The invention relates to apparatus for retaining magnetic particles in self-assembled magnetic particle structures in a liquid flow, and the use of such apparatus in particular in life science, chemistry and microfiltration applications.
The invention concerns in particular an apparatus and a method wherein the magnetic particles are used for selectively capturing target molecules or target particles suspended in and carried by a fluid flowing through a flow-through cell, as is done for instance in clinical chemistry assays for medical diagnostic purposes.
It is known that magnetic particles (‘beads’) embedded in a liquid can be used to carry a probe molecule on their surface that specifically interacts with a complementary target molecule (for example single stranded probe DNA interacting with complementary target DNA). Upon reaction with a molecule to be probed and, for example, using optical or electrochemical measurements, one can determine the amount of target molecules on a bead or within a certain volume containing beads (see for example Hsueh et al., Techn. Digest Transducers '97, p. 175 (1997)). The very interesting point of using magnetic microbeads, is that they can be manipulated using magnetic fields irrespective of fluid motion. In this way one can create an important relative motion of the beads with respect to the fluid and, hence, a large probability of binding a target molecule to a probe molecule fixed on the bead surface. One can then magnetically extract the beads to a place of detection/collection. Historically, beads have been locally fixed by using external magnets or have been transported using mechanically moving external magnets. The latter procedure may be for example used to fabricate mixing devices (Sugarman et al., U.S. Pat. No. 5,222,808 (1992)) and in immuno-assay methods (Kamada et al., U.S. Pat. No. 4,916,081 (1987)).
An elegant way to keep magnetic particles in a fluidic channel was based on an electromagnet consisting of a coil and at least one pair of poles of a magnetic material. Such poles form an inhomogeneous field transverse to the channel which effectively ‘traps’ the particles in regions where the field is strongest (Elsenhans et al., EP 1,331,035-A1). In the latter publication, the prior art of magnetic particle retention devices in microfluidic channels is well described. EP 1,331,035-A1 describes a flexible way of handling magnetic particles, though this requires a complex magnetic apparatus and electrical current manipulation. Specially corrugated pole tips need to be realised to generate the locally inhomogeneous magnetic field.
In another reference, Yellen and Friedman (J. Appl. Phys. 93, 8447 (2003)) describe the use of micropatterned holes in a photoresist layer with at the bottom of the hole, a ferromagnetic thin film. When placed in a magnetic field, the magnetic film focuses the field and a magnetic chain is formed when a magnetic bead-containing solution is placed above the substrate. The idea was to form arrays of bead chains at regular positions of the substrate.
In another study (Forbes et al., IEEE Trans. Magn. 39, 3372 (2003)), these authors propose such ‘magnetic trapping’ by embedding magnetic particles in the wall of a soft polymer microfluidic channel for anchoring magnetic chains over the cross-section of a microchannel in order to resist to a fluidic flow.
Doyle et al. (Science 295, 2237, 2002) and Minc et al. (Anal. Chem. 76, 3770 (2004) have proposed to use magnetic columns, as clustered by a constant magnetic field, for the separation of long DNA molecules. This was the first report of such columns as stationary phases in a chromatography application. Resistance to a fluid flow is zero as a microchannel with homogeneous cross-section is used.
Magnetic particle columns have thus been used in microfluidic channels, but in flow-through channels of homogeneous cross-section, so that the resistance to a fluid flow is minimum.
The invention relates to an apparatus for retaining magnetic particles in self-assembled magnetic particle structures in a liquid flow, of the type comprising a flow-through cell or channel in which magnetic particles are suspendable in a liquid that is flowable through the cell or channel, and means for generating a substantially static magnetic field across the cell or channel such that when magnetic particles are suspended in a liquid in the cell or channel and the magnetic field is applied the particles form magnetic particle structures that are sustained by magnetic forces acting on the particles.
According to the invention, the flow-though cell or channel has along its length transverse large sections alternating with narrow sections. The large sections are periodically distributed along and on either side of the narrow sections, arranged such that in use magnetic particle structures form across the large sections of the cell or channel. In use, the liquid is flowable along the cell or channel through the narrow sections and through corresponding middle parts of the magnetic particle structures in the large sections, the magnetic particle structures being retained by engagement of end parts of the magnetic particle structures in the large sections of the cell or channel.
The alternating large and narrow sections in the flow-though cell or channel permit the formation of chain-like magnetic particle structures using simple magnetic apparatus and good retention of these chain-like magnetic particle structures in the flow-though cell or channel without a need for additional retaining means.
The invention also relates to a corresponding method for retaining magnetic particles in self-assembled magnetic particle structures in a liquid flow, in particular comprising flowing through the cell or channel a fluid carrying molecules or particles to be captured, filtered or activated by the magnetic structures, as well as uses of the apparatus and further features of the apparatus, as set out in the claims and the following description.
The invention will be further described by way of example with reference to the accompanying drawings, in which:
a is a schematic plan view of an apparatus according to the invention;
b shows a detail of
c is a schematic end view of an apparatus according to the invention, looking along the x direction of
d shows a detail of
a is a schematic diagram showing magnetic beads in a microfluidic channel structure of varying width, with no liquid flow and no applied magnetic field;
b is a corresponding diagram still with no liquid flow, but with an applied magnetic field;
c is a corresponding diagram with liquid flow and with an applied magnetic field;
a and 4b are diagrams of two interacting magnetic chains;
a, 5b and 5c show different ways of parallelising the implementation of the invention in microfluidic structures, and
a and 6b show a lay-out and a photograph of an experimental version of the invention.
The magnetic field H is typically comprised between 0.01 Tesla and 1 Tesla.
