WO2014121394A1

WO2014121394A1 - Magnetic nanoparticle assay devices and methods

Info

Publication number: WO2014121394A1
Application number: PCT/CA2014/050077
Authority: WO
Inventors: Scott Tsai; Beau STANDISH; Brian BATTAGLIA
Original assignee: Scott Tsai; Standish Beau; Battaglia Brian
Priority date: 2013-02-06
Filing date: 2014-02-06
Publication date: 2014-08-14

Abstract

Embodiments of the present disclosure provide assay methods and devices for performing assays for the detection of analyte particles using magnetic particles. The magnetic particles are configured to bind to the analyte particles, thereby forming a magnetic particle/analyte particle complex when the magnetic particles are mixed with analyte particles. Magnetic separation is employed to collect both the complexed particles and the uncomplexed magnetic particles. The collected complexed particles and bare magnetic particles may then be collected within a reservoir having a volume configured such that the presence of analyte particles in excess of a pre- selected concentration produces a visually detectable change in fill level of the reservoir. One or more steps of the collection and/or detection step may be performed using a microfluidic device.

Description

MAGNETIC NANOP ARTICLE ASSAY DEVICES AND METHODS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 61/761 ,368, titled "MAGNETIC SOLID-PHASE NANOPARTICLE ASSAY DEVICES AND METHODS" and filed on February 6^th, 2013, the entire contents of which is incorporated herein by reference.

BACKGROUND

The present disclosure relates to devices and methods for the detection of an analyte in a sample.

The World Health Organization's (WHO) toxic threshold

concentration of arsenic in water is 1 0 micro g/L, which is a very low concentration that typically requires highly sensitive detectors. Current laboratory techniques for quantifying such low concentrations of arsenic in water, such as atomic absorption spectroscopy, are time consuming, very expensive, and have a large physical footprint. Using these systems to test, for example, the millions of wells in a country with arsenic contamination problems like Bangladesh, is too time and resource consuming [Rahman et al., Environ. Sci. Technol., 2002].

Previously developed field test kits rely on the reaction of arsine gas with mercuric bromide to form a color complex. It was reported that the kits gave 68% false negative results and 35% false positive results. Toxic arsine gas is also produced during usage [Arora et al., Environ. Geochem. Health, 2009]. Accordingly, there remains a need for a more suitable field testing technique for the detection of arsenic in water.

SUMMARY

Embodiments of the present disclosure provide assay methods and devices for performing assays for the detection of analyte particles using magnetic particles. The magnetic particles are configured to bind to the analyte particles, thereby forming a magnetic particle/analyte particle complex when the magnetic particles are mixed with analyte particles. Magnetic separation is employed to collect both the complexed particles and the uncomplexed magnetic particles. The collected complexed particles and bare magnetic particles may then be collected within a reservoir having a volume configured such that the presence of analyte particles in excess of a preselected concentration produces a visually detectable change in fill level of the reservoir. One or more steps of the collection and/or detection step may be performed using a microfluidic device.

Accordingly, in a first aspect, there is provided a method of detecting the presence of analyte particles in a sample using a microfluidic device; the microfluidic device comprising

a microfluidic channel;

a reservoir adjacent to the microfluidic channel, wherein the reservoir is in flow communication with the microfluidic channel; and

a magnet positioned adjacent to the reservoir for applying a magnetic field within the reservoir and the microfluidic channel;

the method comprising: mixing the sample with magnetic particles configured to bind with the analyte particles;

obtaining a particle suspension comprising complexed particles and uncomplexed magnetic particles, wherein the relative quantities of complexed particles and uncomplexed magnetic particles in the particle suspension depends on the concentration of analyte particles in the sample;

flowing a pre-selected volume of the particle suspension through the microfluidic channel, wherein the complexed particles and the

uncomplexed magnetic particles flowing through the microfluidic channel are deflected into, and collected within, the reservoir, in response to a magnetic field produced by the magnet; and

detecting the presence of the analyte particles within the sample according to a fill level of the reservoir.

In another aspect, there is provided a microfluidic device comprising: a microfluidic channel;

a reservoir adjacent to said microfluidic channel, wherein said reservoir is in flow communication with said microfluidic channel; and

a recess adjacent to said reservoir, wherein said recess is configured to receive a magnet for applying a magnetic field within said reservoir and said microfluidic channel, such that magnetic particles flowing through said microfluidic channel are deflected into, and collected within, said reservoir, in response to a magnetic field produced by the magnet;

wherein contents of said reservoir are visible to a user operating said device for determining the extent to which the reservoir is filled with particles.

