Abstract:
Devices and methods for magnetic flow measurement are provided. Individual analytes are detected in the through-flow, and a dynamic detection of a changing analyte state is carried out, for example with respect to the analyte size or morphology. For this purpose, the analytes to be detected, such as cells for example, are directly marked in the medium surrounding said analytes with magnetic labels and transported through the flow channel of a measuring device comprising at least two magnetic sensors. A characteristic measurement signal is generated by means of the magnetic sensors that are mutually spaced in the flow direction. The magnetic analyte diameter is calculated using the interval between the measurement deflections, and the analyte state can be evaluated using the magnetic analyte diameter.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present patent document is a §371 nationalization of PCT Application Ser. No. PCT/EP2012/064991, filed Aug. 1, 2012, designating the United States, which is hereby incorporated by reference, and this patent document also claims the benefit of DE 10 2011 080 945.7, filed on Aug. 15, 2011, which is also hereby incorporated by reference. 
     
    
     TECHNICAL FIELD 
       [0002]    The present embodiments relate to the technical field of magnetic flow measurement of magnetically marked analytes, and in particular, to magnetic flow cytometry. 
       BACKGROUND 
       [0003]    In the field of analyte measurement, and in particular, cell measurement, microscopy methods and scattered light methods are known for determination of the analyte size and shape, or morphology. By scattered light measurements, for instance, in optical flow cytometry, the cell morphology and the cell diameter are detected by so-called forward or side scattering, or a combination of the two. However, scattered light measurements require the sample preparation that may lead to cell stress, which may thus already modify or destroy the cell during the sample preparation. Therefore, in particular, dynamic changes in the quantity to be determined, such as diameter or morphology that take place within a few minutes, are not accessible with this method. 
         [0004]    By microscopic methods, in turn, cell concentration determination may not be carried out, or may be carried out only with great difficulty. Simultaneous acquisition of the cell concentration and a dynamic change in the cell shape that gives an indication of the cell state is therefore not possible with previously known methods. Especially in the field of analysis in diagnosis and in the so-called life sciences, however, it is important to selectively detect cells from a complex suspension, for instance a blood sample, and to establish the dynamic change thereof. 
       SUMMARY AND DESCRIPTION 
       [0005]    It is an object of the present embodiments to provide a suitable method for dynamic acquisition of analyte changes over time, where the concentration of the analyte in a sample may also be determined. 
         [0006]    In the method according to an embodiment for magnetic flow measurement of an analyte, the following acts are carried out: magnetic marking of analytes in a sample, generation of a gradient magnetic field at least in the acquisition region of a sensor arrangement, and flow generation of the analytes over the sensor arrangement, the flow of the analytes being guided initially over a first magnetoresistive component and subsequently over a second magnetoresistive component. Detection of individual marked analytes, in which at least three measurement excursions are recorded per analyte, is carried out. The measurement excursions are induced by the stray magnetic field of each marked analyte. The at least three recorded measurement excursions form a characteristic measurement signal for single-analyte detection. Subsequently, evaluation of the measurement signal, in which the measurement signal is identified as single-analyte detection with the aid of the measurement excursion sequence, and in which the analyte magnetic diameter is calculated with the aid of the measurement excursion separation, is carried out. The analyte state is evaluated with the aid of the analyte magnetic diameter. 
         [0007]    The method thus uses, in particular, the fact that the magnetic diameter, established by the stray field maximum of the marked analyte, is less than the hydrodynamic or optical diameter, as is determined, for example, with the Coulter counter method. This means that the majority of the stray magnetic field of the immunomagnetic marking extends through the interior of the cell. The analytes are, in particular, cells and may be present in blood samples. By distinguishing the analyte optical diameter from the analyte magnetic diameter, the analyte state may be evaluated since, with the same marking, the magnetic diameter varies with the state. 
         [0008]    The method is used to detect analytes that may assume at least a first state and a second state, the two states being manifested by a change in the analyte magnetic diameter. The state change of the analytes does not simultaneously lead to a change in the analyte hydrodynamic diameter. 
         [0009]    Discrimination between rounded and confluent test cells, for instance, has to date been possible only by elaborate microscopy. By recording the cell magnetic diameter, it is now possible to carry out this discrimination even in a flow measurement. 
         [0010]    Another example is the change of a thrombocyte as a result of its so-called activation. If the thrombocyte when inactive is in the form of a flattened ellipsoid or platelet, the magnetic diameter lies inside the cell. In the activated state, however, when the thrombocyte includes a multiplicity of projections on the surface, the so-called pseudopodia, the cell&#39;s magnetic diameter changes such that the magnetic diameter actually lies outside the hydrodynamic diameter and the magnetic diameter exceeds the hydrodynamic diameter by up to 30%. The method thus has the substantial advantage of being able to discriminate between activated and nonactivated thrombocytes, and at the same time of being able to determine their concentration, for example, in a stabilized whole blood sample. The method thus offers an advantageous possibility for thrombocyte function diagnosis. 
