EP2697639A1

EP2697639A1 - Magnetophoretic analyte selection and concentration

Info

Publication number: EP2697639A1
Application number: EP12724097.6A
Authority: EP
Inventors: Oliver Hayden
Original assignee: Siemens AG
Current assignee: Siemens AG
Priority date: 2011-05-18
Filing date: 2012-05-11
Publication date: 2014-02-19
Also published as: WO2012156324A1; US9400236B2; DE102011076051A1; CN103688165A; US20140087414A1

Abstract

The invention relates to a device and to a method for magnetophoretic analyte selection and concentration. Magnetically marked analytes, in particular cells (16), can be separated out of a sample dynamically in flux, such that they are present in a highly concentrated manner in a reduced sample volume. In particular, the analyte selection can be followed by an analysis (30).

Description

description

Magnetophoretic Analyte Selection and Enrichment The present invention relates to magnetophoretic analyte selection and enrichment.

The analyte are predominantly cells of interest. In the field of cell measurement and cell detection, optical measurement methods, such as scattered light or fluorescence measurement, as well as magnetic detection methods are known in which the cell type to be detected is marked by means of magnetic labels.

In particular, methods are known for magnet-based measurement, in which magnetically marked cells are sorted out by magnetic resonance from a complex cell suspension, for example a blood sample. The magnetic marker he ^¬ follows particular by cell-specific markers are introduced into the complex cellular sample. Magnetophoresis has hitherto been used to sort magnetically labeled cells or generally magnetic particles.

For cell measurement in diagnostics and science, it is necessary to also measure cell types that are present in only very low concentrations in a blood sample, such as, for example, disseminated tumor cells. The loss of cells by the sample preparation is also highly disadvantageous. For the quantification of cell concentrations or for a reliable detection of certain cells, therefore, a previous enrichment of the cells to be determined from a suspension with a complex background is necessary.

Such an accumulation of cells has hitherto been carried out by centrifugation techniques, immunochromatography or magnetic enrichment (MACS). These methods have ge ^¬ Together the disadvantage that the enrichment is static, which means that the cells to a vessel wall or in an ex enriched section of a centrifuge tube. The enrichment factor of these methods is in the range of 10 ¹ to 10 ⁴ and is not sufficiently high. In particular, in centrifugation techniques beyond a mechanical load on the cells is unavoidable.

Previously known magnetophoresis methods for dynamic cell enrichment also have the disadvantage of the low enrichment factor, which does not exceed a lOOfache enrichment. As an example of a known laminar flow magnetophoresis process, the publication by Jung et al. in Applied Physics Letters 93, 223902, 2008. It is an object of the present invention by means of magnetophore retentive analyte selection and enrichment to allow a higher concentration of the analytes to be detected in a sample suspension. The object is achieved by a device according to claim 1. A method for magnetophoretic analyte selection and enrichment is given in claim 11. Advantageous embodiments of the invention are the subject of the dependent claims.

The device according to the invention for magnetophoretic analyte selection and enrichment comprises a flow channel, a first magnetic unit for enrichment and a second magnetic unit for aligning magnetically labeled analytes. In this case, the enrichment and orientation of the analytes is provided in a first channel section. For this purpose, the first channel section comprises the magnetic units. In the second channel section, the separation takes place. The flow channel splits at least two subchannels in the second channel section. In a third channel section, a first of these sub-channels runs. This has a smaller cross-sectional area than the flow channel in the first channel section. This has the advantage that magnetically kierte analytes can be initiated with smaller cross-sectional area ^¬ in this part of channel and thus a much smaller sample volume per section length is in this part channel as in the flow channel with the large cross-sectional area. This reduction in per ^¬ benvolumens a high concentration can be done.