A further understanding of the functional and advantageous aspects of the disclosure can be realized by reference to the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, with reference to the drawings, in which:

Figures 1a and 1 b illustrate an example embodiment for performing an optional pre-concentration step prior to magnetic particle detection of analyte particles in a sample. In Figure 1 a, the sample is mixed with magnetic particles, where the magnetic particles bind to analyte particles present in the sample. In Figure 1 b, a magnetic field is applied to the vessel to collect the bound particles, as well as any bare magnetic particles. After the pre- concentration step, the sample is removed, and the particle complexes and any bare magnetic particles are re-suspended to form a suspension to be used in a subsequent detection step.

Figures 2a-2e illustrate various embodiments for detecting the analyte particles using a microfluidic channel. The suspension of complexed particles (and any bare magnetic particles) is flowed through a microfluidic channel, and the particles are attracted towards a reservoir by an applied magnetic field. The presence and/or quantity of analyte particles is determined by assessing the degree to which the reservoir is filled.

Figure 3 illustrates an alternative example embodiment of a

microfluidic device having a tapered reservoir.

Figure 4 illustrates an example implementation in which a lens is provided above reservoir in order to provide a magnified view of the reservoir. Figure 5a illustrates an example embodiment in which a camera and processor are employed to interrogate the fill level of the reservoir.

Figure 5b illustrates an example embodiment in which an optical beam, a photodetector, and a processor are employed to interrogate the fill level of the reservoir.Figures 6a-5e illustrate alternative embodiments for detecting analyte particles using a microfluidic device, where the particles are collected in a reservoir that is connected to a first microfluidic channel by second and third microfluidic channels.

Figure 7 illustrates an example embodiment in which a plurality of reservoirs are provided serially within the microchannel.

Figure 8 illustrates an example implementation in which flow focusing inlets are included such that hydrodynamic flow focusing may be employed to move the particles towards to the reservoir side of the channel prior to the arrival of the particles at the reservoir.

Figure 9 is an image of an example microfluidic device incorporating a microfluidic channel and a series of magnets.

DETAILED DESCRIPTION

Various embodiments and aspects of the disclosure will be described with reference to details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure.

As used herein, the terms, "comprises" and "comprising" are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms, "comprises" and "comprising" and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not be construed as preferred or advantageous over other configurations disclosed herein.

As used herein, the terms "about" and "approximately" are meant to cover variations that may exist in the upper and lower limits of the ranges of values, such as variations in properties, parameters, and dimensions. In one non-limiting example, the terms "about" and "approximately" mean plus or minus 10 percent or less.

As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially" enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of "substantially" is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result.

Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood to one of ordinary skill in the art. Unless otherwise indicated, such as through context, as used herein, the following terms are intended to have the following meanings:

As used herein, the term "nanoparticle" refers to a particle having an average diameter on the nanometer scale. In some example embodiments, a nanoparticle may be a particle having an average diameter within the range of approximately 1 -10 nm, 10-50 nm, 1 -100 nm, or 1 -1000 nm.

As used herein, the term "microparticle" refers to a particle having an average diameter on the micrometer scale. In some example embodiments, a nanoparticle may be a particle having an average diameter within the range of approximately 1 -10 μηι, 10-50 μηι, 1 -100 μηι, or 1 -1000 μηι.

As used herein, the term "particle" refers to a nanoparticle or a microparticle.

As used herein, "microfluidic device" means a device for containing and manipulating small volumes amounts of fluid, such as volumes less than one milliliter, for example, volumes ranging from 1 μΙ to 1000 μΙ, or even submicroliter volumes, such as volumes ranging from 1 nl to 1000 nl. Such devices may be substantially planar and frequently contain features such as chambers (reservoirs), channels and/or valves. The dimensions of a microfluidic device may be the region of a few cm by a few cm. Some microfluidic devices can be fabricated from a variety of materials, such as glass and polydimethylsiloxane (PDMS). Embodiments of the present disclosure provide assay methods and devices for performing assays for the detection of analyte particles using magnetic particles. The magnetic particles are configured to bind to the analyte particles, thereby forming a magnetic particle/analyte particle complex when the magnetic particles are mixed with analyte particles. Magnetic separation is employed to collect both the complexed particles and the uncomplexed magnetic particles. The collected complexed particles and bare magnetic particles may be collected within a reservoir having a volume configured such that the presence of analyte particles in excess of a pre- selected concentration produces a visually detectable change in fill level of the reservoir. One or more steps of the collection and/or detection step may be performed using a microfluidic device.

