Processing of bound and unbound magnetic particles

Abstract

The invention relates to an apparatus (100) and a method for the processing of magnetic particles (MP) provided in a processing chamber (114) with a binding region (116) to which said magnetic particles (MP) can (specifically) bind. Removal of unbound magnetic particles (MP) from the binding region (116) is achieved by first separating them from the binding region (116) by gravitational forces and then moving them further away by magnetic forces. Gravitational forces can for example be generated by tilting the binding region with a tilting unit (156). The prior separation by gravitational forces prevents that unbound magnetic particles (MP) are captured in a cluster with bound magnetic particles.

Claims

1. A method for processing of magnetic particles (MP, MP), the method comprising: providing magnetic particles (MP, MP) in a processing chamber comprising a binding region; letting magnetic particles (MP) bind to the binding region; separating unbound magnetic particles (MP) from the binding region by gravitational forces (F.sub.g) comprising controllably changing an inclination of the binding region; and generating with a magnetic field generator a magnetic field (H) which moves said separated unbound magnetic particles (MP) further away from the binding region.

2. An apparatus for processing magnetic particles (MP, MP), the apparatus comprising: a processing chamber with a binding region to which magnetic particles (MP) can bind; a magnetic field generator for generating a magnetic field (H) in the processing chamber; and a tilting unit for controllably changing an inclination of the binding region, wherein a light detector is provided for detecting light (L2) coming from the binding region.

3. The apparatus according to claim 2, wherein the binding region is configured to assume a horizontal orientation below or above magnetic particles (MP, MP) present in the processing chamber, while magnetic particles (MP) bind to the binding region.

4. The apparatus according to claim 2, wherein the processing chamber with the binding region is located in an exchangeable cartridge.

5. The apparatus according to claim 2, wherein the binding region is configured to be tilted with respect to the magnetic field generator.

6. The method according to claim 1, wherein the gravitational forces (F.sub.g) act for a time that is sufficient to move unbound magnetic particles (MP) a distance () of more than about two times their diameter (d) away from magnetic particles (MP) bound to the binding region.

7. The method according to claim 1, wherein an intermediate magnetic field is generated prior to the separating of the unbound magnetic particles (MP) from the binding region.

8. The method according to claim 1, wherein the unbound magnetic particles (MP) can be moved to a location outside the binding region.

9. The method according to claim 1, wherein the magnetic field generator comprises a horse-shoe magnet having poles located at the binding region.

10. The apparatus according to claim 2, wherein the magnetic field generator comprises a horse-shoe magnet comprising poles located at the binding region, and at least one pole of the horse-shoe magnet is configured to be activated separately.

11. The apparatus according to claim 2, wherein the magnetic field generator comprises magnets disposed on opposite sides of the binding region.

12. The apparatus according to claim 2, wherein a light source is provided for illuminating the binding region.

13. The apparatus according to claim 2, further comprising: a control unit configured to coordinate the inclination of the binding region and the generating of the magnetic field (H).

14. The apparatus according to claim 2, wherein the apparatus is configured to perform molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and/or forensic analysis.

15. The apparatus according to claim 2, wherein a light source is provided for emitting an input light beam (L1) into the binding region, the input light beam being totally internally reflected at the binding region and leaving the binding region as the light (L2) detected by the light detector.

16. The apparatus according to claim 4, further comprising: an accommodation space configured to receive the exchangeable cartridge.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

(2) In the drawings:

(3) FIG. 1 schematically shows a side view of an apparatus according to the present invention comprising an FTIR detection at the binding region;

(4) FIG. 2 illustrates the effect of a magnetic field acting on bound and unbound magnetic particles at an overhead binding region when gravitational forces act only for a short time;

(5) FIG. 3 shows the situation of FIG. 2 when gravitational forces act for a longer time;

(6) FIG. 4 illustrates the effect of gravitational forces on magnetic particles at an inclined binding region;

(7) FIG. 5 schematically shows a side view of an apparatus according to the present invention comprising a bright-field illumination of the binding region;

(8) FIG. 6 illustrates the apparatus of FIG. 1 when the magnetic field is generated by the top magnet only;

(9) FIG. 7 illustrates the apparatus of FIG. 1 when the magnetic field is generated by the top magnet and one pole of the bottom horse-shoe magnet;

(10) FIG. 8 illustrates the apparatus of FIG. 1 when the magnetic field is generated by one pole of the bottom horse-shoe magnet only.

(11) Like reference numbers or numbers differing by integer multiples of 100 refer in the Figures to identical or similar components.

