Patent Application
20130088221
The invention relates to apparatuses and methods for the detection of magnetic particles in a sample chamber.
Magnetic particles are for example used in biosensors to label components of a sample one is interested in. Typical examples of this application are described in the US 2009/148933 A1. According to this document, the magnetization of superparamagnetic beads vanishes when the external magnetic field is switched off, such that these particles do not agglomerate ([0102]).
Based on this background, it was an object of the present invention to provide means that allow for a more robust and accurate detection of magnetic particles.
This object is achieved by a method according to claim 1 and apparatuses according to claims 4 to 6. Preferred embodiments are disclosed in the dependent claims.
According to a first aspect, the invention relates to a “basic method” for the detection of magnetic particles in a sample chamber, wherein the term “magnetic particles” shall denote permanently magnetic particles or magnetizable particles, particularly nano-particles or micro-particles. In many cases the magnetic particles are used as labels, i.e. they are bound to some target component (e.g. molecule) one is actually interested in. The “sample chamber” is typically a cavity, particularly an open cavity, a closed cavity, or a cavity connected to other cavities by fluid connection channels. The method shall comprise the following steps, which may be executed in the listed or any other appropriate order:
a) The determination of a parameter, called “particle-parameter” in the following, that is related to the amount of magnetic particles in a first detection region. The “amount of magnetic particles” may be expressed by any appropriate definition, including absolute values (e.g. of the total number or total mass of magnetic particles) and relative values (e.g. the number or mass of magnetic particles per unit volume or area).
b) The determination of a parameter, called “cluster-parameter” in the following, that is related to the amount or degree of clustering of magnetic particles in a second detection region. The “degree of clustering” may be expressed by any appropriate definition (e.g. as the average number or mass of magnetic particles per cluster), including any parameter that depends on particle clustering.
The first and the second detection regions are typically sub-regions of the sample chamber. They may be identical, partially overlapping, or distinct. Moreover, they may comprise the whole sample chamber.
c) The evaluation of the particle-parameter based on the cluster-parameter. This evaluation may for example be done automatically in a data processing device, which may be built from dedicated electronic hardware, digital data processing hardware with appropriate software, or a mixture of both.
The method has the advantage that it provides, additionally to the detection of magnetic particles in a sample, an information about a possible clustering of said particles. This information turns out to be important in practice as the outcome of the particle detection process is often affected by the presence of clusters of magnetic particles. A detection method that yields correct results if no (irreversible) clustering of magnetic particles exists may for example yield increasingly impaired results the more clustering of magnetic particles occurs. Determining the degree of such clustering can hence be used to improve the reliability, robustness and/or accuracy of the particle detection.
The information about the particle-parameter and the cluster-parameter can be exploited for different purposes. In a preferred embodiment, a warning signal is generated if the cluster-parameter deviates from a predetermined set of values, i.e. a predetermined “normal range”. The user can thus be informed that exceptional conditions prevail which impair the reliability of the particle detection results and which may for example necessitate a change in the operating parameters.
According to another embodiment, the particle-parameter may be corrected based on the cluster-parameter. This approach requires that some information about the dependence of the particle-parameter on the degree of particle clustering is known, for example from theoretical considerations or calibration procedures.
The “basic method” of the invention requires as an essential prerequisite the determination of a particle-parameter (related to the amount of magnetic particles in a first detection region) and a cluster-parameter (related to the degree of clustering in a second detection region). In the following, various apparatuses are described that can be applied in the method to provide these parameters or at least information from which a particle-parameter and a cluster-parameter can be derived (preferably by pure calculations, i.e. without additional measurements). It should be noted, however, that these apparatuses can also be used for other purposes, too.