Referring now to Figures 1 a-b and 2a-e, a non-limiting example implementation of a particle assay device and method is illustrated. In Figure 1 a, a liquid sample containing analyte particles 100 is mixed with magnetic particles 1 10 in vessel 120. Magnetic particles 1 10 are configured to bind to analyte particles 1 00. After mixing and optional incubation, analyte particles 100 and magnetic particles 1 10 bind to form complexed particles 1 15.

The complexed particles 1 15 and any bare (non-complexed) magnetic particles 1 10 are then collected using magnet 140, and are concentrated as shown at 150 in Figure 1 b.

Although Figure 1 b shows the collection/concentration being performed on the side wall of the vessel 120, it is to be understood that the

collection/concentration may be performed on an external device or implement, such as a sheath that may that may be inserted into vessel 1 20, where the sheath has an internal bore with a removable magnet.

In some embodiments, the magnetic field can be applied in any direction during the magnetic enrichment stage, such that one or more magnets are placed in any location adjacent to the container. For example, in some embodiments, the magnet (or an array of magnets) can encircle at least a portion of the container, so that the particles collect to the side wall of the container. In another example, one or more magnets can also be placed below the container. The one or more magnets can include one or more electromagnets and/or one or more permanent magnets.

The collected particles may be washed, one or more times, while maintaining the application of the magnetic field, prior to performing detection of the analyte. Washing may be beneficial when the initial sample includes additional particulate matter that could otherwise interfere with the detection step. For example, the presence of additional particulate matter could lead to an overestimation of the analyte concentration, and/or false positive test results. In one embodiment, the collected particles are re-suspended and recollected during each wash step, in order to wash away additional particles that are trapped with the magnetic particles during the application of the magnetic field.

The collected particles 150 may then be re-suspended in a liquid or buffer (after removal of magnet 140), thereby providing a concentrated suspension including the complexed particles and bare particles that were collected by magnet 140.

Referring now to Figure 2a, the suspension of particles, or a portion thereof, are then flowed into inlet 210 of a microfluidic device 200. The suspension is flowed through microfluidic channel 220, past reservoir 230, to outlet 215 (or, for example, to a waste chamber or waste reservoir). The suspension can be flowed through microfluidic channel 220 under the application of external pressure, which may be provided by a mechanism or device such as a pump, pipettor, or plunger. In another example

implementation, inlet 210 of the microfluidic device 200 may be connected to the outlet of a vertically-oriented vessel that is configured to receive the particle suspension, such that hydrostatic pressure can be exploited to drive the flow of fluid through microfluidic channel 220.

Magnet 240, which is placed or provided proximal or adjacent to reservoir 230, exerts a magnetic force on any complexed or bare magnetic particles flowing in the suspension, causing the particles to be attracted towards reservoir 230. As shown in Figure 2a, the particles collect within reservoir 230, because both the complexed particles and the bare magnetic particles are attracted via a magnetic force. At least the portion of device 200 that includes reservoir 230 is transparent, or at least partially transparent, such that the fill level of the reservoir may be observed. This may be achieved, for example, by fabricating device 200 such that at least one substrate that confines microfluidic channel 220 is transparent or partially transparent.

Figure 2b shows an example case in which the sample is absent of analyte particles. The particles that are collected within reservoir 230 are thus entirely magnetic particles 250, and only fill reservoir 230 partially.

In Figure 2c, an example case is illustrated in which the sample contains analyte particles. Upon mixing the magnetic particles with the sample, the analyte particles and the magnetic particles form complexed particles, as described above. In the present example, the quantity of analyte particles in the sample is insufficient for complexing with all magnetic particles. Accordingly, some magnetic particles remain bare, and reservoir 230 remains partially filed, but is filled to a level that is greater than in the case illustrated in Figure 2b. As such, provided that reservoir 230 is visible to an observer, the increased fill level of reservoir 230 may be observed, and it may be possible to make a determination that the sample contains analyte particles based on the increased fill level.

Figure 2d illustrates an example case in which the concentration of analyte particles in the sample is such that reservoir 230 is approximately filled. Figure 2e illustrates a case in which the concentration of analyte particles in the sample is such that reservoir 230 is overfilled by the collected particles.

