Patent Application
20210299656
The present invention relates in general to a technique for controlling magnetic nanoparticle distribution in a microfluidic chamber, and in particular to a kit, method and system for constraining magnetic nanoparticles to within spaces between micropillars.
Microfluidic devices offer important opportunities for controlled movements of fluids. Tiny volumes of fluids are advantageous when small amounts of samples or reagents are available, where compact or portable assays are needed, where automation is essential for efficiency, and where fast reaction times are sought, especially in clinical diagnosis and biomedical research. A variety of capillarity, centrifugal, pneumatic, and electrostatic microfluidic devices have been provided to move fluids and perform various types of biochemical assays (which is to be understood broadly, and to include at least Lab on Chip (LOC), micro-total analysis systems (μTAS), organ on chip, and other assaying devices for processing fluid in volumes of less than 10 mL, such as from 10 nL-1 mL). The most basic challenge in microfluidics in general, is to manipulate fluids in a controlled, repeatable manner, to achieve a desired process.
In this context, magnetization of particles can be very useful for controlling interactions between particles and a fluid. For example, immunomagnetic separation is a widely recognized approach for sample preparation.1-3 Separation of target species, such as cells, may be achieved using immunomagnetic particles (i.e. magnetic nanoparticles conjugated to capture probes, such as antibodies) that bind specifically to cell surface receptors. Conventional immunomagnetic separation is typically performed in tubes with several commercially available kits, including MACS® system (Miltenyi Biotec, USA), CellSearch System (Veridex, USA), and Dynabeads MPC separator series (Life technologies, USA). Furthermore, many applications call for chemical interaction of samples with functionalized superparamagnetic particles for sensitive detection of analytes, including food- and water-borne testing, blood testing, pharmacological testing, and clinical and biological testing.
In the past decade, microfluidic-based approaches that leverage magnetism have also emerged as viable, high throughput, low cost alternatives.4,5 “When brought into a microfluidic channel, nano- and micro-particles offer a relatively large specific surface for chemical binding.”4 Open or “empty” microchannels in microfluidic structures can be loaded with packed beds of functionalized particles or particle suspensions to profit from an even larger surface-to-volume ratio, an enhanced interaction of reactive surfaces with fluids passing by, and an improved recuperation of reaction products.4 Thus packed beds and bead suspensions are both known in the art. Packed beds may be porous enough to offer relatively low resistance to the flow while retaining the particles well enough to prevent (or reduce to a satisfactory level) their entrainment in the flow. However, the engineering required to achieve these is non-trivial.
Using magnetic nanoparticles MNPs offers some advantages over larger particles. Herein a MNP refers to nanoparticles: with a size in the range of 1-500 nm, preferably in the range of 5-250 nm or 20-200 nm; having magnetic moment per unit mass of 0.5 to 1 Am2/Kg, such as about 0.67 Am2/Kg and a saturation field of about 100-1000 kA/m, such as 500 kA/m, as can be produced with one or more superparamagnetic domains within each particle; and having a surface that is effective to avoid agglomeration (such as via electrostatic or steric repulsion). MNPs have higher surface areas, for higher interaction potential, no magnetic remanence and lower inertia and hydrodynamic drag, for fast response in a fluid.
It would be desirable in the art to improve control over spatial distributions of MNPs so that smaller amounts of nanoscale powders can be effective to interact with suspended or dissolved species within a microfluid. While the nanoparticles can be distributed randomly within a magnetic field, there is limited ability to control distribution and movement of the beads, because of the substantial limits on spatial and temporal variation of the magnetic fields within microfluidic chambers.
Given the complexity of magnetic field generators and their control apparatus; the need to vary, apply and remove the magnetic field during microfluidic processes; and the preference for avoiding contamination and cleaning issues by reusing microfluidic chips (which can otherwise be made very inexpensively), it is cost effective to avoid integration of controllable magnetic field generators within the chip. Most microfluidic methods rely either on (i) positioning a magnet in the vicinity of the microfluidic channel6,7 where the magnet provides both magnetic field and magnetic field gradient for the magnetic capture process, or (ii) using a magnet as a field generator and soft magnetic elements integrated into (or in the immediate vicinity of) the channel for the magnetic field gradient (high gradient magnetic separation—HGMS).