A first apparatus for an application in the “basic method” comprises the following components:
a) A magnetic field generator for generating a magnetic field in the sample chamber, wherein said field has different inclinations with respect to a binding surface of the sample chamber in a “first field region” and a “second field region” of the binding surface, respectively. The magnetic field generator may for example comprise one or more permanent magnets and/or electromagnets that can selectively be controlled. The different inclinations of the magnetic field in the first and second field region are preferably assumed simultaneously, though in general they may also be assumed sequentially (i.e. during partially overlapping or even distinct time intervals). Moreover, the first and the second field region are preferably distinct, though in general they may also be partially overlapping or even identical (wherein the magnetic fields with different inclinations have to be applied sequentially in those parts of the field regions that overlap). Furthermore, the “binding surface” shall in general be an interface between the sample chamber and an adjacent component at/near which the magnetic particles can reside. As its name indicates, there will preferably be some kind of linkage or binding between said “binding” surface and the magnetic particles.
b) A sensor element for detecting magnetic particles (separately) in the first and in the second field region, wherein the sensor element produces detection signals corresponding to its detection results.
c) An evaluation unit for relating the detection signals of the first and the second field regions that are provided by the aforementioned sensor element to each other. The evaluation unit may be realized in dedicated electronic hardware, digital data processing hardware with associated software, or a mixture of both. The relating of the detection signals may for instance comprise the calculation of their ratio, difference, or any other function of these two variables.
Related to the described first apparatus is a method comprising the following steps:
a) Generating a magnetic field in the sample chamber, wherein said field has different inclinations with respect to a binding surface of the sample chamber in a first field region and a second field region of the binding surface, respectively.
b) Detecting magnetic particles in the first and the second field regions.
c) Relating the detection signals originating from the first and the second field region to each other.
The described first apparatus and the corresponding method allow for the manipulation of magnetic particles by a magnetic field and for the detection of these particles at the binding surface. A specific feature is that the applied magnetic field has at least two different inclinations in two regions of the binding surface and that magnetic particles in these regions are separately detected, which allows to relate the resulting detection signals to each other. It turns out that this approach yields valuable additional information about the conditions at the binding surface. In particular, it is possible to obtain information about a possible (irreversible) clustering of the magnetic particles, because said clustering is sensitive to the direction of the magnetic field. Hence it is possible to derive a “particle-parameter” and a “cluster-parameter” from the detection signals.
It should be noted that the “detection regions” of the “basic method” and the “field regions” occurring in the above apparatus are different concepts. In a typical embodiment, the first and second detection regions are identical and correspond to the union of the first and the second field region. This means that the “particle-parameter” and the “cluster-parameter” are determined for the same sub-region of the sample chamber by means of a differentiation of this sub-region into a first and a second field region.
A second apparatus for an application in the “basic method” comprises the following components:
a) A magnetic field generator for generating a magnetic field in the sample chamber that is oblique to the binding surface in a “first” field region of a binding surface of the sample chamber. In this context, a magnetic field is considered to be “oblique” to a surface if it is not parallel to that surface, i.e. if the angle a between the field and the surface fulfills 0°α≦90°.
b) A sensor element for detecting magnetic particles in said first field region, wherein the sensor element produces detection signals corresponding to its detection results.
c) An evaluation unit for relating the detection signals obtained before and after a permanent switch-off of said magnetic field. In this context, the switching-off of a magnetic field is considered to be “permanent” if its duration is longer than a predetermined time interval that is related to the relaxation and diffusion processes at the binding surface. For magnetic particles with a diameter of about 500 nm, a “permanent” switch-off may typically be assumed if its lasts for more than one minute, preferably longer than two minutes. For larger (more heavy) particles (e.g. 1000 nm beads) the required minimal times may be shorter because the larger gravitational force will drive the particles quicker to the surface.
The second apparatus may optionally comprise the features of the first apparatus (in this case the terms “first field region” may refer to the same region). In general, explanations given above with respect to the first apparatus apply analogously also to the second apparatus.
Related to the described second apparatus is a method comprising the following steps:
a) Generating a magnetic field in a sample chamber that is oblique to a binding surface in a “first” field region of the binding surface.
b) Detecting magnetic particles in the first field region.
c) Relating the detection signals obtained before and after a permanent switch-off of said magnetic field.
The second apparatus and the corresponding method allow for the manipulation of magnetic particles by a magnetic field and for the detection of these particles at the binding surface. A specific feature of the second apparatus and method is that the magnetic field shall be oblique to the binding surface and that the detection signals before and after the action of this field are detected and related to each other. It turns out that this approach provides valuable additional information about the conditions at the binding surface, particularly about a possible (irreversible) clustering of magnetic particles. Again, this information can be used to derive a “particle-parameter” and a “cluster-parameter”.