DETAILED DESCRIPTION OF EMBODIMENTS

(12) Platforms for the specific detection of marker molecules in bodily fluids are for example provided by the Magnotech technology developed by the applicant. An example of a marker molecule is troponin-I (cTnI) for the detection of cardio-vascular disease. The detection technique is based on immuno-assays in combination with the optical detection of superparamagnetic nanoparticles (beads) on the surface of a cartridge. Many platforms use Total Internal Reflection (TIR) illumination by creating an evanescent optical field near the surface. This technique is surface sensitive and in principle free of interference from the nanoparticles in the bulk.

(13) FIG. 1 schematically shows a section through a first embodiment of a (sensor) apparatus 100 that applies the aforementioned technology and is further designed according to the present invention. The apparatus 100 comprises (a) a reader 150 with an accommodation space for an exchangeable cartridge 110 and (b) said cartridge 110.

(14) The apparatus 100 is used for the detection of target components comprised in a sample fluid (e.g. blood) that fills a processing chamber 114 of the cartridge. The cartridge 110 is composed of a transparent base part 111 which borders the processing chamber 114 at its bottom side and which provides a processing surface 115. The side walls of the processing chamber 114 are constituted by an intermediate layer 112, for example a tape into which openings for the processing chamber and associated fluidic channels (not shown) have been cut. The processing chamber 114 is covered at its top side by a (e.g. plastic) cover 113.

(15) At least one binding region 116 is located on the processing surface 115. It comprises capture probes, for example antibodies, to which certain substances can specifically bind. These substances may particularly be magnetic particles MP with probes (antibodies) on their surface that have (specifically) captured target components of interest from the sample medium in the processing chamber 114.

(16) FIG. 1 further shows a magnetic field generator, here comprising a horse-shoe magnet 153 (with two poles 153a, 153b) below the binding region 116 and a top magnet 154 above it. The poles of these magnets may individually be controlled by a control unit 155 of the reader 150 for generating a magnetic field B in the processing chamber 114 by which the magnetic particles MP can be manipulated.

(17) FIG. 1 further indicates a light source 151 for emitting an input light beam L1 into the cartridge 110. This input light beam is totally internally reflected at the binding region 116 and then leaves the cartridge 110 as an output light beam L2 towards a light detector 152. These light beams can be used to detect target components of the sample fluid that are specifically bound to magnetic particles MP and the capture probes of the binding region 116. Further details of this assay and the optical detection of target components by frustrated total internal reflection (FTIR) may be found for example in the WO 2008/115723 A1, which is incorporated into the present text by reference.

(18) In order to create a fast reaction, the amount of probes (antibodies) to capture the target molecules (e.g. a cardiac marker) needs to be high and as a consequence the amount of magnetic particles in the bulk needs to be high. This causes several problems:

(19) (a) Due to incorrect alignment of the cartridge and scattering at impurities in the cartridge material itself (e.g. tiny air bubbles, scratches), a fraction of the incoming light may be scattered into the volume of the cartridge instead of being confined near the surface. This causes bulk illumination. Because of scattering at the large amount of magnetic particles in the bulk (the bead cloud), also light from the bulk is therefore detected. This raises the background level and decreases the contrast of the image. Due to the non-uniform distribution of the bead cloud, the background will also be non-uniform.

(20) (b) The evanescent illumination field decays exponentially with the distance to the surface. Typically the decay length is of the order of 100 nm. The exponential decay causes the intensity of the magnetic particles on the surface to be very sensitive to the height above the surface. Although the height-dependent intensity can give useful information (e.g. about the bond length), it also causes that only a fraction of the magnetic particles on the surface can be detected. By using bright-field illumination from the top of the cartridge as an additional means instead of TIR illumination, the height dependence can be eliminated. However due to the large amount of free nanoparticles, the bright-field illumination is hampered.

(21) (c) During the magnetic washing (i.e. a magnetic field removes unbound particles from the surface), vertical clusters of magnetic particles are attached to bound magnetic particles. Information about the length of these vertical clusters can improve the performance of the detection. However due to the short decay length of the evanescent field, the size of these vertical clusters is not visible. By using bright field illumination (e.g. in combination with a varying focus depth of the objective lens), an estimate of the vertical cluster size can be obtained. However due to the large amount of free magnetic particles, the bright-field illumination is hampered.

