Manufacturing of a biosensor cartridge

Abstract

The invention relates to a processing device (110) and a method for manufacturing such a device. In a preferred embodiment, a mixture of magnetic particles (MP), a matrix material, and a volatile carrier is deposited onto binding sites (112) of a reaction surface (113). The deposited mixture is then dried while the magnetic particles (MP) are pulled away from the reaction surface (113) by a magnetic field (B). Thus unspecific binding of magnetic particles to the binding sites can be prevented.

Claims

1. A method for manufacturing a processing device comprising magnetic particles, said method comprising the following steps: a) depositing a liquid mixture comprising magnetic particles, a matrix material, and a volatile carrier onto a surface of the device; b) pulling the magnetic particles of the mixture away from said surface to a desired location within the mixture; and c) drying the mixture by removing the volatile carrier.

2. The method according to claim 1, wherein the magnetic particles of the mixture are pulled by a magnetic field in step b).

3. The method according to claim 2, wherein a non-zero gradient of the magnetic field is oriented substantially perpendicular to said surface.

4. The method according to claim 1, wherein said surface is a reaction surface which is at least locally coated with binding sites that can specifically bind target substances.

5. The method according to claim 1, wherein said surface is treated with a blocking substance that binds to binding points before the depositing step a).

6. The method according to claim 1, wherein the volatile carrier comprises an aqueous liquid.

7. The method according to claim 1, wherein the magnetic matrix material comprises at least one material selected from the group consisting of sucrose, salt, buffer components, blocking components and assay reagents.

8. The method according to claim 1, wherein that the matrix material is water soluble.

9. The method according to claim 1, wherein that the magnetic particles are disposed upstream of a target location with respect to the intended flow of a fluid in the processing device.

10. The method according to claim 9, wherein that the target location comprises binding sites that can specifically bind target substances.

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 illustrates the deposition of a droplet comprising magnetic particles on a reaction surface and the pulling of said magnetic particles away from the surface by a magnetic field;

(4) FIG. 2 schematically illustrates a cartridge obtained after completion of the procedure shown in FIG. 1;

(5) FIG. 3 shows photos of magnetic beads bound to reaction surfaces in tests using different manufacturing methods;

(6) FIG. 4 is a diagram representing the counts (vertical axis) of bound magnetic particles in the tests of FIG. 3.

(7) FIG. 5 is a picture taken from a bright field microscope, showing the dried spot with chains of magnetic particles after having used the present invention.

(8) FIG. 6 is a picture taken from a bright field microscope, showing the dried spot without any chain of magnetic particles without having used the present invention.

(9) Like reference numbers refer in the Figures to identical or similar components.

DETAILED DESCRIPTION OF EMBODIMENTS

(10) The specific detection of marker molecules in bodily fluids may for example be achieved in biosensor platforms such as the Magnotech technology or a Single Bead Detection 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 magnetic particles (e.g. super-paramagnetic nanoparticles or beads) on a reaction surface of a cartridge. The mentioned platforms use Total Internal Reflection (TIR) illumination by creating an evanescent optical field near the surface.

(11) In a typical embodiment of the aforementioned technologies, the cartridge may consist of a base-part (consisting of fluidic structure, reaction chambers, reactive spots and optical detection), laminate (for closing the cartridge and placing the magnetic beads in the reaction chamber), and a blood housing (for filtering the cell fraction from the plasma fraction in a blood sample). The magnetic beads are disposed on top of the laminate, which is placed on top of the cartridge in such a way that the beads are inside the reaction chamber. To reduce the cost of the cartridge and to simplify the production process, the beads can alternatively be placed on top of a reactive spot that is present on the base-part in the reaction chamber.

(12) In the WO 2009/024922 A1 (which is incorporated into the present application by reference), a method is described for storing magnetic beads close to a reactive site using a magnetic field to make sure the beads are close to the sensor surface for fast reaction and interaction. In some applications, it is observed however that there is a (typically non-specific) interaction of the magnetic beads with the base-part during the drying process. The non-specifically bound magnetic particles may later reduce the level of detection (LoD) and the dynamic range.

(13) In order to address the aforementioned issues, an embodiment of the present invention proposes pulling magnetic beads away from the reaction surface, for example by applying a magnetic field on a droplet comprising magnetic beads during drying or by sedimentation (i.e. gravity). This will position the magnetic beads to the top of the droplet preventing interaction with the reaction surface. A glassy state of the dried bead droplet may prevent the magnetic beads from interacting with the reaction surface after the magnetic field has been removed. Moreover, the configuration of the magnet usually creates chains of magnetic beads substantially parallel to the reaction surface which are pulled upward away from the surface (see FIG. 5 in comparison with FIG. 6). Said chains can be detected after drying, e.g. in bright field microscope images, and allow for a distinction of the resulting dried body from a body obtained by ordinary procedures.