U.S. Pat. No. 7,601,265 to Rida et al. (Rida) teaches methods for manipulating magnetic micro-/nano-particles with magnets. The embodiments of
Rida teaches that chains of superparamagnetic particles are densified and held together. A resulting risk is obstruction of flow through the tube or microchannel, which is obviously problematic for achieving high surface area required for high fluid interaction potential. The manipulation of magnetic particles is challenging for flow through arrangements of the magnetic particles, because too strong a flow tends to result in loss of particles, and too strong a magnetic field reduces fluid permeability, and does not necessarily provide a high surface area.
By temporally varying the magnetic field (high frequency AC), Rida teaches agitating the “chains” of particles to produce a “vortex rotational dynamic” that provides a more efficient homogeneous distribution of the magnetic particles over the cross-section of the flow channel, even with a lower density of magnetic particles, and permits a more efficient interaction between the magnetic particles and target particles carried by a liquid flowing through the cell. Rida still requires a fairly high number of particles, and is limited by the spatial and temporal control over magnetic fields within microfluidic channels.
It appears that Rida has no appreciation for the effect that use of MNPs (as opposed to larger particles in the nanometer or micrometer range), can have on the ability to produce chains thereof, given that they disclose (C1;L50) that any magnetic particles can be used. The Sinclair article referred to by Rida reveals that all particles are micron-sized with the one exception: “Miltenyi Bioteck manufactures the smallest beads on the market—a mere 50 nm in diameter.” C6,L12 of Rida shows a preference for magnetic particles with a diameter of 2-5 microns.
It will be appreciated that using larger diameter particles (micron scale) with high magnetic moments will enable effective control over the particles with weaker external fields ceteris paribus, but at the cost of efficiency of binding with targets. It is known that when trapping micron scale targets, such as cells and bacteria, micron-scale particles do not provide sufficient surface area to volume capture area, and mobility to provide sufficient interaction probability with a sample stream. Thus nanoparticles are preferred, but these are harder to control magnetically. The corrugated poles provide the limit of control of magnetic field gradients available, without some kind of magnetic bodies within microfluidic channels of microfluidic devices.
High gradient magnetic separation (HGMS) is a field of study most closely related to the present invention. Proposed HGMS approaches to improve magnetic gradients within microfluidic chip include embedding patterned soft magnetic materials in microscopic elements to create local distortions of an externally applied magnetic field and thus generate stronger magnetic gradients. These can, for example, be used for efficient separation of magnetic vs. non-magnetic targets in microfluidic devices, or for immunomagnetic separation. HGMS devices typically use ferromagnetic wires for their desirable dimensions, and their demonstrable ability to create strong magnetic gradients when subjected to external magnetic fields.8 For example, the device described by Inglis et al. used ferromagnetic stripes recessed into a silicon substrate to alter the flow of magnetically labeled cells by magnetizing the stripes by an externally applied field.9,10 Magnetically labeled cells were attracted to the strips and tended to follow the strip direction, while unlabeled cells did not interact with the strips and followed the direction of fluid flow. Magnetic species can be trapped in separated channels by the high gradient magnetic regions created by arrays of small wires.11 Finally, repulsive modes in HGMS devices can also be used for diamagnetic targets.12
One of the problems with HGMS devices reported in the literature relate to the difficulties in releasing (cleaning) captured magnetic particles13, and in particular, the difficulty in preventing the magnetically labeled particles (cells) from permanent adhering to the magnetic elements. In the device designed by Inglis et al., a significant number (˜50%) of magnetically labeled cells either stuck permanently to the nickel strips or were not sufficiently attracted to the stripes to be separated.9,10
To alleviate this problem, pillars on silicon substrates for HGMS have been formed of permalloy or other soft magnetic materials.14 A good trade-off has not been found between strong enough magnetism for effective retention of magnetically labelled particles, and timely release thereof thereafter.
For complex biological assays, the release of magnetically labelled particles is highly desirable for further downstream processing. However, the reported devices had difficulty releasing captured material due to the device design that is focused on creating magnetic field gradients as high as possible to maximize the capture forces, using solid magnetic wires21-23 as they provide maximum perturbation effects and non-uniformities (gradients) in the applied field. However, in addition to the poor control of the capture regions due to an attractive capture force present everywhere on the surface of the wires/pillars,24 the large amounts of magnetic material employed usually possess strong remnant magnetization. This creates significant capture forces that persist after removal of the external magnetic field which makes the release challenging.