In the first field region of the first or second apparatus, the magnetic field preferably includes an angle of more than about 10° with respect the binding surface. In this case the magnetic field is sufficiently oblique to the binding surface to reveal clustering effects of magnetic particles.
In the second field region (if present), the magnetic field preferably includes an angle of less than about 10° with respect to the binding surface, more preferably of less than about 5°. Such a magnetic field can be considered as substantially being parallel to the binding surface in the second field region. Diffusion related effects of the clustering of magnetic particles are minimized in this configuration.
A third apparatus for an application in the “basic method” comprises the following components:
a) A “particle detection unit” for detecting magnetic particles in the first detection region of the sample chamber. The particle detection unit typically generates a signal (e.g. an electrical signal) that is associated to the detection result, representing for example the amount of detected magnetic particles as some analogue value. This signal may hence directly correspond to the particle-parameter required by the method of the invention.
The particle detection unit may apply any appropriate detection principle, for example optical, magnetic, mechanical, acoustic, thermal and/or electrical. Most preferably, the particle detection unit will be surface sensitive, i.e. detect magnetic particles only within a limited region close to the surface of the sample chamber.
b) A “cluster detection unit” for detecting the degree of clustering of magnetic particles. Again, the cluster detection unit typically generates a signal (e.g. an electrical signal) that is associated to the detection result and that may directly correspond to the cluster-parameter required by the method of the invention.
There are different ways how the presence of clusters can be detected. In a preferred embodiment, the cluster detection unit may for example comprise a light source and a light detector that are arranged to measure the light transmission in the second detection region. For non-spherical clusters comprising a plurality of magnetic particles, for example chains of magnetic particles that are aligned to an external magnetic field, the light transmission will typically depend on the occurrence and degree of clustering. A transmission measurement thus provides an appropriate means to determine a cluster-parameter.
In the aforementioned case, the cluster detection unit may preferably comprise at least one reflective and/or at least one refractive interface that is encountered by light on its way from the light source to the light detector. A reflective surface (mirror) can for example be used on one side of the sample chamber to reflect light back towards the side of the light source, thus allowing to arrange the light source and the light detector on the same side of the sample chamber. Similarly, refractive windows on sides faces of the sample chamber may be used to redirect light such that the light source and/or the light detector can be arranged at convenient locations.
The method and the apparatuses according to the invention may preferably comprise a magnetic field generator, for example a permanent magnet or an electromagnet, for generating a magnetic field in the sample chamber that acts on the magnetic particles.
The magnetic field generator may preferably comprise a horse-shoe magnet. This provides configurations of a well-defined behavior with which a magnetic field of different inclinations can readily be generated (e.g. having a larger inclination near the pole tips and an approximately parallel direction between them). Additionally or alternatively, the magnetic field generator may comprise a magnet that is positioned opposite to a sensor element.
A magnetic field in the sample chamber may serve various purposes, which usually comprise an interaction with the magnetic particles. In many important applications, the magnetic field is configured in such a way that it generates forces on the magnetic particles, particularly forces that are attractive towards a binding surface. To this end, the magnetic field will usually have a gradient to produce the desired direction of the force. The attraction of magnetic particles towards a binding surface can be used to accelerate their migration from the whole sample volume into much smaller regions where they are detected. During the action of a magnetic field, clusters or chains of magnetic particles are usually formed, which may persist after the field has been switched off. This (undesirable) irreversible clustering can be dealt with by the determination of the cluster-parameter.
According to a further development of the embodiment with a magnetic field generator, the magnetic field is modulated, particularly switched on and off repetitively. Hence the effect of the magnetic field on magnetic particles alternates with periods during which the particles are free from external magnetic influences. If the magnetic field exerts a force on the magnetic particles, this means for example that a forced movement of the particles alternates with free (diffusion controlled) migration. It should be noted that the switching-off of the magnetic field that is meant in this context is usually shorter than the “permanent” switching-off defined above.
The frequency of the repetitive switching-off of the magnetic field preferably ranges between about 10 Hz and about 0.1 Hz (wherein the period of this switching is defined as the time between two consecutive switching-on events).