(22) In order to address the above problems, it is proposed to remove the unbound beads (magnetic particles) after or possibly during the reaction from the processing chamber. Due to the nature of the superparamagnetic beads they will form chains when an external magnetic field is applied. This can hamper the removal of all the unbound magnetic beads from the field of view because a fraction of them will be magnetically coupled to the bound beads on the binding region. One solution is to still remove the large excess of beads leaving only a small part still bound to the surface bound beads. When applying a magnetic field perpendicular to the surface the beads will form chains in the same orientation allowing an easy detection.

(23) The cartridge also can be tilted or even turned upside down to allow sedimentation of the unbound beads from the functionalized surface. When thereafter the magnetic fields are applied the unbound beads will form only chains with other unbound beads, thus removing all the unbound beads from the beads bound to the surface. This will be explained in more detail in the following.

(24) Experiments have shown that the binding process in a setup which has been put upside down can be done equally well when the actuation protocol is slightly adapted. The advantage of using the setup upside down is that the gravitational force can improve the washing process.

(25) This is illustrated in FIG. 2a), which sketches the situation directly after the binding process. Bound magnetic particles MP and free magnetic particles MP are present in close proximity because the binding process uses a magnetic force to keep all the particles near the binding region 116.

(26) In FIG. 2b), a magnetic washing field H is switched on to move the free particles away from the surface. However, actually magnetic clustering occurs: due to the magnetic interactions between the magnetic particles, a large number of free beads will couple magnetically to the particles bound to the surface. Therefore these free beads cannot be removed. The presence of these free beads near the bound beads distorts the (optical) signal of the bound beads (e.g. the intensity of the bound bead is altered).

(27) FIG. 3 illustrates how this problem can be solved by shortly using a gravitational force. FIG. 3a) corresponds to the starting situation of FIG. 2a).

(28) According to FIG. 3b), gravitational force F.sub.g is used directly after the binding process to move the free magnetic particles MP away from the bound magnetic particles MP of the binding region 116 without having an interaction force between them.

(29) Once the free magnetic particles have moved some distance from the bound particles (typically a distance of 5-10 m is sufficient), the magnetic washing force F.sub.m can be used to move the free beads further away from the surface. This is shown in FIG. 3c).

(30) Because the distance between the bound beads and the free beads is large enough, the magnetic interaction becomes negligible (the magnetic interaction force F.sub.m decreases with the 4th power of the distance between the particles). The surface contains only bound particles without free particles attached to them. The (optical) signal is then a better representation for the number of bound beads on the surface.

(31) Typically one needs approximately a distance between the bound and the free beads of approximately 5 times the bead diameter d, i.e. 5 d. Then the magnetic attraction force between the beads is low enough to prevent magnetic coupling.

(32) Some numerical examples are as follows:

(33) For beads with a diameter of d=500 nm, sedimentation velocity is about 100 nm/s. The distance to travel is then 5.500 nm=2500 nm. The time the gravitational force has to work is therefore 2500 nm/(100 nm/s)=25 s.

(34) For d=1000 nm beads, a velocity of 400 nm/s, the distance to travel is =5000 nm, with a required time of 5000/400 s=12.5 s.

(35) So the time frame depends on the bead diameter and of course the density of the bead (in the above calculation a typical density of 1.8 g/cm.sup.3 was used).

(36) When using gravity to remove unbound magnetic beads from the binding region, an improvement may be achieved by first switching on an intermediate magnetic field shortly. This will form chains of magnetic beads, particularly of unbound beads to bound beads. Then the gravitational force is allowed to do its work. Due to the (vertical) alignment of the chains and the hydrodynamic coupling between them, they will fall faster than isolated magnetic beads. Finally the magnetic field is switched on again to attract the beads away from the binding region.

(37) With the setup in the normal position, i.e. the processing surface 115 and the binding region 116 being oriented horizontally below the processing chamber 114, the gravitational force can also be used to remove free, unbound magnetic particles from the printed spot (binding region 116) by means of a sideways translation. This is illustrated in FIG. 4. By tilting the surface 115, a component of the gravitational force F.sub.g directed along the surface will become available. This force component will move free particles MP away from the bound particles MP in the spot. Similar to FIG. 2 a magnetic force cannot achieve this effect because the magnetic interactions between bound and free particles which will keep the free beads near the bound beads in the spot. This effect is more pronounced when the number of bound beads in the spot is larger.

(38) In the apparatus 100 of FIG. 1, the above principles can be realized with the help of a tilting unit 156 that is indicated in the Figure as one foot of the apparatus 100 which can be varied in height, controlled by the control unit 155. Accordingly, the whole apparatus 100 with the binding region 115 can be tilted with respect to gravity (z-direction).