Accordingly, in some embodiments, the fill level of reservoir 230 may be employed to determine the relative quantity of analyte particles in the sample. In some embodiments, the analyte concentration may correspond to a cutoff concentration of a quantitative assay. For example, the size and/or concentration of the magnetic particles may be selected to cause reservoir 230 to become filled at a pre-selected concentration that is relevant to a threshold detection level. An observer performing an assay using device 200 may therefore determine whether or not the concentration of the analyte particles in the sample exceeds the threshold concentration according to whether or not reservoir 230 is filled.

Although the shape of reservoir 240 is shown as rounded in Figures 2a-2e, other reservoir shapes may be employed in order to improve the sensitivity of detection and/or the dynamic range of detection. For example, in one embodiment, a rectangular reservoir geometry may be employed.

In another embodiment, the reservoir geometry may be configured to amplify the sensitivity of detection for analyte concentrations that are near to a pre-selected concentration. For example, this may be achieved by tapering or necking the reservoir geometry at a fill level that corresponds to the preselected concentration, such that a change analyte particle concentration produces a locally enhanced relative change in the fill level of the reservoir. Such an embodiment is illustrated in Figure 3, in which microfluidic device 300 includes microfluidic channel 320, reservoir 330, and adjacent magnet 340, where the width of reservoir 330 is tapered in a region 335 near microfluidic channel 320.

In another embodiment, one or more optical lenses may be provided on, embedded within, or provided above (or below) the microfluidic device, such that the reservoir region, or at least a portion the reservoir region, is optically magnified when viewed or imaged from above or below the device. For example, the one or more lenses may optically magnify a region of the reservoir associated with an assay cutoff. The magnified region may also be selected to magnify graduation indicators on or within the device. It will be understood that the lens or lenses may be formed or provided according to a wide variety of embodiments. In one example embodiment, one or more of the lenses may be a conventional refractive lens. In another example

embodiment, one or more of the lenses may be a Fresnel lens. In one example implementation, a lens may be integrally formed within a the microfluidic device during its fabrication.

In another example implementation, a lens may be formed by placing a polymerizable liquid solution on a surface above the reservoir, and curing the solution to create a magnifying lens over the reservoir, to improve the visualization of the change in fill level of the reservoir. Such an example implementation is illustrated in Figure 4, where magnifying lens 360 is shown formed above the reservoir.

While the preceding example employed a pre-concentration step prior to detection, as illustrated in Figures 1 a and 1 b, it is to be understood that in some applications (i.e. in some clinical or industrial settings), it may not be necessary to perform the pre-concentration step. For example, if the analyte concentration is particularly high, and if the sample matrix is relatively free from potentially interfering particulate matter, then the pre-concentration step may not be necessary.

It is to be understood that while the illustrated example embodiments provided herein relate to visible, label-free detection based on the fill level of the reservoir, the detection may be performed by direct operator observation and assessment, or by automated assessment. In the later case, and as illustrated in the example embodiment shown in Figure 5a, a camera 420 may be employed to record an image of the reservoir 430 of microfluidic device 400, and the fill level of the reservoir may be determined by image analysis, such standard image processing packages or routines that are known to those skilled in the art, via processor 450. In some embodiments, a quantitative or semi-quantitative determination of the concentration of analyte particles in the sample may be made using pre-determined calibration data, such as a dose-response curve or a look-up table.

In other another example embodiment, automated determination of whether or not a pre-selected analyte concentration has been exceeded in the sample may be performed by passing an optical beam through the reservoir. For example, as shown in Figure 5b, optical beam 455 could be transmitted from light source 460 through reservoir 430, (or reflected off the bottom of the reservoir) at a location where the optical transmission would be impeded by the presence of collected particles when the analyte particle concentration exceeds the pre-selected concentration. The interruption and/or attenuation of the optical beam, as detected by monitoring the power transmitted through the reservoir via photodetector 470 and processor 480, could be employed to determine whether or not the concentration of analyte particles in the sample exceeds the pre-selected concentration.

In other example embodiments, non-optical detection methods may be employed for the determination of the analyte particle concentration. For example, two or more electrodes located within the reservoir may be employed to measure electrical properties of the reservoir, such as electrical impedance, and to relate the measured electrical properties to the presence and/or concentration of the analyte particles in the sample.