The duty cycle of the repetitive switching-off of the magnetic field preferably ranges between about 20% and about 90%, wherein said duty cycle is defined as the duration of the “on” interval with respect to the whole switching period (comprising both the “on” and “off” interval). Via the duty cycle it can be controlled how much time the magnetic particles have to freely migrate without the influence of a magnetic field. If clustering of the magnetic particles occurs in the magnetic field and if the clusters are oriented oblique to a binding surface, the available time for a field-free migration determines if distal magnetic particles or sub-clusters have enough time to reach the binding surface by diffusion or not. The value of the duty cycle may hence crucially influence the outcome of the detection procedure. This dependency can for instance be used to determine if the clustering is reversible (no sub-clusters occur after switching-off of the magnetic field) or irreversible (sub-clusters occur).
The described switching of the magnetic field may particularly take place during the detection of the particle-parameter and/or the cluster-parameter. As explained above, clusters of magnetic particles that form due to the action of a magnetic field will usually disintegrate due to thermal motion as soon as the magnetic field is switched off. In case of a permanent or irreversible clustering, the magnetically formed clusters of particles may however persist for whatever reason (e.g. due to chemical bindings). The action of a modulated magnetic field can therefore be used to distinguish between reversible and irreversible clustering of magnetic particles and to determine a degree of (irreversible) clustering.
The aforementioned distinction between reversible and irreversible clustering may be inferred from a variety of different measurements. Most preferably, the detection signal of the cluster detection unit may be evaluated with respect to a local relative amplitude, i.e. with respect to the relative difference between the nearest local maximum and the nearest local minimum at some point in time, wherein said local extrema are assumed when the magnetic field the switch on or off, respectively.
In a preferred embodiment of the invention, the sample chamber comprises a binding surface that is covered with binding sites for the magnetic particles. It should be noted in this context that the magnetic particles may often contain specifically bound target components (e.g. biomolecules) from a sample to be examined, and that the binding to the binding sites may occur via these target components. The binding sites may accordingly be “specific” in the sense that they only bind magnetic particles of a certain population, particularly magnetic particles that comprise the mentioned target components.
The detection of magnetic particles, if it is part of the method or the apparatuses, may be achieved by any suitable method or principle, for example by optical, magnetic, mechanical, acoustic, thermal and/or electrical measurements. The detection signal will typically be an electrical signal or a computer generated signal (resulting e.g. from image processing) representing a scalar value that is related to the amount of magnetic particles in the corresponding sensing region.
The invention further relates to the use of the apparatuses described above for molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and/or forensic analysis. Molecular diagnostics may for example be accomplished with the help of magnetic beads or fluorescent particles that are directly or indirectly attached to target molecules.
These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. These embodiments will be described by way of example with the help of the accompanying drawings in which:
Like reference numbers or numbers differing by integer multiples of 100 refer in the Figures to identical or similar components.
The invention will in the following be described with respect to biological or healthcare applications, comprising for example the detection of DNA (molecular diagnostics) and proteins (immuno-assays), both important markers for all kinds of diseases in the human body. Immuno-assay techniques may use small (super)paramagnetic beads to selectively capture the biological markers of interest. Subsequently the magnetic beads can couple to specific antibody sites on the surface, followed by a registration of the beads for the final detection. Based on this platform detection instruments can be developed for decentralized measurements such as the roadside testing of Drugs-Of-Abuse in saliva or the Point-Of-Care testing of cardiac markers in human blood at the physicians place.
As an example of the aforementioned instruments,
The carrier 110 comprises a sample chamber 111 in which a sample fluid with target components to be detected (e.g. drugs, antibodies, DNA, etc.) can be provided. The sample further comprises magnetic particles MP, for example superparamagnetic beads, wherein these particles MP are usually bound as labels to the aforementioned target components (for simplicity only the magnetic particles MP are shown in the Figures). The bottom interface between the massive part of the carrier 110 and the sample chamber 111 is formed by a surface called “binding surface” 112. This binding surface 112 may optionally be coated with capture elements, e.g. antibodies, which can specifically bind the target components.