While very high flow rates and associated drag forces may improve release of trapped particles, the removal in low flow regions and stagnation points on the pillars are particularly challenging, especially using only a single unidirectional liquid flush. Various strategies, such as coating the magnetic material with PDMS,18 have been employed to improve magnetic release, which further complicates the process of making these MNPs, and increases the cost of these devices.
Magnetizable nickel coated posts in microfluidic channels have advantages over solid wires. Deng et al. showed a simple process based on electrodeposition of Ni integrated with PDMS microfluidic devices.17 The same process was later employed by Yu et al., and Liu et al., to integrate Ni pillars for magnetic capture of cells.18-20 Specifically, microtransfer molding of a PDMS chip was used17 to form the posts, and electrodeposited nickel coatings were applied to the posts (7 μm high and 15 μm in diameter, or an aspect ratio of 0.5:1). After the nickel coatings were deposited, an external permanent magnet was used to magnetize the posts. The magnetic field generated was ˜40 kA/m. Both transverse and axial magnetic fields were used. The device is proposed for trapping MNPs and separation of MNPs from a fluid.
While this could potentially decrease the fabrication costs, the process suffers from low-throughput. In addition to the difficulty of integrating soft magnetic materials in microfluidic channels, the devices reported in the literature suffered from small channel sizes and low density of magnetic microstructures.17-20 This typically results in low flow rates, low MNP capture capacity and limits use in higher throughput applications where a large number of magnetic beads have to be processed.
A more recent disclosure by Applicant in 201525 teaches magnetizable nickel-coated pillars in microfluidic channels for producing magnetic field variations that form MNP capture and MNP depleted regions. Higher aspect ratio nickel coated pillars (3:1 prior to coating and ˜3.5:1 after coating) with thinner (2 μm) nickel coating, and a 100 kA/m field were shown to produce magnetic field gradients in a denser array of pillars, suitable for trapping nanoparticles. Local magnetic fields surrounding the pillars are examined and mapped out, and modeling shows depletion and capture areas. While this disclosure shows a possibility for using field variations from magnetizable arrayed pillars,
Accordingly there remains a need for improved techniques for controlling MNP distribution in microfluidic chambers of microfluidic chips, and in particular to a method of distributing the MNPs in a spatially constrained region that extends between the magnetic integrated in the microfluidic chamber, especially one that maintains the distribution while a sample is flowed across the region. The need remains for a better trade-off between magnetic capture strength, and quick and reliable demagnetization for release. By flowing a sample through a MNP cloud of this distribution, improved interaction with the MNPs is possible with: lower incubation time, higher capture efficiency, using fewer MNPs, or under higher flow rates of the sample.
Applicant has demonstrated that with a dense, high aspect ratio array of pillars coated with magnetizable material, arranged in rows within a microfluidic chamber, the rows aligned with an externally generated magnetic field of sufficient strength, distribution of MNPs to form a cloud region substantially limited to row spaces between the pillars, that a density of the MNPs across this space is sufficient that there are no visible gaps in the cloud between the row's pillars, and that no visible gaps appear, even when subjected to moderate flow thereacross.
In some embodiments of the invention, a process for forming such a chamber in a microfluidic chip, with improved cost effectiveness, is provided by producing the pillars on an insert, and bonding the insert in a microfluidic chip.
In some embodiments of the invention, the coated pillars are used both as magnetic capture features, for creating field gradients to control MNP cloud distribution, as well as fluidic obstacles, to distort fluid streamlines, and force the biological target species to interact with the functionalized nanoparticles in the nanoparticle cloud. By aligning the rows of pillars to a small angle with respect to stream lines, a controlled interaction with particles of a given range of sizes and densities can be preferentially induced into crossing through the cloud regions. Furthermore, as particles passing through the cloud regions are moving substantially parallel with the cloud regions, a dwell time within the cloud region is increased compared with substantially orthogonal traversal. Even within the cloud region, differences in MNP concentrations are expected. Where the flow is highest (furthest from the pillars, floor and ceiling, or near the centre) is also the most depleted of MNPs. The trajectories of particles in the stream in this layout discourages flow through the depleted regions, and favours flow through higher MNP concentration paths. This may reduce time and complexity of the assay by avoiding the need for diffusion-based incubation and mixing.