Thus, in particular a three-dimensional accumulation and managers is effected tion magnetically labeled analytes by suitable arrangement of the magnetic units which permits ^¬ be required reduction of the sample volume by the Kanalge ^¬ ometrie or possible. Due to the narrowing of the Ka ^¬ Nals in flow direction enrichment factors may be achieved greater 100th

In an advantageous embodiment of the invention, the first sub-channel in the third channel portion has a cross ^¬ sectional area which is less than half the cross-sectional area ^¬ of the flow channel in the first channel section. In particular, it is less than 1/10 of the cross- ^{sectional area of} the flow channel in the first Kanalab ^{¬ section} . For example, this first subchannel is on

Microfluidic channel. Preferably, analyte selection and enrichment is performed on cells in complex media, such as blood samples. The analyte, ie the cells have varying diameters between 1 and 20 ym. Typically, white blood cells measure 7 to 12 ym in diameter. For such described cell selection and accumulation, cell types with diameters around 3 μm, such as platelets, and cell types with diameters between 8 and 12 μm are of particular interest. For example, CD4 + cells have a diameter of around 7 ym. But even within a cell type, the diameter varies. Tumor cells typically have a diameter of 10 to 20 ym. In addition to cells, in particular magnetically marked polymeric beads, so-called beads with a typical diameter of 100 nm to 20 ym, or even small analytes, such as Vi ^¬ ren enriched.

Preferably, an analyte sample is passed through a much wider microfluidic channel, which is 10 to 100 times the diameter in diameter. In principle, channel diameters of 10 ym to 10000 ym can be realized. In this case, the lower dimension, ie the minimum diameter, is limited by the analyte diameter and the upper dimension is limited by the condition that a laminar flow should be able to prevail in the channel.

In a further advantageous embodiment of the invention, a second sub-channel has a cross-sectional area and / or a plurality of second sub-channels have a Gesamtquer ^¬ sectional area, which is sufficiently large abzuzutransportieren a Probenvo ^¬ lumen, through the flow channel in the first channel section at the sub-channels in the third channel section arrives. This larger second subchannel thus takes on the task of removing the existing sample volume from which the labeled analytes have been selected. This derivation of the sample volume is advantageous and of essential importance for the functionality, since otherwise there could be speed gradients in the sample flow and, for example, disturbing turbulences.

In particular, the cross-sectional area of the second Teilka ^¬ Nals and / or the total cross-sectional area of the plurality of two ^¬ th partial channels is so large that the flow behavior of the product is not substantially affected benvolumens. That is, the second sub-channel or the plurality of second sub-channels are designed such that the enrichment and orientation is not disturbed magnetic ^¬ table labeled analytes in the sample volume. The diameter of the second sub-channel is preferably between 100 μm and 10000 μm.

In an advantageous embodiment of the invention, the first sub-channel runs in the third channel section in the same Direction as the flow channel in the first channel section. This means that no kinking of the flow direction takes place for the sample to be selected. Only the part of the sample volume which is not to be selected is deflected out of this original direction of the first channel section. This arrangement in turn has advantages regarding the flow behavior.

In a further advantageous embodiment of the invention, the first magnetic assembly is arranged in the first channel section, characterized that a magnetic gradient field it is ^¬ zeugbar, which accumulates magnetically labeled analytes within the flow channel at the channel floor.

In particular, the channel bottom of the first Teilka- Nals is in the third duct section on the same level as the Ka ^¬ nalboden of the flow channel in the first channel section. The turn ^¬ ser planar course of the channel bottom has passed through the sections to cause no adverse embedding ^¬ flussung the flow advantage.

In a further advantageous embodiment of the invention, the second magnetic unit is arranged in the first Kanalab ^{¬ section} , characterized in that magnetically marked analytes are aligned within the flow channel along an axis which continues in the third channel section within the first sub-channel. That is, the magnetically labeled analytes are aligned on a Ach ^¬ se in the first channel section, which will be straight in the first sub-channel.

In the third channel section may still be provided a channel feed to the first sub-channel. Such a channel feed to the channel section of the analyte separation has the advantage that a second mark can be fed to the analyte via it. For example, other markers can be supplied to the analyte via the Kanalzu ^¬ guide and are connected via, for example, antibodies. Such a channel feed is of particular advantage if the first subchannel b

read analyte directly a measuring device, in particular for cells, feeds. This is in particular also covered by the third channel section. For example, the device for analyte selection and enrichment also comprises an analyte detection or analyte counter, in particular with a magnetoresistive sensor which is arranged at the end of the second channel section or in the third channel section.