In another example embodiment, a quantitative or semi-quantitative

(e.g. binned) determination of the concentration of analyte particles in the sample may be made visually by providing visual indicators on or adjacent to the reservoir. For example, graduation indicators may be provided that allow for the approximate visual determination of the concentration of the analyte particles within the sample. Referring now to Figures 6a-6e, an alternative embodiment of a microfluidic device 500 is shown in which reservoir 51 0 is connected to (i.e. in fluid communication with) first microfluidic channel 505 through second 520 and third 530 microfluidic channels. Magnet 540 causes magnetic particles (bare or complexed) to be deflected into second microfluidic channel 520 and to be collected in reservoir 51 0. Third microfluidic channel 530 may be beneficial in providing a path back to first microfluidic channel 505 for excess fluid that is drawn towards reservoir with the magnetic particles. This design may be advantageous in providing an asymmetric visual signature when reservoir 310 is overfilled, based on the filling of second microfluidic channel 520, as shown at 550 in Figure 6e.

In some embodiments, the suspension may be re-circulated through device 500 one or more times in order to achieve improved capture of the suspended particles by reservoir 510.

It is to be understood that the magnet need not be permanently affixed to microfluidic device. For example, in some embodiments, the microfluidic device may include a recess, slot, opening, aperture or other configuration suitable for receiving the magnet. In one embodiment, the magnet may be a rare-earth permanent magnet, such as an Nd₂Fei₄B permanent magnet, or other types of permanent magnets. In a non-limiting example, the

magnetization of the magnets can range from approximately 1 0⁵ to 10⁷ A/m, the magnet height may range from approximately 10 μηι to 1 0 mm, and the width and length may each vary from approximately 1 mm to 40 mm. In another embodiment, the magnet may be an electromagnet.

In another embodiment, the microfluidic device may include, or be interfaced with, two or more magnets. For example, in some embodiments, a plurality of magnets may be included adjacent to the microfluidic channel.

For example, one or more additional magnets may be placed upstream of the collection reservoir, adjacent to the microfluidic channel, in order to pull the bare magnetic particles and the complexed particles closer to the reservoir-side of the channel.

In another example implementation, two or more reservoirs may be included in a serial fashion within the microfluidic channel, as illustrated in Figure 7. The figure shows an example implementation with three reservoirs 510, each having an associate magnet 540.

In another embodiment, hydrodynamic flow-focusing may also be employed to move the particles toward the reservoir-side prior to the particles arriving at the reservoir. For example, flow-focusing may be in the form of additional sheath flow of buffer solution from the side opposite to the reservoir, adjacent to the microchannel. In such an example implementation, the sheath flow can be applied via an inlet on the opposite side of the reservoir, adjacent to the microchannel.

Another example implementation is illustrated in Figure 8 in which two inlets are provided. As shown in the Figure, flow focusing buffer may be provided from two sheath flow inlets 560 located adjacent to the microfluidic channel, at a location that is upstream of the reservoir 510. In such an embodiment, flow focusing with two sheath flows helps align the particles close to the center of the channel before they are magnetically deflected to the reservoir. This prevents particles from contacting and sticking to the side walls of the channel. In one example implementation, the relative flow-rates of the sheath-flow inlets may be tuned to position the particles such that their alignment is closer to the reservoir/magnet, to improve the uptake.

It is to be understood that in some embodiments, the magnetic particles are ferromagnetic particles such as magnetite (Fe₃0₄; an iron oxide). Non-limiting examples of other ferromagnetic materials for forming magnetic particles include Fe, Fe₂0₃ (magnetic phases), Ni, Co, Gd, Dy, Fe₃C, FeBe₅, Cu₂MnAI, Cu₂Mnln, Au₂MnAI, Fe₂B, MnAs, MnBi, MnB, CrTe, Cr0₂, CrBr₃, EuO, and GdCI₃. Magnetic particles may be provided in a core-shell configuration, for example, in which a magnetic core is capped by a non- magnetic shell. In other examples, the magnetic particles may be

paramagnetic, superparamagnetic particles, and/or diamagnetic particles.

As noted above, the magnetic particles are configured to bind with the analyte particles in order to facilitate detection (and to facilitate the optional pre-concentration step shown in Figures 1 a and 1 b). In some embodiments, the magnetic particles may naturally bind to the analyte particles, without further modification.

In one embodiment, the magnetic particles are magnetite

nanoparticles, and the analyte particles are arsenic nanoparticles. In such a case, the magnetite particles will attract and bind the arsenic nanoparticles without further modification. The magnetite particles may have an average diameter of approximately 5-20 nm, and more particularly, approximately 10 nm.

In other embodiments, the magnetic particles may be modified to bind with the analyte particles. For example, the magnetic particles may be modified to include a molecular recognition element that is suitable for binding the analyte particles. Non-limiting examples include ligands, proteins, antibodies, aptamers, nucleic acids, and nucleic acid analogs.