By controlling a thickness of the magnetic material coating on the pillars, remanence of the magnetic field can be low, allowing for a satisfactory magnetic field surrounding the pillars, with suitable depletion and capture areas, but also providing for a fast and reasonably complete release of the particles in a moderate flow once the magnetic field is removed.
By providing an array of the rows, each row having a respective alignment with respect to the others with a respective stagger, the magnetic field being movable into position to alignment with a plurality of different rows in different directions. The different rows in different directions can consist of all of the same pillars, or different overlapping subsets of pillars can be used for different directions. By arranging for two or more differently directed rows, and by moving external magnets between different aligned positions, or moving different magnets or sets of magnets towards and away from the chamber, a rapid movement of the MNPs can be performed to permit mixing or interaction without any other fluid motion. Thus magnetic stirring can be performed with enhanced interaction probability, in what is otherwise a stagnation chamber.
A copy of the claims as filed are incorporated herein by reference.
Accordingly controlling superparamagnetic nanoparticle distribution in a microfluidic chamber of a microfluidic chip is provided, where: at least one row of at least 3 magnetically coated pillars are provided in a wall of the chamber, the pillars having a minimum separation with neighbors of 0.2-500 μm, an aspect ratio greater than 2:1, and a mean diameter of 1-1000 μm, where a polyline connects centres of the pillars; and a fluid is contained in the chamber surrounding the pillars, the fluid suspending superparamagnetic nanoparticles (NPs) that are self-repellant to reduce agglomeration. The control is provided by: applying a magnetic field to the chamber using magnets that are outside of the microfluidic chip, the magnetic field having a local field line that is at least 75% aligned with each segment of the polyline, wherein the NPs, pillars, and thickness of the magnetic coating of the pillars, are selected so that the NPs are substantially distributed between the pillars in that at least one of the following obtains: a NP density at every point between two adjacent pillars of a single row is at least 50% higher than the NP density midway between two adjacent rows; a NP density at every point between two adjacent pillars of a single row is at least 50% higher than the NP density a distance normal to the polyline equal to a mean separation of the pillars; a mean NP density in inter-pillar spaces between adjacent pillars is at least 10 times higher than a mean NP density within the chamber; a magnified view from a direction in which end faces of the pillars are in view, there are no visible gaps in the NP density between adjacent pillars of a single row, and visible gaps across at least 80% of the chamber away from the rows.
Preferably at least ⅓ of the NPs have a surface or subsurface coating for electrostatically, sterically, or chemically repelling like particles, and the NPs are surface functionalized to selectively bond to a target analyte. Preferably the NPs are distributed substantially only between the pillars in that at least 80% of the NPs are retained within one or more strips centred on the polylines, with a strip thickness of twice a mean diameter of the pillars.
The pillars may be coated with one of: a soft magnetic shell of thickness of 0.1-20 μm, composed of a nickel-based alloy; and a soft magnetic shell of thickness of 0.1-20 μm, composed of a nickel-based alloy coated with a gold passivation layer.
Controlling may further comprise flowing a sample fluid through the chamber across the NP distribution for NP analyte capture while the magnetic field is applied. If so, the wall may include at least 3 rows that form a two-dimensional Bravais lattice of the pillars, with one of the primitive vectors of the lattice being oriented at an angle between 1° and 15° with respect to the liquid flow through the chamber. If so, the magnetic field may be oriented: in a direction that minimizes an inter-pillar space between adjacent pillars of row; in a direction of one of two primitive vectors of a two-dimensional Bravais lattice of defined by the at least one row; or in a flow direction through the chamber, which is oriented at an angle between 1° and 15° with respect to one of two primitive vectors of a two-dimensional Bravais lattice of defined by the at least one row.
Controlling may further comprise removing the magnetic field after the sample flowed through the chamber, and flushing the NPs to a detection chamber. Flushing may be accomplished only with fluid dynamics, and without magnetic guidance, or a density or spatial distribution of the NPs is increased within the detection chamber by mechanical, flow, magnetic or ultrasonic filtration.