(14) FIG. 1 schematically illustrates a section through the base-part 111 of a cartridge 110 according to an embodiment of the aforementioned concepts. The base-part 111 has a reaction surface 113 that extends in x,y-direction and carries a detection spot with binding sites 112 for target molecules (usually the reaction surface features a plurality of such detection spots with binding sites for the same or for different target molecules). Moreover, a droplet D of a mixture comprising magnetic particles MP, a matrix material, and a volatile carrier (e.g. water) has been deposited on the binding sites 112.

(15) The Figure further illustrates a magnetic field generator, here a permanent magnet 120, that is arranged (in z-direction) above the droplet D with its axis extending in x-direction parallel to the reaction surface 113. The distance of the magnet 120 from the reaction surface 113 is adjusted such that a magnetic field B is generated within the droplet D that is substantially parallel to the reaction surface and that has a field gradient substantially perpendicular to said surface (i.e. pointing in z-direction). Due to said field gradient, the magnetic particles MP are attracted towards the magnet 120, i.e. pulled away from the reaction surface 113, and collect at the top surface of the droplet D. Thus a contact between the magnetic particles MP and the binding sites 112and hence an undesired binding of the magnetic particles MP to the binding sites 112is prevented.

(16) In typical embodiments of a cartridge, the distance between the magnet 120 and the magnetic beads MP is smaller than about 10 mm to properly pull the magnetic beads to one side of the droplet. The distance should however not be too small (e.g. <1 mm) in order to prevent magnetic bead migration outside the droplet.

(17) A magnetic field perpendicular to the reaction surface (i.e. running in z-direction) could also be used. However, the resulting chains of magnetic particles will then usually occupy a larger depth within the droplet perpendicular to the reaction surface (i.e. in z-direction). When applying a magnetic field that is parallel to the reaction surface (as shown in FIG. 1), the magnetic bead chains will occupy a smaller depth, which is preferred in a droplet with limited thickness perpendicular to the reaction surface (the diameter of the droplet D typically ranges between about 50 m and about 100 m).

(18) When the configuration of FIG. 1 is maintained for some time, the volatile carrier of the droplet D will eventually evaporate. This process can optionally be supported by a (moderate) increase of temperature of the droplet D and/or a ventilation of dry air along the droplet D.

(19) Optionally, an additional blocking step may be applied to further decrease the chance that a magnetic bead will bind non-specifically to the reaction surface. The blocking step may particularly comprise the addition of an inert protein (e.g BSA, Caseine) which (reversibly) binds to (unspecific) binding points both inside and outside the binding spots. Because the area inside the binding spots may contain proteins (e.g. antibodies as the binding sites 112), this is essentially already blocked.

(20) FIG. 2 illustrates the situation after a complete removal of the volatile carrier. A solid mass has formed that comprises a body of the matrix material MX immediately above the reaction sites 112 and a layer of magnetic particles MP within a top part of said matrix material. Thus the spatial separation between magnetic particles MP and the binding sites 112 has been permanently fixed. Moreover, the typically glassy body of the matrix material MX prevents degradation of the binding sites (e.g. proteins, antibodies). A laminate or cover 114 is finally attached to the base-part 111 in order to close the reaction chamber(s), channels etc. of the cartridge 110 (these details are not shown in the Figures).

(21) In an alternative embodiment, the laminate or cover 114 is applied onto the base-part 111 before the drying and before or during or after the application of said magnetic field. In that case, the cover 114 may be further arranged to keep or attach the magnetic particles MP onto its surface, optionally through the application of an appropriate magnetic field attracting the magnetic particles MP towards the surface of the cover 114 (and away from the surface of the base-part 111). In this embodiment, the magnetic particles may be applied in the liquid mixture or droplet D on the surface of the cover 114 (instead of on the base-part 111). Thereafter, one may assembly the cover 114 with the base-part 111, apply a magnetic field, during or just after this assembly, to pull the magnetic particles MP of the mixture D to a desired location of the mixture D (e.g. towards the surface of the cover 114), and finally dry. In an alternative, the droplet D can be inserted between the cover 114 and the base-part 111 already assembled one to the other, and the invention is implemented thereafter.