(39) FIG. 5 schematically shows a side view of a sensor apparatus 200 according to a second embodiment of the present invention. The sensor apparatus 200 comprises a reader 250 and an exchangeable cartridge 210. The cartridge 210 has a processing chamber 214, filled with a fluid comprising magnetic particles MP, and a binding region 216. Moreover, a tilting unit 256 is symbolically indicated with which the cartridge 210 can controllably be tilted with respect to the reader 250.

(40) The reader 250 comprises a bottom magnet 253 and a top magnet 254 (washing magnet), which now both are horse-shoe magnets. This configuration offers the opportunity for a light source 251 directly above the binding region 216 while moving the magnetic beads MP away from the light path. Thus a bright field illumination of the binding region 216 is achieved which can be used with a high NA objective lens 252 directly below the binding zone (cf. WO 2011/036634A1).

(41) In summary, the above approach is characterized by the following features:

(42) 1. Having the binding region with the antibodies in an orientation such that gravity directs particles substantially away from the region.

(43) 2. Applying gravity (no magnetic force) for a sufficiently long time for the magnetic particles to travel more than several bead diameters d from the region.

(44) 3. Applying a magnetic force directing the particles substantially away from the detection area (defined as the area where the particles still interfere with the detected signal, e.g. by scattering).

(45) Removal of unbound magnetic particles can additionally or alternatively be done by the available electromagnets, using the top magnet 154 (washing magnet) in combination with one of the pole tips 153a, 153b of the bottom magnet (horse-shoe magnet 153) to create an off-center magnetic field direction pulling the beads from the processing chamber 114. It is also possible to bring the beads back to the reaction area by either using only the top magnet or by the combination of both the bottom and top magnet.

(46) Accordingly, the present invention relates to a method to remove unbound beads from a processing chamber having one or more of the following features: Unbound beads are removed from a processing chamber by means of magnetic forces. Unbound beads are removed from a processing chamber by creating a magnetic field trap that is outside the field of view by using one of the pole tips of the horse-shoe magnet. The contrast of the image is enhanced by removing unbound beads. A bright field illumination is enabled by removing unbound beads. A bright field illumination is enabled by partially removing unbound beads, i.e. by creating chains of the (limited) amount of unbound beads linked to the bound beads on the surface. The (partial) removal of unbound beads is enabled by using gravitation force (e.g. in a configuration where top and bottom magnet are reversed).

(47) FIGS. 6-8 illustrate a preferred procedure of generating and applying magnetic fields in the processing chamber 114 to achieve some of the above objectives. This procedure starts after the usual magnetic actuation (designed to have the optimal specific binding of superparamagnetic beads to the surface) and an optional intermediate step of particles separation by gravitational forces.

(48) According to FIG. 6, the unbound beads are pulled away from the field of view by first pulling them to the top of the processing chamber 114 by the top (wash) magnet 154, which removes them from the processing surface but not from the bulk above the surface.

(49) According to FIG. 7, the beads can then be moved to the side of the processing chamber by using both the top magnet 154 and one of the pole tips 153b of the horse-shoe magnet. In addition the use of the top magnet may force the small amount of free beads to form chains on top of the already bound beads making them invisible for detection.

(50) According to FIG. 8, the beads can then be pulled to the side of the chamber, by using only one of the pole tips 153b of the horse-shoe magnet. Only due to the creation of magnetic fields that are almost parallel to the surface the unbound beads will form chains with the bound beads that are also in parallel to the surface hampering the detection.

(51) Optionally, one can alternate between one of the pole tips of the horse-shoe magnet 153 and the top magnet 154 to ensure that the beads are removed from the field of view.

(52) In summary, the invention relates to an apparatus and a method for the processing of magnetic particles provided in a processing chamber with a binding region to which said magnetic particles can (specifically) bind. Removal of unbound magnetic particles from the binding region is achieved by first separating them from the binding region by gravitational forces and then moving them further away by magnetic forces. Gravitational forces can for example be generated by tilting the binding region with a tilting unit.

(53) The basic idea behind using the gravitational force is to prevent the magnetic attachment of free beads to the bound beads on the surface. Generally when the washing magnet is switched on, bound and free beads are magnetized and the free beads will magnetically couple to the bound beads. The gravitational force allows the free beads to move away from the bound beads and thus prevents that unbound magnetic particles are captured in a cluster with bound magnetic particles.

(54) While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word comprising does not exclude other elements or steps, and the indefinite article a or an does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.