In particular, the enrichment and guiding the magnetically labeled analytes by the nature of the magnetic marker can be influenced and / or by the Durchflussgeschwindig ^¬ ness which is established in the apparatus. The flow rate, in turn, can be adjusted by the microfluidic dimensioning. Be carried out Furthermore, the Anreiche ^¬ tion and management can be optimized in that the magnetically tophoretischen guides tung with respect to their magnetic permeability and with regard to the angle to Flussrich-, in which they are arranged, so that a stringent alignment of magnetically labeled analyte he follows. The magnetic gradient field which is generated by the first magnetic means, in particular ^¬ sondere a further screw for optimization of enrichment of a particular Analytsorte, including a particular cell type.

For example, a device may also have a cascaded arrangement of a plurality of magnetophoretic enrichment and selection paths described, i. a series of several, especially different enriched enrichment and selection routes.

In the method according to the invention for magnetophoretic analyte selection and enrichment, a flow of a sample with magnetically-labeled analytes is generated, the magnetically-labeled analytes of this sample are dynamically enriched and aligned in a magnetic gradient field, so that the magnetically labeled analytes are concentrated in a partial volume of the sample, this partial volume is separated dynamically from the üb ^¬ membered volume of the sample. Such a dynamic enrichment and above all concentration of the analytes in a sample, which is in particular a Zellsus ^¬ pension, has the advantage over previous methods that the analyte, so in particular the cells, stress can be concentrated so high without mechanical stress, that they can be quantified and measured.

In an advantageous embodiment of the invention, the enrichment and alignment of the magnetically-labeled analytes in a flow channel is carried out three-dimensionally in such a way that enrichment takes place at the channel inner wall by means of a first magnetic unit and alignment takes place along an axis by means of a second magnetic unit , The axis runs in the flow direction along the channel inner wall. This dreidimensio ^¬ tional enrichment and orientation has the advantage that the selected analytes can be forwarded to a sub-channel that can accommodate a much lower volume than the enrichment flow channel.

For example, the selected analog LYTEN be in the process, which are in particular cells, other markers supplied ^¬ leads. In particular, the additional markers have antibodies which can bind to characteristic isotopes on the cell surface. This step has the advantage that the cells magnetically marked for selection can be provided with further markers, which are necessary, for example, for a further cell measurement. In particular, the thus ^¬ additionally labeled cells are fed directly to a further cell measurement. In particular, the enrichment and Ausrich ^¬ tion and the subsequent selection by a device according to the invention takes place by a cell sample is injected into this. The described dynamic enrichment has the great Before ^¬ part that the cells can be stress-free, in particular by mechanical ^¬ specific load, a secondary analysis supplied. For example, after the selection, a fluorescence labeling can be carried out and a microscopy or flow cytometry can be carried out with low marker consumption.

Since the selection and enrichment coupled so directly to a demand following work or study step who can ^¬, advantageously reduces the analysis time and there must be no sample transfer. This in turn has the advantage of lossless working, as far as the loss of labeled analytes, eg for concentration determinations, as well as a reduction of the consumables, eg pipette tips.

1 shows a cross section through a channel 10, which is traversed from left to right by a suspension 15, for example a blood sample. The flow direction is indicated by an arrow 40, that is, on the left side of the channel 10 there is at least one inlet for the sample 15 and on the right side of the channel 10 is located to ^¬ least one outlet. The suspension 15 contains at least one sort of cells 16 which carry magnetic markers. These magnetically marked cells 16 are first deflected in the left portion of the channel 10 by a permanent magnet 20, which is mounted below the channel bottom, to the channel bottom and thereby enriched at the channel bottom. These Ab ^¬ section of the channel 10 with the permanent magnet 20 is also called enrichment route 11. This enrichment route serves further to align the magnetically labeled cells 16. For this purpose, above the channel bottom, that on the channel inner wall or, alternatively, embedded in the channel base further magnetic means 21 intended. For the alignment of the magnetically marked cells 16, in particular ferromagnetic strips 21 are suitable, which can only be seen in cross section in FIG. 1, as guide lines. The strips 21 run into the plane of the drawing, so to speak.