         [0011]    Thus, in the method, for example, a state change that is a change in the cell morphology is evaluated, and/or a state change that is attributable to a change in the cell geometry is evaluated, as described in the examples mentioned above. 
         [0012]    In an advantageous embodiment, a plurality of subsequent measurements are carried out in the method in order to detect individual analytes, so that a dynamic change in the analyte state is recorded with the aid of its magnetic diameter. The method thus has the advantage of not only detecting an analyte state at a single time but of tracking the analyte state over a period of time. The immunomagnetic marking of the analytes, which may be cells, is used in the method so that a dynamic change in the cell state over a time range of from one second to one hour may be observed. Furthermore, the method has the advantage of also determining the cell concentration as a function of time. 
         [0013]    For example, the plurality of successive measurements is carried out by guiding the flow of the analytes over a plurality of sensor arrangements, each of the plurality of sensor arrangements having a first magnetoresistive component and a second magnetoresistive component. Alternatively, the sample is guided several times in succession over one or more sensor arrangements. 
         [0014]    The method thus offers the advantage not only of being able to detect an analyte state just once, but of dynamically recording and evaluating its variation over time. This may also be contributed to by the magnetic flow measurement, since in this way, other than the addition of magnetic markers, scarcely any sample preparation that would stress or even destroy the cells has to be carried out. Marked cells may be observed in the second to hour range with the described method. 
         [0015]    In the method, advantageously, the magnetic marking is carried out with magnetic nanobeads, e.g., superparamagnetic nanobeads. Nanobeads have, for example, hydrodynamic diameters of between 10 nm and 500 nm. Magnetic nanobeads that have magnetite or maghemite as material are advantageous. The marking is carried out, for example, such that the occupation density of the markers on the analyte surface is between 10% and 90%, for example, depending on the cell surface and the epitope number on the cell surface. The material magnetite, for example, has a saturation magnetization of about 80 to 90 (A·m 2 )/kg. Depending on the proportion of the material, or if another material is contained in the nanobeads, the superparamagnetic labels have a saturation magnetization of between about 10 and 60 (A·m 2 )/kg. Such magnetic marking is advantageous since the stray magnetic field, caused by the marking in the gradient magnetic field, extends predominantly through the interior of the cell, and in particular the maxima of the stray field lie inside the cell diameter. In this way, the method additionally allows that even analytes directly following another analyte, which move over the sensor arrangement, may be read individually. In the case of a stray magnetic field that for the most part lies outside the cell, a signal overlap of the magnetoresistive signal occurs that no longer allows unambiguity of single-analyte detection. 
         [0016]    In another advantageous embodiment, the flow speed of the sample in the method is adjusted such that the analytes are guided over the magnetoresistive components with a constant speed. For example, by adjustment of the flow speed, the analytes are caused to roll over the magnetoresistive components. 
         [0017]    In another advantageous embodiment, a preparation act is carried out in the method where the channel inner wall is modified such that the state of the analytes to be detected is altered upon contact with the channel inner wall. In particular, this state change may be activation of cell analytes. Thrombocytes are one example of this. If thrombocytes are being detected by the described method, the thrombocytes may be activated upon contact with a suitably prepared channel inner wall that leads to a change in the cell morphology. As already described, the thrombocyte changes from a flat ellipsoid to a structure having very many projections from the surface. Such a state change is manifested clearly in the magnetic radius and may be detected by the described method. 
         [0018]    Statistical distributions of diameters may be recorded by the described method. Even in the case of only one analyte type to be detected being present, the analytes have different hydrodynamic diameters and different magnetic diameters. 
         [0019]    The device according to an embodiment for flow measurement includes a flow channel, at least one magnetic unit that is arranged below the channel bottom of the flow channel and is configured in order to generate a gradient magnetic field that permeates the volume enclosed by the flow channel, at least one cell measurement instrument having at least two magnetoresistive components, the magnetic unit being configured in order to induce a gradient field having a magnetic field strength of between 1 mT and 500 mT, each magnetoresistive component being arranged at a distance of between 0.2 μm and 40 μm from another magnetoresistive component, and the channel inner wall being configured such that the adhesion force of the analytes with the surface of the channel inner wall is less than the shear force for flow speeds of between 0.1 mm/s and 10 mm/s. 