The sample fluid, after flowing through the chamber, may travel through a second chamber bearing a respective wall with pillars and a fluid suspending at least one second NP distribution with NPs functionalized to selectively bond to at least one second analyte, where a single magnetic field applies fields across the chamber and the second chamber concurrently. The chamber and second chamber may be stacked horizontally, for example on separately bonded and aligned microfluidic chips.
The pillars may have a mean separation of 1-100 μm, an aspect ratio greater than 3:1, and a mean diameter of 10-300 μm, the NPs may be electrostatically charged to prevent agglomeration; the magnetic field may have a local field line that is at least 90% aligned with the segments of the polyline, and has a magnetic field strength of at least 110 kA/m across this local field line; and during the application of the magnetic field, the NPs may be distributed substantially only between the pillars in that at least 80% of the NPs are retained within one or more strips centred on the polylines, with a strip thickness of twice a mean diameter of the pillars.
The pillars may have a mean separation of 20-80 μm, an aspect ratio greater than 5:1, and a mean diameter of 20-150 μm; the NPs may be electrostatically charged to prevent agglomeration; the magnetic field may have a local field line that is at least 90% aligned with the segments of the polyline, and have a magnetic field strength of at least 110 kA/m across this local field line; and during the application, the NPs may be distributed substantially only between the pillars in that at least 85% of the NPs are retained within one or more strips centred on the polylines, with a strip thickness of twice a mean diameter of the pillars.
The at least one row of at least 3 magnetically coated pillars further comprises an array having at least 2 axes, along each of which axes the pillars are arranged at least one row of at least 3 pillars with a minimum separation with neighbors of 0.2-500 μm, further comprising applying the magnetic field alternately along the axes to redistribute the NPs.
Further, a microfluidic device is provided, the device comprising: a microfluidic chip with at least one wall of a microfluidic chamber, the wall supporting at least one row of at least 3 micropillars, where the micropillars of the row: are arrayed to form a polyline; have mean diameters of 1-1000 μm; have mean separations of 0.2-500 μm; have aspect ratios greater than 2:1; and are composed of a low susceptibility material coated with a soft magnetic material; a generator adapted to apply a magnetic field of at least 110 kAmp/m across the at least one row; and a support comprising a holder for the microfluidic chip in at least one prescribed position and orientation, and a registration feature for registering the generator in a position in which a field line of the magnetic field is at least 75% aligned with the polyline.
The microfluidic device may further comprise a sample introduction chamber, an analyte detection chamber, and a sample flush reservoir, the sample introduction chamber coupled to an ingress of the microfluidic chamber by an inlet channel, the microfluidic chamber coupled to the reservoir by an outlet channel, and the microfluidic chamber coupled to the detection chamber by a NP channel.
Each of the at least one wall of the microfluidic chamber, may be provided as an insert into an opening within a patterned microfluidic chip.
The soft magnetic coating comprises a soft magnetic shell of thickness of 0.1-20 μm, composed of a nickel-based alloy to ensure a low remanence.
The microfluidic device may comprise a plurality of the microfluidic chambers on one or more microfluidic chips, and the support comprises a holder for the one or more microfluidic chips in prescribed positions and orientations, and the registration feature registers the generator in a position in which one or more field lines of the magnetic field generated are at least 75% aligned with each of the respective polylines of the respective walls of the microfluidic chambers.
The at least one row of at least 3 magnetically coated pillars may comprise an array having at least 2 axes, along each of which axes at least one row of at least 3 pillars are arranged with a minimum separation with neighbors of 0.2-500 μm, the holder comprises a plurality of registration features for registering the generator in respective positions in which field lines of the magnetic fields are at least 75% aligned with the axes.