After the alignment and enrichment section 11, the separation of the magnetically marked cells 16 follows from the remaining suspension 15. In this selection region 12, there are, so to speak, several outflow directions 40, which can be better seen in the plan view in FIG. However, what the cross-section in FIG. 1 illustrates is that the channel 10 at the end of the separation section 12 tapers to form a microfluidic channel 13 through which essentially only the magnetically marked cells 16 flow in a small sample volume 15. Furthermore, in the area of the microfluidic channel 13, a detection device 30 is schematically shown which, for example, uses microscopy or flow cytometry.

Facility can be. This coupled analysis has the advantage of being able to anticipate stress selected and enriched Zel ^¬ len 16th 2 shows a plan view of the channel 10 with its three sections, the alignment and accumulation section 11, the separating section 12 and the Mikrofluidikstrecke 13. On the left side of the channel 10 in the area at the Anreiche ^¬ approximately path 11, the ferromagnetic guide lines 21 arranged in a herringbone pattern, which lead the magnetically marked cells 16 to the channel center. This concentration thus happens in the plane. At the same time, the magnetically marked cells 16 are approximated to the channel bottom by the permanent magnet 20, which is not visible in the drawing and is mounted below the channel 10, which covers the third dimension in the enrichment. These enriched and aligned cells 16 then flow in the region of the separation section 12 into the microfluidic channel 101. This comprises a much smaller sample volume 15 than the remaining channel 10. In addition to the microfluidic channel 100, the cell sample 15 can also flow to the left and to the right thereof, which is indicated by the three flow directions with arrows 40. The sections 101 run left and right of the Microfluidic channel 100 y-shaped away from the central flow ^¬ direction. The entire volume of channel 15 and the Kanalgeo ^¬ geometry is designed so that it can not come to turbulence in the flow 40 especially in the area of the separation 12, which could interfere with the magnetic enrichment and orientation. Accordingly, the drainage regions 101 must include a sufficiently large sample volume 15, so that the taper on the microfluidic channel 100 can be compensated. The marking of the three areas of the enrichment path with A, the microfluidic path with B and the lateral discharge routes with C is chosen such that the front view in FIG. 3 is understandable.

This front view of Figure 3 illustrates non-yardstick according to the different cross sections of the Anreiche ^¬ approximate flow channel A, microfluidics 13, 3 and the Ab ^¬ flow stretch C left and right of the microfluidic B. The schematic representation is to illustrate that the

Microfluidic channel 100 is closely chosen such that the magnetically-labeled cells can 16 a large part of the channel volume 15 take a ^¬, that are highly concentrated and thus, coupled to these microfluidic channel 100 Analytikvorrich ^¬ tung 30 ensures a substantially highly reliable Einzelzellde- tektion.

In turn, Figure 4 shows a plan view of the through ^¬ flow channel 10. Similarly to the embodiment in Figure 2 duri ^¬ fen of the flow channel 10 rungs- with the magnetic enrichment and alignment path in the first section 11 and the sub-channel 100 into which the magnetically-labeled cells 16 be guided along a common axis. In the second channel section 12, in which the separation of the magnetically marked cells 16 takes place from the suspension 15, the partial channels 101 extend perpendicularly away from the flow channel 10. These in turn have a much higher pulp ^¬ te than the sub-channel 100. The channel 100 is part insbeson ^¬ particular a microfluidic channel. In addition to the vertical routing of the sub-channels 100, the majority of the original Pro benflüssigkeit record 15, this embodiment shows yet another change from the y-shaped flow channel 10 in Figure 2, namely the channel feeders 31, which meet on both sides of the sub-channel 100. These are designed in particular for supplying additional markers 19. After the magnetically marked cells 16 have been introduced into the sub-channel 100 by the enrichment at the channel bottom and by the magnetophoretic alignment along the magnetic guide lines 21, they are thus provided with further markers 19, which prepare the cells 16 for a further cell measurement 30. Via the microfluidic channel 100, the dual-labeled cells 18 are directed into a cell measuring device 30. For example, the additional markers 19 may be fluorescent markers and, correspondingly, the cell measuring device 30 may be a fluorescence detector.