         [0020]    In an advantageous embodiment of the device, the flow channel is configured in respect of channel diameter and surface condition of the channel inner wall such that, with a predetermined gradient magnetic field and predetermined flow speed, shear forces acting on the analytes are less than 1 g/(cm·s 2 ) and/or the Reynolds number is less than 2000. 
         [0021]    Advantageously, each magnetoresistive component is arranged at a maximum distance from another magnetoresistive component so that the analytes pass over the cell measurement instrument essentially with a constant speed. The maximum separation between magnetoresistive components is, for example, less than 40 μm, or less than 30 μm. In this way, a sufficient signal amplitude with a constant external magnetic field is possible. 
         [0022]    In certain embodiments, the magnetoresistive components are giant magnetoresistance (GMR) sensors. The magnetoresistive components are, in particular, arranged in a Wheatstone bridge circuit. At least two magnetoresistors may be interconnected as a half-bridge. The advantageous use of a Wheatstone bridge is known, for example, from the patent application DE 10 2010 040 391.1. 
         [0023]    In particular, the described device may also have a magnetophoretic enrichment path, as is known for example from the patent application DE 10 2009 047 801.9. 
         [0024]    The scope of the present invention is defined solely by the appended claims and is not affected to any degree by the statements within this summary. The present embodiments may obviate one or more of the drawbacks or limitations in the related art. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0025]      FIG. 1  depicts a side view of one embodiment of a sensor arrangement. 
           [0026]      FIG. 2  depicts a diagram of one embodiment of a measurement signal. 
           [0027]      FIG. 3  depicts a diagram of one embodiment of an analyte distribution. 
           [0028]      FIG. 4  depicts a schematic representation of one embodiment of inactive thrombocytes. 
           [0029]      FIG. 5  depicts a schematic representation of one embodiment of activated thrombocytes. 
           [0030]      FIG. 6  depicts a side view of one embodiment of a flow channel with inactive analytes. 
           [0031]      FIG. 7  depicts a similar view as  FIG. 6  of one embodiment of a flow channel with activated analytes. 
           [0032]      FIG. 8  depicts a similar view as  FIGS. 6 and 7  of one embodiment of a flow channel, the stray magnetic fields of the analytes being shown. 
       
    
    
     DETAILED DESCRIPTION 
       [0033]      FIG. 1  shows a detail of a flow channel  10  that leads from left to right in the figure. The channel bottom  11  of the flow channel  10  carries two magnetic sensors  20   a ,  20   b,  represented in a very simplified way as rectangles in the side view that are flowed over successively in the flow direction  40 . The flow direction  40  is indicated by two velocity arrows v from left to right in the flow channel. The two sensor elements  20   a,    20   b  lie at a center distance Δx from each other that may, for example, be adapted to the analyte type to be detected and, in particular, to the analyte diameter d. Analytes  30   a,    30   b,    30   c  that move in the flow direction  40 , are also represented as circles of different diameter d in the flow channel  10 . A curved arrow  41  indicates that the analytes  30   a,    30   b,    30   c  rotate while moving forward in the flow  40 . In particular, the analytes  30   a,    30   b,    30   c  roll over the channel bottom  11 . 
         [0034]    The sensor elements  20   a,    20   b  are in particular magnetoresistive components, that is to say the sensor elements  20   a,    20   b  are in particular magnetoresistors that are connected to one another in a Wheatstone bridge circuit. When two sensors are used, the sensors form in particular a half-bridge. An advantage of the interconnection is the magnetoresistive signal MR that may thereby be generated, represented in a signal/time diagram in  FIG. 2 . 
         [0035]    In the time profile of the passage of an analyte  30   a,    30   b,    30   c  over the two sensor elements  20   a,    20   b,  a peak sequence of at least three, or for example even four peaks P 1 , P 2 , P 3 , P 4 , as shown in  FIG. 2 , is generated. In this case, the first peak P 1  occurs when the analyte  30   a,    30   b,    30   c  just reaches the first sensor  20   a,  and the second peak P 2  occurs when the analyte  30   a,    30   b,    30   c  has just passed over the first sensor  20   a.    
         [0036]    This position is also indicated in  FIG. 1  by a dashed line P 2  extending perpendicularly to the channel bottom  11 . The third peak P 3  in turn occurs when the analyte  30   a,    30   b,    30   c  just reaches the second sensor  20   b,  as is likewise shown in  FIG. 1  again by a dashed line P 3  extending perpendicularly to the channel bottom  11 . Lastly, the fourth excursion P 4  occurs when the analyte  30   a,    30   b,    30   c  has also passed fully over the second sensor  20   b.    