The apparatus 100 comprises a light source 121 (e.g. a red 650 nm LED) for emitting an “input light beam” L1 into the carrier 110. The input light beam L1 arrives at the binding surface 112 at an angle larger than the critical angle of total internal reflection (TIR) and is therefore totally internally reflected as an “output light beam” L2. The output light beam L2 leaves the carrier 110 and is detected by a light detector, e. g. by the light-sensitive pixels of a camera 131. The light detector 131 thus generates detection signals representing the amount of light of the output light beam L2 (e.g. expressed by the light intensity of this light in the whole spectrum or a certain part of the spectrum). An evaluation unit 132 receives the detection signals from the light detector for further processing (evaluation, recording etc.).
The apparatus 100 further comprises a magnetic field generator 140 for controllably generating a magnetic field B at the binding surface 112 and in the adjacent space of the sample chamber 111. The magnetic field generator may for example be realized by an electromagnet 140 with a coil and a horse-shoe core having two pole tips 141 and 142. It may optionally comprise further magnetic units, for example a (e.g. cylindrical) magnet above the cartridge 110 (not shown), which commonly generate a magnetic field by superposition. With the help of the generated magnetic field, the magnetic particles MP can be manipulated, i.e. be magnetized and particularly be moved (if magnetic fields with gradients are used). Thus it is for example possible to attract magnetic particles MP to the binding surface 112 in order to accelerate the binding of the associated target component to said surface.
The described apparatus 100 applies optical means for the detection of magnetic particles MP and the target components one is actually interested in. For eliminating or at least minimizing the influence of background (e.g. of the sample fluid, such as saliva, blood, etc.), the detection technique should be surface-specific. As indicated above, this is achieved by using the principle of frustrated total internal reflection. This principle is based on the fact that an evanescent wave propagates (exponentially dropping) into the sample chamber 111 when the incident light beam L1 is totally internally reflected. If this evanescent wave then interacts with another medium having a different refractive index from water like the magnetic particles MP, part of the input light will be coupled into the sample fluid (this is called “frustrated total internal reflection”), and the reflected intensity will be reduced (while the reflected intensity will be 100% for a clean interface and no interaction). Further details of this procedure may be found in the WO 2008/155723 A1, which is incorporated into the present text by reference.
A problem with which an apparatus of the kind described above has to deal is that body-fluids like saliva and blood (or plasma) show large differences in physical and chemical properties from patient to patient. Preferably the assay and the actuation techniques used in a bio sensor should be robust against these variations.
A related problem is the irreversible clustering of the magnetic particles. Because a magnetic field B is used to attract the magnetic particles MP towards the binding surface 112, the magnetic particles MP become magnetized and also start attracting each other. Chains of magnetic clusters are formed. This effect can be clearly observed under a microscope with sufficient magnification. The chains are held together by the magnetic forces which the magnetic particles MP exert onto each other (the so-called bead-bead interactions). When the magnetic actuation field B is switched off, the magnetic particles are not magnetized anymore (if they are superparamagnetic) and the magnetic forces which hold together the chains disappear. Under normal circumstances the chains disintegrate into individually moving beads again. This process is referred to as “reversible clustering” or magnetic clustering: the magnetic field causes magnetic clustering but once the magnetic field is switched off, the clusters disappear again.
In contrast to reversible or magnetic clustering, also irreversible clustering is possible. There are various ways of irreversible clustering: irreversible clustering can take place in the absence of a magnetic field (cf. colloid chemistry) or it can be triggered by the presence of a magnetic field. In the case of irreversible clustering, the clusters do not disintegrate to individually moving beads when the magnetic field is switched off. The amount of irreversible clustering is strongly dependent of the composition of the body fluid and can vary a lot from patient to patient. The real mechanism for this irreversible clustering is not yet understood.
In general, the signal measured by an apparatus like that of
In the following, various approaches will be disclosed which determine a “cluster-parameter” that is related to the degree of (irreversible) clustering. The cluster-parameter can for example be used to emit a warning if the measured signal is probably not reliable, or to correct measurement results.
In
In order to understand the proposed solution, first the process of binding functional magnetic particles MP to the binding surface 112 will be explained in a little more detail with reference to
As a starting point it is assumed that the sample fluid above the measurement spot contains magnetic particles MP (typically superparamagnetic beads) with target molecules attached to them. It will not further be described here how this incubation reaction is carried out. In order to measure the concentration of magnetic particles with a target molecule, these particles have to bind to antibodies printed on the binding surface 112. These antibodies specifically catch the target molecules. The kinetics of this binding reaction between the target and the antibody can by speeded up by enhancing the concentration of targets near the surface. This is done by means of the magnetic field B. When the magnetic field B is switched on, a magnetic force (perpendicular to the binding surface) drives the magnetic particles MP from the liquid towards the binding surface 112 where the antibodies are. This process is called the “attraction phase” of the actuation cycle.
