration signal is used to set / reset the plurality of magnetic sensors.

7. 3. The apparatus of claim 1, wherein the magnetic sensor has a sampling rate of about 0.05 kHz, 0.1 kHz, 0.5 kHz, 1 kHz, 5 kHz, 10 kHz, 15 kHz, or 20 kHz.

8. The apparatus of claim 1 or 2, wherein the magnetic sensor has a sampling rate of about 100 kHz to about 200 kHz.

9. 3. The device according to claim 1, wherein at least the sensing zone is provided on an upper surface of a circuit board.

10. 10. The device of claim 9, further comprising a magnetic field generator or an electric field generator, the magnetic field generator or the electric generator being provided on a surface of the circuit board at a location on the top surface of the circuit board corresponding to the detection zone.

11. The apparatus of claim 9 , wherein the circuit board comprises multiple layers.

12. 10. The apparatus of claim 9, wherein the circuit board comprises at least one top layer, a ground plane layer, and a bottom layer, and a plurality of circuit layers.

13. 10. The apparatus of claim 9, wherein the circuit board comprises a data transmission layer configured to protect the signals being transmitted from the one or more magnetic sensors from electromagnetic interference generated by other components of the circuit board and / or a magnetic field generator.

14. 14. The apparatus of claim 13, wherein the data transmission layer is located between upper and lower layers and upper and lower ground planes.

15. 10. The apparatus of claim 9, wherein the circuit board comprises a plurality of magnetic field transparent windows, each transparent window defining a portion of the circuit board that is free of a copper layer, the transparent window corresponding to an area of ​​the circuit board underlying each magnetic sensor.

16. Approximately 1cm ２ ~Approx. 25cm ２ 3. The device according to claim 1, wherein the device has a detection surface area of

17. The device of claim 16, wherein the sensing surface comprises between about 6 and about 24 magnetic sensors.

18. 3. The apparatus of claim 1, wherein the array of magnetic sensors is densely packed.

19. 3. The device of claim 1 or 2, comprising a housing that houses at least one circuit board.

20. 20. The device of claim 19, wherein the housing includes a built-in display configured to render diagnostic output obtained from the circuit board.

21. 20. The apparatus of claim 19, wherein the housing with an integrated display and at least one circuit board is configured to perform the operations of a lab-on-a-chip device.

22. 20. The apparatus of claim 19, wherein the housing with a built-in display and multiple circuit boards arranged in parallel is configured for operation as a lab-on-bench device.

23. 20. The apparatus of claim 19, wherein the housing is configured to be controlled by a user interface in lab-on-chip and lab-on-bench device modes.

24. 3. The apparatus of claim 1 or 2, wherein the controller is configured to controllably bias one or more of the sample introduction device, field generator, array of sensors, amplifier, and / or filter.

25. 3. The apparatus of claim 1, wherein the controller is configured to control the bias of the sample introduction device.

26. The device of claim 1 or 2, wherein the magnetizable particles have a particle size of about 1 nm to about 100 nm.

27. The device according to claim 1 or 2, wherein the magnetizable particles have a particle size of about 0.5 μm to 5 μm.

28. 27. The apparatus of claim 26, wherein the controller biases the particles through generation of an external force, the external force acting to increase any inter-particle, particle-solvent, or binding forces.

29. 28. The apparatus of claim 27, wherein the controller biases the particles through generation of an external force, the external force acting to completely counteract any inter-particle, particle-solvent, or binding forces.

30. 3. The apparatus of claim 1, wherein the sample introduction device biases the particles relative to the sensor.

31. The circuit board is approximately 5 cm ２ ~Approx. 100cm ２ 10. The device of claim 9, wherein the device is sized at

32. 10. The device of claim 9, wherein the sensing surface spans between about 10% and about 50% of the surface of the circuit board.

33. 3. The device of claim 1, further comprising a sensor for detecting the orientation of the device so that the device can be operated in any orientation.

34. 34. The device of claim 33, wherein the sensor that detects the orientation of the device comprises one or more of a gyroscope sensor, an inertial measurement unit, and an accelerometer.

35. 3. The apparatus of claim 1, wherein the one or more magnetic sensors are analog sensors.

36. The apparatus of claim 1 or 2, wherein the one or more magnetic sensors comprise one or more of a magnetoresistive sensor, a Hall effect sensor, and a fluxgate sensor.

37. a signal processing module, the signal processing module comprising: an amplifier for amplifying the signal from the one or more magnetic sensors; an analog-to-digital converter, and ・Power supply 3. The apparatus of claim 1, further comprising one or more of:

38. 3. The apparatus of claim 1 or 2, wherein the sample introduction device is removable.

39. 3. The apparatus of claim 1 or 2, wherein the sample introduction device is integrated into the apparatus.

40. 3. The device of claim 1, wherein the detection zone comprises a plurality of wells.

41. 3. The device according to claim 1 or 2 as a multiplex design.

42. 42. The apparatus of claim 41, wherein the plurality of channels are arranged in a cross-hatch configuration.

43. 3. The device according to claim 1 or 2 as a parallel simplex design.

44. 42. The apparatus of claim 41, wherein the plurality of channels are arranged in a non-crosshatch configuration.

45. 3. The device of claim 1, wherein the plurality of wells are pre-loaded with a binding complex.

46. 46. ​​The apparatus of claim 45, wherein the binding complex is provided in a gel in the sample introduction device.

47. 46. ​​The device of claim 45, wherein the binding complexes are provided with complementary surface chemistries to promote complex-to-complex binding upon input of analyte.

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