         [0037]    That is to say, the time separation Δt between the second peak P 2  and the third peak P 3 , as indicated in the diagram in  FIG. 2 , is correlated with the distance ΔP between the two sensors  20   a,    20   b  by the flow speed v. Furthermore, this peak separation Δt also depends on how large the analyte  30   a,    30   b,    30   c  is. The larger the analyte diameter d is, the faster the analyte  30   a,    30   b,    30   c  is transported in the flow channel  10 , and the shorter is the time Δt that elapses between the second and third peaks, or the associated positions P 2 , P 3 . By the characteristic measurement signal MR with four measurement excursions P 1 -P 4 , single-analyte detection is also possible. The flow speed v may, for example, be determined from the time separation of the first peak P 1  and the third peak P 3 . 
         [0038]    Lastly,  FIG. 3  shows a further diagram that shows the distribution of the analytes around their measured diameter d. The diameter d is indicated in micrometers. The cell distribution N M  is plotted on the left-hand axis as a function of the magnetic cell diameter d measured as described above, and the cell distribution N C  that is based on the optical or hydrodynamic diameter of the analyte, as determined in particular by the Coulter method, is plotted on the right-hand axis of the diagram. Here, it is immediately clear that the two diameters, determined by the magnetic measurement or by the Coulter measurement, do not match. The magnetic diameter is in this case, for example, about 2 μm less than the Coulter diameter. This shift is attributable to the fact that, when passing over the sensor units  20   a,    20   b,  it is not the actual analyte or cell diameter d that is responsible for the measurement excursions P 1 -P 4 , but the edge  26  of the stray magnetic field  24 , or its maximum  26  of the x component of the stray field  24  that is recorded by the sensors  20   a,    20   b.    
         [0039]    Although the magnetic marking is applied externally on the analyte surface, the stray magnetic field maximum may also lie inside the cell  32 ,  34 . The magnetic field lines  24  of the magnetically marked cells  32 ,  34  are, for example, shown in  FIG. 8 . The differently lying magnetic diameters d M  are shown in  FIGS. 6 and 7 . In this case, it is not only the magnetic marking, but in particular also the shape and surface of the analyte  32 ,  34  that play a role as to whether the magnetic diameter d M  lies inside or outside the analyte  32 ,  34  to be detected. 
         [0040]      FIGS. 4 and 5  show very simplified schematic representations of cells  32 ,  34  that may in particular constitute thrombocytes. In a first state  32 , these are compact ellipsoids as represented in  FIG. 4 , and in a second state  34  the thrombocytes are very irregularly shaped and have a multiplicity of extending projections, so-called pseudopodia. These represent a very great surface enlargement of the cell  34 . 
         [0041]    As shown in the subsequent  FIGS. 6 and 7 , for the same cell and the same magnetic marking, the diameter d M  of the stray magnetic field is altered by the state change from inactive platelets  32  to activated thrombocytes  34 . In the case of the compact ellipsoids  32 , the magnetic diameter d M  lies inside the cell, while in the case of the activated cell  34  with the projecting pseudopodia, the maximum  26  of the stray magnetic field  24  moves outward, so that the magnetic diameter d M  becomes greater than the actual cell diameter d. 
         [0042]      FIGS. 6 to 8  again show the flow channel  10  with the channel bottom  11  and the sensor arrangement  20   a,    20   b  as in  FIG. 1 . The magnetic unit  22 , a permanent magnet, is arranged below the channel bottom  11 . In the surface extent of the magnetic unit  22 , the magnetic unit  22  is in particular large enough so that a homogeneous gradient magnetic field that enriches the magnetically marked cells  32 ,  34  on the channel bottom  11 , may be generated throughout the entire volume of the flow channel  10 . 
         [0043]    Lastly,  FIG. 8  once more shows the cross section through the flow channel  10  in a similar way to  FIGS. 6 and 7 . Magnetically marked cells  32 ,  34  and the magnetic field lines  24  of the stray magnetic field, generated by the magnetic marking of the cells  32 ,  34 , are indicated. Of this stray field  24 , the sensors  24   a,    24   b  detect in particular the x component. The x direction is indicated by the diagram by the side of the flow channel  10 , and points in the direction of the flow  40 . 
         [0044]    It is to be understood that the elements and features recited in the appended claims may be combined in different ways to produce new claims that likewise fall within the scope of the present invention. Thus, whereas the dependent claims appended below depend from only a single independent or dependent claim, it is to be understood that these dependent claims may, alternatively, be made to depend in the alternative from any preceding or following claim, whether independent or dependent, and that such new combinations are to be understood as forming a part of the present specification. 
         [0045]    While the present invention has been described above by reference to various embodiments, it should be understood that many changes and modifications may be made to the described embodiments. It is therefore intended that the foregoing description be regarded as illustrative rather than limiting, and that it be understood that all equivalents and/or combinations of embodiments are intended to be included in this description.