An essential aspect of the aforementioned processes is that, although all magnetic particles (with or without target) are attracted by the magnetic field B towards the binding surface, only a small fraction of these particles will actually be in contact with the surface and be able to bind. A first fraction of magnetic particles will bind to the originally empty surface but as soon as a certain surface coverage of magnetic particles is reached, the following arriving magnetic particles will magnetically cluster to the already bound beads. Because the magnetic field normally makes a certain angle a with the binding surface 112 (this angle a being dependent on the location between the poles 141, 142 of the magnet), the distal beads in a cluster CL are outside of the evanescent light field and are invisible to the measurement system. Under a continuous magnetic field B, the fractional surface coverage is about 10% at the center C of the magnet and even lower near in the regions P at the pole tips 141, 142 of the magnet (cf.
In order to allow the magnetically clustered beads to reach the binding surface 112 and bind, the magnetic field B is switched off after the first attraction phase. In this case the clusters CL can disintegrate into individually moving beads of which a part can reach the binding surface by diffusion. This is called the “diffusion phase” of the actuation cycle. Another fraction of the beads will diffuse into the liquid channel. This fraction can be brought back to the surface by switching the magnetic field B on again. By repetitively switching the magnetic field B on and off (the total actuation sequence consists of many actuation cycles), finally all magnetic particles have the possibility to bind to the binding surface 112. This is the essence of the described pulsed actuation protocol.
From the previous it will be clear that the largest part of the detection signal (90%) is generated through the process of disintegration of the clusters into individual magnetic particles and the diffusion of free magnetic particles towards the binding surface.
The main question is now how to discriminate between a low level of signal due to a low target concentration and a low level due to the effect of irreversible clustering. Normally the surface concentration of bound magnetic particles is measured in the center C of a horseshoe magnet. At this position the highest signals are measured and the reproducibility between spots is the best. However, this approach is also less sensitive to effects of diffusion if a pulsed actuation consisting of a magnetic field on and off is considered because the distance of the formed chains to the binding surface is rather short. In order to better see the effects of diffusion, it is proposed to create a larger average distance between the chains and the binding surface.
The direction of the magnetic field lines B as generated by the horseshoe magnet 140 is more or less in parallel to the binding surface 112. This is exactly true at the center position C of the electromagnet 140 (cf.
When no irreversible clustering appears, the signals at the center position C and the pole tip position P will be more or less the same.
In the measurements shown in
Whenever irreversible clustering appears, the diffusion of the cluster fragments is slowed down because the fragments are larger than single magnetic particles and there is more hydrodynamic resistance from the fluid.
In the measurements shown in
The magnetic force generated by the horseshoe magnet 140 is directed mainly perpendicular to the binding surface 112. The transport of magnetic particles to the center area C of the magnet is more or less equal to the transport of magnetic particles to the pole tip areas P. So basically both areas collect the same amount of beads. When finally the magnetic field is permanently switched off and one waits until the diffusion process has been completed, one would expect the same amount of signal at the pole tip position P and the center position C. In practice this is not always seen because beads also diffuse away from the surface. However, in the case of the reversible clustering it is observed that switching off the pulsed actuation with the 90% duty cycle, gives an extra contribution in the signal near the pole tip (cf. curve “P” in
In summary, the measurement of detection signals near the pole tips P of the actuation magnet, where the magnetic field has an inclination with respect to the binding surface, can provide information about the irreversible clustering behavior when compared to the detection signal near the center C of the magnet, where the magnetic field is approximately parallel to the binding surface. The ratio between the detection signal at the pole tips and the detection signal at the center is a measure for the amount of irreversible clustering. Thus a check can be build into a handheld device. A warning can be given by the evaluation unit 132 when irreversible clustering appears to indicate that the measured signal is probably not reliable.