Further, a kit is provided, the kit comprising: the microfluidic device, and a fluid suspending superparamagnetic nanoparticles (NPs), the fluid being injectable into the microfluidic channel, wherein: the NPs are self-repellant to reduce agglomeration, and applying the magnetic field to the chamber with the magnet in registered position, with fluid in the microfluidic channel, substantially distributes the NPs between the pillars in that pillars in that at least one of the following obtains: a NP density at every point between two adjacent pillars of a single row is at least 50% higher than the NP density midway between two adjacent rows; a NP density at every point between two adjacent pillars of a single row is at least 50% higher than the NP density a distance normal to the polyline equal to a mean separation of the pillars; a mean NP density in inter-pillar spaces between adjacent pillars is at least 10 times higher than a mean NP density within the chamber; a magnified view from a direction in which end faces of the pillars are in view, there are no visible gaps in the NP density between adjacent pillars of a single row, and visible gaps across at least 80% of the chamber away from the rows.
A kit is provided comprising the microfluidic device, and a fluid suspending superparamagnetic nanoparticles (NPs), the fluid being injectable into the microfluidic channel, wherein the NPs: have a surface or subsurface coating that makes at least ⅓ of the particles electrostatically or chemically repel like particles; and are surface functionalized to selectively bond to an analyte.
In either kit, the microfluidic device may have a plurality of microfluidic chambers, and a plurality of fluids are provided each suspending respective NP that are surface functionalized for selectively bonding to respective analytes. The magnetic field may be oriented: in a direction that minimizes an inter-pillar space between adjacent pillars of row; in a direction of one of two primitive vectors of a two-dimensional Bravais lattice of defined by the at least one row; or in a flow direction through the chamber, which is oriented at an angle between 1° and 15° with respect to one of two primitive vectors of a two-dimensional Bravais lattice of defined by the at least one row.
Furthermore, a microfluidic chip insert is provided for insertion in a microfluidic chip to form a chamber, the insert comprising at least one wall for the chamber, the wall defining at least one row of at least 3 pillars, where the pillars: are arrayed to form a polyline; have mean diameters (d) of 1-1000 μm; have mean separations of 0.2-500 μm; have aspect ratios greater than 2:1; and comprise a soft magnetic coating; and the polyline meets an edge of each pillar where the extent of the pillar is d or greater.
The polyline may meet the pillars at points where the extent of the pillar is strictly greater than d. The insert may comprise two ledges perpendicular to the wall defining sidewalls of the chamber, and a flow direction defined between the two sidewalls, wherein the wall includes at least 3 rows of the pillars that form a two-dimensional Bravais lattice, with one of the primitive vectors of the lattice oriented at an angle between 1° and 15° with respect to the flow direction.
Further features of the invention will be described or will become apparent in the course of the following detailed description.
In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which:
Herein a technique is described for distributing MNPs to form a cloud region between magnetic micropillars of a microfluidic chamber in a microfluidic chip. Applicant has demonstrated that the cloud distribution can be maintained while a sample fluid passes through the cloud. Advantageously, the magnetic micropillars can be coated with a thickness of magnetic material that permits low remanence magnetic actuation.
The shape of micropillar 10 and core 12 is an equilateral triangle, and an average circle Cavg of the micropillar 10 is shown having a diameter davg. For any shape, an average circumference can be calculated, for example by computing the radial coordinates of the perimeter about a centre of the micropillar 10, which will vary between two positive values minimum radius rm and maximum radius rm, and can be estimated by analysis of magnified images or by optical, mechanical, hydrostatic or hydrodynamic inspection.
The shape of the cross-section of the micropillar 10 can have substantially any form. The pillars do not absolutely have to have a constant cross-section shape, area or dimension as a function of height, and can generally taper slightly for easier demolding if that is the forming route for the micropillar cores 12. That said, a mass of the coating 14 at all elevations from a base of the micropillar 10 is uniform enough to provide continuity of the magnetic field. This mass is preferably distributed substantially completely around the core 12 (at least) at most elevations, to ensure good attachment of the coating 14. If electroless plating is used to deposit the coating 14, the mass deposited at every elevation is proportional to the perimeter at that elevation, and so a perimeter of the pillars may be reduced by less than 10% from base to highest elevation, even if tapered. If a higher taper is required, it can be achieved with a gradual transition of a core base having a more circular form and a top having shape with a higher perimeter to area ratio.
As shown in
A space between the pillars is bounded by a cross-hatched area on a wall 15 and the pillars 10.