(22) When the cartridge 110 is used, a liquid sample (not shown) will be filled in the reaction chamber, dissolving the magnetic particles MP and the matrix material MX above the binding sites 112. The detection of target components in the sample (or whatever assay is intended) can then proceed.

(23) During usage of the cartridge 110 the problem may arise that if the filling of the cartridge is slow the magnetic beads MP are pulled to the outlet of the reaction chamber, i.e. away from the binding sites 112. Instead of depositing the droplet with the matrix material MX with the magnetic beads MP above the binding sites 112, these components may therefore optionally be disposed at a location upstream of the binding sites 112 to compensate for the displacement during filling. Additionally or alternatively, the positioning of the droplet may substantially be left as it is, but the distribution of the magnetic beads in the droplet is changed such that the beads concentrate in a sub-region located upstream. This can be achieved in a modification of the arrangement of FIG. 1 by pulling the beads in the bead droplet by a magnetic field towards the inlet of the reaction chamber (which may e.g. be at the left side of the processing device in FIGS. 1, 2) during the drying process. During the filling of the cartridge, the beads are then transported towards the outlet again, however they now end up above the binding sites 112.

(24) The aforementioned approach can also be used when magnetic beads are applied to the cover 114 (top of the chamber), where the bead re-dispersion is also important. A droplet of matrix material, volatile carrier, and magnetic beads may hence also be deposited on the cover 114 and dried while a magnetic field pulls the magnetic particles to an upstream position within the droplet.

(25) In a concrete embodiment of the described procedures, magnetic beads MP have been dosed in a quantity of about 50 nl on the base-part 111 on top of an antibody spot 112. The magnetic beads were comprised in a solution containing buffering components, salts and sucrose, among other things. After dosing the magnetic beads were dried for about 30 min at about 37 C. During this drying process an external magnetic field was applied on a distance of about 5 mm with a magnet that creates a field that is parallel to the reaction surface. After the drying phase the magnetic field was removed and the cartridge was processed using the normal procedure.

(26) The reduction of non-specific binding was measured for different preparation procedures using a single bead detection technology. This technology provides a surface specific image showing the amount of magnetic beads bound to the surface. The amount of magnetic beads can directly be translated to a FTIR signal change.

(27) FIG. 3 shows four images of reaction surfaces obtained by single magnetic bead imaging during a washing phase of a blank sample (plasma pool containing no target analyte). Each dark spot in the images corresponds to a magnetic bead bound to the reaction surface. The preparation conditions for the four images were as follows: Image A (top left) shows a reaction surface obtained without any blocking treatment and without application of a magnetic field during drying. Image B (top right) shows a reaction surface obtained without any blocking treatment but with application of a magnetic field during drying. Image C (bottom left) shows a reaction surface obtained with blocking treatment and without application of a magnetic field during drying. Image D (bottom right) shows a reaction surface obtained with blocking treatment and with application of a magnetic field during drying.

(28) The images of FIG. 3 clearly show that applying a magnetic field during the drying phase reduces the non-specific interaction time of the magnetic beads with the reaction surface. When applying a blocking step next to the magnetic field, the amount of non-specific interaction is further reduced and brought back to a level which is usually only seen when the magnetic beads are dosed on the laminate, so have no interaction with the surface during drying.

(29) FIG. 4 shows in a diagram the results of a magnetic bead count for the four different configurations A, B, C, and D shown in FIG. 3 (the vertical axis indicating the counted number of beads). The hatched bars in_Sp correspond to a magnetic bead count (corrected for area) inside the region of interest (ROI) of the printed antibody spots. The white bars out_Sp correspond to the area outside the printed spots.

(30) The magnetic bead count is for the configuration of test D on par with the bead count seen when the magnetic beads are dosed on the laminate and thus when there is no interaction of the magnetic beads with the surface during processing.

(31) In comparison to a technology in which magnetic beads are provided on a lid of the cartridge, the described approach has an advantage when measurements in a dirty matrix (i.e. blood) are made. This is because placing the magnetic beads on top of the reaction/binding surface reduces variation and increases assay performance. This is due to the fact that the blood-cells make the transportation of the beads from the laminate to the reaction/binding surface difficult.

(32) In summary, embodiments of the invention have been described in which a mixture of magnetic particles, a matrix material, and a volatile carrier is deposited onto binding sites of a reaction surface. The deposited mixture is then dried while the magnetic particles are pulled away from the reaction surface by a magnetic field. Thus unspecific binding of magnetic particles to the binding sites can be prevented.

(33) 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. A single processor or other unit may fulfill the functions of several items recited in the claims. 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.