In
As already explained above, sample fluids like human plasma seem to contain interfering factors that cause the irreversible aggregation (“clustering”) of the magnetic particles MP, which leads to a decreased assay performance. In the case where the analyte is cardiac troponin I for example, this can lead to a false negative result. A way of accurately determining the amount of clustering in a magnetic particle assay would therefore be valuable, either as a control (e.g. to disqualify the outcome of a particular measurement if the amount of clustering exceeds a certain threshold) or as a calibrator: if the relationship between the amount of clustering and the decrease in assay performance is known, the obtained outcome could be corrected and thereby resulting in more accurate measurements.
In the apparatus 200, the aforementioned objective is achieved by the provision of a “cluster detection unit” 260 that allows to determine a “cluster-parameter” related to the degree of clustering of magnetic particles MP in a “second detection region” DR2. In short, light is transmitted through the cartridge 210 containing the sample chamber 211 with a fluid sample in which magnetic nanoparticles MP are dispersed. In the apparatus 200, this is realized in the most straightforward way by placing a light source 261 on one side of the cartridge 210 and collecting the light transmitted through the second detection region DR2 at the other side by a detector 262 (e.g. an image sensor). It is observed that when the magnets 241, 242 are switched on, the intensity recorded by the cluster detection unit 260 increases. When the coils are switched off and the particles redisperse into a random pattern, the intensity decreases again. As will be explained in more detail below, the intensity changes allow to determine the desired “cluster-parameter” (degree of clustering).
The arrangement of the cluster detection unit 260 puts some limitations on the use of the top coil 243, which comprises no core material to allow the passage of light.
If it is preferred to analyze only a small portion of a cartridge, this could be done by using a microscope objective at the same or the opposite side as the bottom coils. Besides monitoring changes in the transmitted light intensity, it is of course also possible to position a detector outside the primary light path of the light source, thereby collecting only the scattered light. At low particle concentrations, this could be more favorable as it is difficult to detect very small changes at a high light intensity.
In the apparatuses 200, 300, and 400, the detector 262, 362, or 462 which collects the light is connected to a control unit (not shown) with software which can power the magnetic coils and record the intensity measured by the detector.
A typical recording of measured transmission intensity I is shown in
It is observed that when the magnetic particles exhibit (irreversible) clustering, the chains formed during the action of a magnetic field do not (fully) redisperse. While the diagram of
Especially the relative amplitude at a given time t proves to provide a robust way of measuring the amount of clustering in a particular sample, this relative amplitude Irel being defined as
I
rel=100%·(local maximum−local minimum)/local minimum.
As can be seen, the higher the percentage BP of clustering-inducing second sample, the lower the signal amplitude S (which means that there is more clustering). It is clear from
1. As a control: if a sample displays clustering above a certain threshold, the measurement is disqualified and the apparatus returns an error message. This is very important to exclude false negatives.
2. As a calibrator: when it is know how a certain amount of clustering leads to a diminished interaction of magnetic particles with the surface (and therefore a decreased end signal), it is possible to correct for this decreased interaction and multiply the result of the signal change (S) with a factor dependent on the amount of clustering (Irel).
Besides for the unwanted clustering described above, it should be noted that the proposed technique is also able to measure the clustering induced by the presence of a target. As an example, magnetic particles coated with an antibody directed against a first epitope of the cardiac troponin I (cTnI) molecule were mixed with magnetic particles coated with an antibody directed against a second epitope of cTnI. Finally, also cTnI (at a concentration of 800 pM) was added and the complete mixture was analyzed in a cluster assay. As a control, the mixture of both magnetic particles alone (without cTnI) was also analyzed.
Measurement data (comparable to
Because this assay format does not require the binding of the particles to a surface in which often a large part of the particles cannot participate in forming the molecular sandwich, this is a highly efficient assay format. Although 800 pM is still a relatively high concentration, it can be seen from
While the invention was described above with reference to particular embodiments, various modifications and extensions are possible, for example:
Finally it is pointed out that in the present application the term “comprising” does not exclude other elements or steps, that “a” or “an” does not exclude a plurality, and that a single processor or other unit may fulfill the functions of several means. The invention resides in each and every novel characteristic feature and each and every combination of characteristic features. Moreover, reference signs in the claims shall not be construed as limiting their scope.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10166839.0 | Jun 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2010/055495 | 11/30/2010 | WO | 00 | 12/18/2012 |