The micropillars 10 have uniform height h greater than 2 μm, and less than 2 mm, and an aspect ratio (AR) given by h:davg of 2:1 or greater, more preferably 3:1, 5:1 or even 10:1. The aspect ratio is important for providing high throughput with low hydrodynamic resistance, and increasing a volume of the space between the pillars as a fraction of the volume of the chamber. The pillars preferably extend between two opposite walls of the chamber. The high aspect ratio improves a uniformity of the magnetic field gradients.
Note that microfluidic rheology also plays a part in the preferred layouts of these micropillars 10: as flow through these micropillars 10 will be laminar, a velocity gradient will naturally form between the micropillars 10 with slowest flow nearest the micropillars 10, and fastest midway therebetween. By selecting an arrangement and profile of the microfluidic pillars 10, this gradient can be minimized, to improve capture efficiency. Thus while a flat bottom surface 15 of a wall of a microfluidic chamber is shown in
It is noted that a shape and distribution of the micropillars 10 can be chosen to improve control over the nanoparticle distribution between neighboring pillars. In general, the shape and the orientation of the pillars is designed to create “anchor” points for the nanoparticle regions: i.e. spots where high magnetic capture forces (high gradient fields) are coincident with the stagnation points in the microfluidic flow. This allows the nanoparticle regions to extend from one anchor point to another anchor point with minimum depletion caused by the flow between the pillars.
While the elongated structures of
Approximations to any of these micropillar structures may be used, particularly those that are more easily patterned, more reliably patterned, or that exhibit least consequences to MNP distribution under applied field of imperfect forming.
It will be noted that while each of the arrays of micropillars shown consist only of one kind (shape, size, orientation), and further that the spacing and arrangements have been exemplified by only a few arrangements, as long as the micropillars are of a satisfactory size, shape, and separation, and have sufficient soft magnetic material, they will collectively define capture and depletion regions that cooperate with adjacent capture and depletion regions of neighbouring micropillars, to permit (with the application of a sufficiently strong magnetic field) the retention of MNPs in a cloud configuration, to resist a modest fluid throughput.
An electroless plating process may be applied to metallize the substrate 19 with the soft magnetic metal coatings of previously described composition. Other coating techniques that form consistent distributions of the soft magnetic material can alternatively be used, including bath/immersion or solvent based deposition techniques similar to electroless plating with controlled surface adhesion, and mechanical insertion of coated non-magnetic rods or threads through the substrate 19 by a template, die, or registered machine. The former technique may offer better anchoring of the micropillars, while the latter techniques may avoid metallization of floor of the insert 20, reserving the metal for where it is needed for gradient field generation. The floor acts like an in-plane magnetized thin film, with minimal effect on the magnetic field within chamber. Magnetically isolated pillars can be produced by electrodeposition through porous membranes followed by gently removing (dissolving) the membranes afterwards. Side walls of the insert for the chamber may be defined in the insert, if alignment and integration with fluid paths of the chamber of the chip can be arranged. An advantage of coating side walls adjacent to the rows is the formation of cloud regions between pillars and the walls, and thus extending all the way across the chamber. In this way a large number (31 shown in the present example, but any other number is possible) of inserts 20 can be formed. The metallized substrate is diced to produce the inserts.
A slot 29 is provided in the chip 22 for aligning a magnet with the inserts, such that magnetic field lines are substantially aligned with the rows of micropillars in each of the 4 magnetic chambers.
While irrelevant to the drawing, it will be appreciated that the chip 22 conventionally has a top cover bonded thereto, that would typically be transparent. If so the transparent cover has holes aligned with MNP loading inlets 26, or suitable puncture films, for loading via a syringe or dropper in one of the various ways known in the art. Likewise ports or air holes in fluid communication with the detection chambers 28 are open to ambience, or may be subjected to a negative pressure, in order to imbibe the MNPs in a fluid (typically liquid) carrier 30.
It will be appreciated that other routes for producing a magnetic chamber 21 in a microfluidic chip 22 with the requisite micropillars 10 that have soft magnetic coatings over non-magnetic cores, can alternatively be used, and so some aspects of the present invention begin with
The MNP fluid carrier 30 moves into the magnetic chamber 21, for example under the action of capillarity, centrifuge, or a pressure differential between the respective port and the loading inlet 26. A higher surface area of the magnetic chamber 21 as a result of the micropillars naturally improves the capillarity attraction of the MNP fluid 30, and preferably encourages a coverage of the micropillar array from edge to edge of the insert 20, which defines the boundaries of the chamber 21. Surface tension and the capillary effect may be sufficient to draw the carrier 30 over the micropillar array, for a suitable fluid, and otherwise vacuum pressure at ports of the chip may be required.