The micro-channel 5 is contained in a microchip 4 that can be loosely placed (or fixed) in a recess 3 between the magnets 1,2. The microchip 4 is made of a plate of plastics material, or any other suitable non-magnetic material that has no magnetic shielding effect on the magnetic field, this plate having therein a central longitudinal channel 5 that has inlets and outlets 6,7 for connection to an external supply of liquid containing magnetic particles and other components, depending on the end use. The flow of liquid in the micro-channel 5 can be produced by a hydrodynamic or electrokinetic (electrophoretic and electro-osmotic) pumping mechanism, not shown.
The apparatus is typically used with magnetic particles 12 in the range of the nanometer to a few micrometers. The size and the nature of the magnetic particles or beads 12 can of course vary for different applications. Typically, the chains 15 of magnetic particles are used to selectively capture target molecules, filter the liquid flowing in the micro-channel or catalyze chemical reactions at the surface of the magnetic chains.
As shown in
In
Of course, the dimensions of the large and narrow sections 8,9 of the channel 5 can be changed as well as their periodicity. Typically, the transverse width of the narrow sections 9 is between 1 μm and 100 μm, and the transverse width of the large sections 8 is between 1 μm and 10 μm.
The large section 8 of the micro-channel 5, when viewed from the top, can be rectangular, triangular or round tapered shapes.
The invention implements a very simple solution for forming and retaining the magnetic particle structures 15, by providing a microfluidic channel 5 with varying cross section perpendicular to the flow.
If desired or necessary, the micro-channel 5 can comprise sub-structures along the channel axis for enhanced retention of the magnetic chains 15, for example, micro-pillars (like the pillars shown in
The chains 15 of magnetic particles are formed at the position of the largest section b, for two reasons. First of all, a longer chain is characterised by a smaller magnetic demagnetisation factor in the direction of the field H and hence forms a magnetic object with lower magnetostatic energy than a shorter chain. This explains the situation of
where m is the dipole moment of a magnetic bead 12 induced by the field, μ0 is the magnetic permeability of the vacuum, r the distance between the particle centres and θ the angle between the applied magnetic field and the line joining the beads centres.
When assuming the distance between the particles fixed (spherical particles), the tangential contribution to the total retained force is defined as:
which give a maximum tangential force for θ=45°. Suppose that there is not a single magnetic chain 15 formed within the cavity of dimension b, but a larger magnetic structure 15 containing N coupled uni-dimensional magnetic chains, this force can be multiplied by N and doubled as the chain 15 is held from the two sides. Therefore the total tangential magnetic force can be approximated by:
F
θtot
=N·2Fθ Equation 3
When a chain 15 is formed, and exposed to a viscous drag force (which can be generated by a pressure, capillary or electroosmotic-based force), one can estimate the dragging force, by assuming the chain 15 as a cylinder:
where α is the cylinder length and β its radius, η the dynamic viscosity of the fluid and νflow the flow velocity.
A chain 15 will resist to the flow without rupture, as long as the tangential force is higher than the dragging force, i.e. Fθtot>Fνisq,cylinder for a stable chain. For example, with 1.5 μm diameter beads, a channel dimension of a=50 μm and b=70 μm, N=4, the Fθtot calculated is 1.66×10−10 N in a field of 0.12 T. In these conditions, a 50 μm length and 5 μm diameter cylinder formed by the magnetic beads will resist up to a 1 mm/s flow velocity.
Following the same argument, an array of cavities along the channel 5 will lead to the formation of a long-scale periodic structure 15 (made by many cylinders formed by the beads) inside the channel (
Furthermore, the magnetic retention force within a channel 9 can be increased by adding pillar-like structures or posts 19 in the channel (
A classical electrophoresis microchip with structured microchannels, flanked with two permanent magnets can be used for realisation of this device.
This invention can be used in various fluidic microsystems. Magnetic chains 15 by virtue of their high interaction with the liquid can capture or retain molecules, cells or particles carried by the liquid. The magnetic beads 12 can be coated with specific chemical groups, and have the role of probe for specific molecules.
By choosing proper chemical groups (for example a well know single strand of a DNA or RNA molecule), one can capture the complementary strand. Therefore, due to the high specific interactions between the probe molecule on the bead and the target molecule carried by the flow, specific DNA or RNA molecules can be isolated from a complex mixture of molecules (a cell lyses solution for example). In a similar way, the magnetic chains 15 will capture target molecules from solution containing these molecules even in very low concentrations. One can later recover and concentrate the target molecules from the beads in a smaller volume. The molecules can then be used for analysis or PCR, for example.
Another promising application of the invention can be magnetic bead bio-sampling like use in a sandwich immunoassay. In this case, magnetic beads coated with specific antibodies would be used. A solution containing antigens will flow through the beads, and only the target antigens will be immobilized on the surface of the coated beads, while other antigen and undesired molecules will be washed out. The captured antigens can be released (and concentrated in a same way as above for the DNA or RNA), or labelled secondary antibodies can be flown over the beads and incubated with the immobilised antigens. Then, detection can be performed on-chip before washing the beads and starting another assay. The invention can be also used with any analytical procedure that requires interaction between antibody-coated beads and antigens. On-chip protein digestion can also be performed.
Another interesting field of use of the invention can be in a microreactor. It is well known that a large amount of chemical reactions can take place in a microchip with a catalyst (atoms or small molecules) fixed on the walls of the microchip. The molecules used for catalysing the chemical reactions can be immobilised on magnetic beads. Due to the high interactions between the reagents and the catalyst on the bead (which leads to a complete reaction), its flexibility and the very small amount of reagents used (typically few hundreds of nanoliters to few tens of microliters), the invention can have an important impact in pharmacology research and development.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP2006/050459 | 1/26/2006 | WO | 00 | 6/7/2010 |