It will be understood that the design parameters, such as the size of the reservoir, the average diameter of the magnetic particles, and the

concentration of the magnetic particles, will vary among different applications and settings. In one example embodiment, these parameters may be determined experimentally in order to obtain filling of the reservoir for a given analyte particle concentration. For example, one or more parameters may be varied while performing test assays with a standard sample having a known concentration equal to that of the desired threshold concentration in order to determine the parameters that are suitable for obtaining filling of the reservoir.

In one example embodiment, in which the analyte particles are arsenic nanoparticles and the magnetic particles are magnetite

nanoparticles, the size of the reservoir may be designed in such a way that if it is completely filled with particles, it indicates that the sample had 10 μg/L of arsenic (corresponding to the WHO threshold concentration for toxicity). Accordingly, if a user performs a test and observers that the reservoir is only partially filled, the user can conclude that the water sample had an arsenic concentration below the toxic level.

In one embodiment, the design of the channel dimensions may be based upon a particle deflection parameter that was described previously [S. S. H. Tsai et ai, Lab Chip, 201 1 ],

_ χα²Μ²

Ω~ - - r\u₀l_c

where the dimensionless deflection parameter Ω is employed to determine how large the particle deflections are going to be. Here χ is the magnetic susceptibility of the particle or cluster, a is the particle/cluster radius, M is the magnetization of the permanent magnet, η is the viscosity of water, u₀ is the average speed of the flow in the channel, and l_c is the characteristic length-scale of the channel.

The volume of the reservoir can also be estimated based on the following expression:

where V_r is the full volume of the reservoir, V_s is the volume of the fluid that will be placed onto the microfluidic lab-on-a-chip device, Ci and C₂ are the post re-suspension magnetite and the toxic threshold concentration of arsenic, in units of g/L. Finally, pi and p₂are the densities of the magnetite and arsenic nanoparticles, respectively. The prefactor 3/5 accounts for the voids in a random close-packed system.

For example, the densities of magnetite and arsenic are approximately 1500 kg/m³ and 4000 kg/m³ respectively. Therefore, if the magnetite and arsenic nanoparticles are pre-concentrated to Ci = 10 g/L and C₂ = 1 g/L, respectively (assuming that magnetite nanoparticles are supplied at ten times the toxic arsenic concentration), and re-suspended in a 1 mL water solution, then the volume of the on-chip reservoir should be approximately 1 0 \xL.

It is noted that when applied to the detection of arsenic, this present embodiment does not involve chemical reactions, does not produce toxic gases and has the potential for being low cost with mass production and the use of inexpensive plastics like PMMA for the chip. Such an example device is shown in Figure 9, where a PMMA microfluidic device 600 is shown incorporating a microfluidic channel 610 and several magnets 620. The example magnets 620 were Nd₂Fei₄B permanent magnets, with dimensions of 9.5 x 4.8 x 1 .6 mm, and a magnetization out of plane (up) at 1 0⁶ A/m.

Although the example embodiments disclosed above employ magnetic nanoparticles for the detection of analyte nanoparticles, it is to be understood that alternatively embodiments may employ microparticles. For example, in some embodiments, the magnetic particles may have a dimension on a micron scale. Similarly, the analyte particles may be particles having a dimension on the micron scale.

It is to be understood, however, that the devices and methods according to the present disclosure may have increased and/or optimal sensitivity when the size of the magnetic particles is on the same scale as the size of the analyte particles. This provides a particle system in which the presence or absence of either type of particle has a substantial effect on the total spatial region filled by a collection of both particles. For example, in one example embodiment, the average diameter of the magnetic particles is selected to be approximately equal to the average diameter of the analyte particles. In another example embodiment, the average diameter of the magnetic particles is selected to be on the same order of magnitude as the average diameter of the analyte particles. In another example embodiment, the average diameter of the magnetic particles is selected to be larger than the average interstitial gap between close-packed analyte particles. In another example embodiment, the average diameter of the magnetic particles is selected such that the average interstitial gap between close-packed magnetic particles is smaller than the average diameter of the analyte particles. In cases in which the diameter of the analyte particles is not monodisperse, the diameter of the magnetic particles may be selected to correspond to a statistical size measure associated with the analyte particle. For example, in one example embodiment, the magnetic particle diameter may be selected to be approximately equal to the average diameter of the analyte particles, if known. In another example embodiment, the magnetic particles may be selected to have a size range, or polydispersity, that is approximately equal to the measured, estimated, or expected size range or polydispersity of the analyte particles.