Claims

claims

1. An apparatus for magnetophoretic analyte selection and enrichment with

a flow channel (10),

a first magnetic unit (20) for enrichment and

a second magnetic unit (21) for aligning magnetic labeled analytes (16),

wherein in a first channel portion (11) the magneti ^¬ rule units (20, 21) are arranged in a second duct section (12) of the flow channel (10) into at least two channels (100, 101) splitting, and (in a third channel portion 13 ) a first sub-channel (100) has a smaller cross-sectional area than the

Flow channel (10) in the first channel section (11).

2. Device according to claim 1, wherein in the third channel section (13), the first sub-channel (100) has a cross-sectional area which is less than half of

Cross-sectional area of the flow channel (10) in the Ers ^¬ th channel section (11), in particular less than one-tenth of the cross-sectional area of the flow channel (10) in the first channel section (11).

3. Device according to one of claims 1 or 2, wherein a second sub-channel (101) has a cross-sectional area and / or a plurality of second sub-channels (101) have a total ^¬ cross-sectional area, which is sufficiently large to transport a sample volume from the

Flow channel (10) in the first channel section (11) at the sub-channels (100, 101) in the third channel section (13) arrives. 4. The apparatus of claim 3, wherein the cross-sectional area of the second sub-channel (101) and / or the total cross-sectional area of the plurality of second sub-channels (101) is so large that the flow behavior of the sample volume is not significantly affected, so that the Anreiche ^¬ tion and alignment of magnetically labeled analytes (16) is not disturbed in the sample volume.

Device according to one of the preceding claims, wherein the first sub-channel (100) in the third channel portion (13) in the same direction as the flow channel (10) in the first channel portion (11).

Device according to one of the preceding claims, wherein the first magnetic unit (20) is arranged in the first Kanalab ^{¬ section} (11), characterized in that a magnetic ^¬ cal gradient field can be generated, which magnetically labeled analyte (16) within the flow channel (10). accumulates at the channel bottom.

Device according to claim 6, wherein the channel bottom of the first sub-channel (100) in the third channel section (13) is at the same height as the channel bottom of the flow channel (10) in the first channel section (11).

Device according to one of the preceding claims, wherein the second magnetic unit (21) in the first Kanalab ^{¬ section} (11) is arranged, characterized in that magnetically mar ^¬ labeled analytes (16) are aligned within the flow channel (10) along an axis, which in drit ^¬ th channel portion (13) within the first sub-channel (100) extends further.

Device according to one of the preceding claims with a channel feed (31) to the first sub-channel (100) in the third channel section (13).

Device according to one of the preceding claims, which includes the third channel portion (13) has a Analytmesseinrichtung (30) which is ausges ^¬ taltet particular for cell measurement.

A method for magnetophoretic analyte selection and enrichment in which a flow of a sample of magnetically labeled analyte (16) is generated, wherein the magnetically labeled analytes (16) are dynamically enriched and aligned in a magnetic gradient field such that the magnetically labeled analytes (16) in a partial volume of the sample are concentrated, this partial volume is dynamically separated from the remaining volume of the sample.

The method of claim 11, wherein the enrichment and alignment of the magnetically labeled analytes (16) in a flow channel (10) is three-dimensional, such that by means of a first magnetic unit (20), the enrichment takes place at the channel inner wall and by means of a second magnetic unit ( 21) the alignment takes place along an axis, wherein the axis runs in the flow ^¬ direction along the channel inner wall.

13. The method according to any one of claims 11 or 12 in which the selected analyte more markers are supplied.

14. The method according to any one of claims 11 to 13 in which an analyte sample is injected into a device according to one of claims 1 to 10.

15. The method according to any one of claims 11 to 14 in which, after the analyte selection, a measurement of the analytes.