After loading, a magnetic field is applied with a permanent magnet 33 having one pole inserted within the registration slot 29, as shown in
As shown in
While there are several arrangements that may make favourable use of bump arrays of magnetic coated pillars in accordance with the present invention, particular attention is drawn to arrangements of the pillars that are parallel to the stream line, or parallel to the deviated bump path that is defined by the angle. An orientation of the magnetic field substantially perpendicular to both of these will have equal probability of capturing particles above and below the critical size. An orientation of the magnetic field substantially parallel to the stream line will increase probability of binding target particles following the bump path, and an orientation of the magnetic field in the direction of the bumped path will preferentially interact with zigzagging particles, such as biological cells. As other particles size selected to paths, and the paths are not equally encouraged to pass through the MNP-dense inter-pillar spaces, particles that are not encouraged to pass do not interact with the MNPs, which can be efficient for selecting MNP interactions.
An illustrative trace 31 of a single particle as it approaches, and passes between the micropillars 10 is shown. The particle typically interacts with the array by a weaving motion as it approaches the spaces between the micropillars 10 prior to and after passing through the space between two micropillars (assuming the row shift fraction is less than ½). The weaving motion is somewhat akin to motion of a bump array of a deterministic lateral displacement array. Not only does the particle have increased probability of capture by the MNPs during the crossing, but also before and after. The typical trace 31 brings the particle much closer to stagnation points near a periphery of the micropillars than a substantially normal flow through approach. Comparatively, the zig-zagging particles which alternate between stream lines, remain preferentially directed towards the stream direction furthest from the pillars. Furthermore, during the pass through the inter-pillar space, a probability that a particle will remain within a central part of the spacing between the micropillars, where a flow is fastest and a density of the MNPs is lowest, is much greater with the substantially normal flow through approach.
It should also be noted that the trace gives a false impression that the speed of the particle is uniform. The speed of the particle is mostly determined by the flow speed of the carrier liquid, which varies according to laminar flow lines. While inertia may cause some acceleration of the particle with respect to the fluid flow during a deceleration or acceleration of the liquid nearing the pillar array, the particle will dwell near the periphery of the micropillars, and thus the time and location of the particle throughout the trace 31 is expected to offer a much higher probability of interaction, resulting in a higher capture efficiency process.
A magnetic capture device and apparatus, and it's fabrication has been described25, the entire content of which is incorporated herein by reference, including the supplementary information material. The magnetic capture device was filled with a 500 ng/ml concentration dispersion of superparamagnetic iron-oxide core silica shell nanoparticles. For present purposes, the NPs used were equivalent to NPs available from a variety of commercial suppliers. The chamber was 30 mm×17 mm. A pair of permanent magnets was initially placed at the opposite edges of the device. The magnets were 1 cm×5 cm×10 cm at a distance of 5.8 cm from each other. They generated a substantially uniform magnetic field within the capture region. We expect the graphs showing the magnetic field along different directions (for example
The obtained magnetic cloud was subsequently used for capture and release of fluorescently labeled heat-killed bacteria. Initially, a pair of permanent magnets was positioned perpendicularly to the flow within the microfluidic chamber, and the chamber was filled by flowing, at a flow rate of 25 μl/min, 50 μl volume of 500 ng/ml concentration of a dispersion of anti-listeria antibody functionalized superparamagnetic iron-oxide core silica shell nanoparticles similar to those described previously25. This allowed formation of inter-pillar cloud regions throughout the microfluidic chamber. Subsequently, 1 ml volume of fluorescently labeled heat-killed Listeria monocytogenes, at a concentration of 10E4 bacteria/ml, was flowed through the microfluidic chamber at a flow rate of 100 μl/min. The flow was perpendicular to the inter-pillar regions. Following 10 minutes of the flow, the magnetic field was removed, and the captured species were released from the chamber with buffer wash for three minutes.
Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2019/056616 | 8/2/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62714391 | Aug 2018 | US |