In an additional embodiment, the magnetic enrichment process could be used as a method to detect analyte molecules (for example, but not limited to, heavy metals, biomolecules , organic and inorganic compounds) that are too small to produce a measurable volume change itself, but where the volume change in the reservoir is facilitated via an intermediate particle that is configured to bind to analyte molecules, thereby forming analyte particles, which in turn bind to the magnetic particles. Thus, when the analyte is present, complexed particles are formed including the magnetic particle, one or more analyte molecules, and the intermediate particle. The intermediate particle is selected to have a size that produces a detectable change in the volume of the reservoir when the analyte is present, thereby effectively "amplifying" the size of the analyte.

For example the magnetic particle could have active binding sites (e.g. ligands or receptors) that bind to a first molecular recognition site or epitope present on the analyte, , and the intermediate particle could also have active binding sites (e.g. ligands or receptors) that bind to a second molecular recognition site or epitope present on the analyte, such that one or more analyte molecules bind to both the intermediate particle and the magnetic particle in a sandwich assay configuration. The binding may be based on specific molecular recognition.

An example of an intermediate particle is a polymer sphere, such as a polystyrene sphere. In some embodiments, the intermediate particle may have a pre-selected color or opacity in order to facilitate visible or optical detection of the reservoir fill level. For example, when the analyte is attached to both the magnetic particle and a coloured polystyrene sphere, it can be collected in a particular region of a microfluidic device via magnetic attraction, where the colored polystyrene sphere could be easily visible without the need for complex detection techniques. It is noted that for more complex interactions, multiple detection techniques involving both chemical and/or visible methods such as white field illumination could be used to identify a reaction of interest.

The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the claims are not intended to be limited to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure.

Claims

THEREFORE WHAT IS CLAIMED IS:

1 . A method of detecting the presence of analyte particles in a sample using a microfluidic device;

the microfluidic device comprising

a microfluidic channel;

the method comprising:

mixing the sample with magnetic particles configured to bind with the analyte particles;

2. The method according to claim 1 wherein the magnetic particles are magnetic nanoparticles.

3. The method according to claim 2 wherein the magnetic nanoparticles are magnetite nanoparticles.

4. The method according to claim 2 or 3 wherein the analyte particles have a natural affinity for the magnetic nanoparticles.

5. The method according to any one of claims 2 to 4 wherein the analyte particles having a natural affinity for the magnetic nanoparticles are arsenic nanoparticles.

6. The method according to any one of claims 1 to 5 wherein the magnetic particles comprise a molecular recognition element for binding with the analyte particles.

7. The method according to any one of claims 1 to 6 wherein a volume of the reservoir is selected such that the reservoir is filled with particles when a concentration of analyte particles within the sample approximately equals a threshold concentration.

8. The method according to claim 7 wherein the threshold concentration is a threshold for toxicity.

9. The method according to claim 8 wherein the analyte particles are arsenic nanoparticles, and wherein the threshold is approximately 10 ug/L.

10. The method according to any one of claims 1 to 9 wherein the particle suspension is obtained by:

mixing, in a vessel, the sample and the magnetic particles, such that at least a portion of the magnetic particles bind with the analyte particles to form the complexed particles;

magnetically separating the complexed particles and the uncomplexed magnetic particles from the sample; and

adding the complexed particles and the uncomplexed magnetic particles to a volume of liquid to form the particle suspension.

1 1 . The method according to claim 10 wherein the volume of the liquid is selected such that the particle suspension is concentrated.

12. The method according to claim 1 1 further comprising washing the complexed particles and the uncomplexed magnetic particles prior to forming the particle suspension.

13. The method according to any one of claims 1 to 12 wherein the detection of the analyte particles is performed visually.

14. The method according to any one of claims 1 to 12 wherein the detection of the analyte particles is performed by: imaging the reservoir; and

processing the image to determine the fill level of the reservoir.

15. The method according to any one of claims 1 to 12 wherein the detection of the analyte particles is performed by:

passing an optical beam through a portion of the reservoir;

detecting the transmitted power of the optical beam ; and

determining the fill level of the reservoir according to the transmitted power of the optical beam.

16. The method according to any one of claims 1 to 15 wherein the microfluidic device further comprises one or more focusing fluid inlets upstream of the reservoir, wherein at least one of the flow focusing fluid inlets is positioned on a side of the microfluidic channel that is opposite to that of the reservoir, the method further comprising:

flowing a flow focusing fluid into the microfluidic channel via the flow focusing fluid inlets.

17. The method according to claim 16 wherein the microfluidic device comprises a single flow focusing inlet, and wherein the flow focusing fluid is provided such that the complexed particles and the uncomplexed magnetic particles are biased towards the reservoir.

18. The method according to claim 16 wherein the microfluidic device comprises a two flow focusing inlets on either side of the microfluidic channel, and wherein the flow rates of the flow focusing fluid provided to the two flow focusing inlets is controlled such that the complexed particles and the uncomplexed magnetic particles are biased towards the reservoir.

19. The method according to any one of claims 1 to 18 wherein the microfluidic device comprises one or more additional reservoirs serially disposed within the microfluidic channel, each additional reservoir having associated therewith an additional magnet, such that the complexed particles and the uncomplexed magnetic particles may be retained in two or more reservoirs.

20. The method according to any one of claims 1 to 18 wherein the analyte particles comprise:

one or more analyte molecules bound to an intermediate particle, wherein the intermediate particle has a size selected to produce a detectable change in the fill level of the reservoir when a sufficient concentration of analyte molecules are present, thereby amplifying the size of the analyte molecules.

21 . The method according to claim 20 wherein the intermediate particles are configured to bind to a first recognition site of the analyte molecules, and wherein the magnetic particles are configured to bind to a second recognition site of the analyte molecules.

22. A microfluidic device comprising: a microfluidic channel;

23. The microfluidic device according to claim 22 wherein a volume of said reservoir is selected such that, when performing an assay based on binding of magnetic particles to analyte particles and flowing a controlled volume of sample through the microfluidic channel, a fill level of the reservoir is dependent on a concentration of analyte particles within the sample.

24. The microfluidic device according to claim 23 further comprising markings suitable for assisting in the determination of the concentration of the analyte particles based on the fill level of said reservoir.

25. The microfluidic device according to claim 23 wherein the reservoir is filled with particles when a concentration of analyte particles within the sample approximately equals a threshold concentration.

26. The microfluidic device according to claim 25 wherein the analyte particles are arsenic particles, and wherein the threshold concentration is a threshold for toxicity.

27. The microfluidic device according to claim 26 wherein the threshold is approximately 1 0 ug/L.

28. The microfluidic device according to any one of claims 22 to 27 wherein a portion of the reservoir has a narrowed width.

29. The microfluidic device according to claim 28 wherein the portion of the reservoir with the narrowed width is located at or proximal to a fill level associated to a threshold concentration.

30. The microfluidic device according to any one of claims 22 to 29 further comprising a flow mechanism in flow communication with said microfluidic channel.

31 . The microfluidic device according to claim 30 wherein said flow mechanism is a vertical cylinder having a lower outlet in flow communication with an inlet of said microfluidic channel, wherein a height of said vertical cylinder is selected to provide sufficient hydrostatic pressure for producing flow within said microfluidic channel.

32. The microfluidic device according to any one of claims 22 to 31 further comprising the magnet.

33. The microfluidic device according to claim 32 wherein said magnet is a rare-earth magnet.

34. The microfluidic device according to claim 33 wherein said rare-earth magnet comprises Nd2Fe12B.

35. The microfluidic device according to any one of claims 22 to 34 further comprising one or more additional magnets positioned upstream from the reservoir for pulling magnetic particles closer to the reservoir-side of said microfluidic channel.

36. The microfluidic device according to any one of claims 22 to 35 further comprising one or more additional reservoirs serially disposed within said microfluidic channel, each additional reservoir having associated therewith an additional magnet.

37. The microfluidic device according to any one of claims 22 to 36 further comprising a lens that is positioned to magnify at least a portion of said reservoir.

38. The microfluidic device according to claim 37 wherein the lens is integrally formed within the microfluidic device.

39. The microfluidic device according to claim 37 wherein the lens comprises a polymer lens formed above said reservoir on a surface of said microfluidic device.

40. The microfluidic device according to any one of claims 20 to 39 further comprising an imaging device positioned to image at least a portion of said reservoir.

41 . The microfluidic device according to any one of claims 20 to 39 further comprising:

a light source positioned to pass an optical beam through said reservoir; and

a photodetector positioned to detect the optical power within the optical beam after it passes through said reservoir.

42. The microfluidic device according to any one of claims 20 to 41 further comprising one or more focusing fluid inlets provided upstream of the reservoir, wherein at least one of the flow focusing fluid inlets is positioned on a side of the microfluidic channel that is opposite to that of the reservoir.