WO2008142492A1

WO2008142492A1 - Method for detecting label particles

Info

Publication number: WO2008142492A1
Application number: PCT/IB2007/052938
Authority: WO
Inventors: Jeroen Hans Nieuwenhuis
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2007-05-22
Filing date: 2007-07-24
Publication date: 2008-11-27

Abstract

The invention relates to a method and a microelectronic sensor device for the detection of target components comprising label particles, for example magnetic nano-particles (1). The method comprises the determination of at least one 'reference value' (Sbg, Ssat) before and/or after target components have been admitted to a binding surface (12), and the measurement of a 'signal value' (Ssig) after unbound target components have been removed from the binding surface (12). The reference values may particularly comprise a background reference value (Sbg) measured while no target components are at the binding surface (12), and a saturation reference value (Ssat) measured while a maximal amount of target components is attracted to the binding surface (12). The reference values (Sbg, Ssat) can be used to correct the signal value (Ssig), to determine a reliability measure, and/or to detect error states. A movement of the target components is preferably achieved by electrical or magnetic fields (B) acting on their labels (1), while the measurement of target components at the binding surface (12) is done with an independent, for example optical measurement principle.

Description

METHOD FOR DETECTING LABEL PARTICLES

The invention relates to a method and a microelectronic sensor device for the detection of target components, for example biological molecules, comprising label particles.

From the WO 2005/010543 Al and WO 2005/010542 A2 a microsensor device is known which may for example be used in a microfluidic biosensor for the detection of molecules, e.g. biological molecules, labeled with magnetic beads. The microsensor device is provided with an array of sensors comprising wires for the generation of a magnetic field and Giant Magneto Resistances (GMR) for the detection of stray fields generated by magnetized beads. The signal of the GMRs is then indicative of the number of the beads near the sensor. A problem of this and similar measurement principles is that their accuracy can be affected by uncontrollable external influences like sample contamination, production tolerances and the like.

Based on this background it was an object of the present invention to provide means for the detection of target components comprising label particles with an improved and/or controllable reliability.

This object is achieved by a method according to claim 1 and a microelectronic sensor device according to claim 14. Preferred embodiments are disclosed in the dependent claims.

According to its first aspect, the invention relates to a method for the (qualitative or quantitative) detection of target components which comprise label particles and which are contained in some sample, particularly a sample fluid. The target components may for example be biological substances like molecules, complexes, cell fractions or cells. The term "label particle" shall denote a particle (atom, molecule, complex, nanoparticle, microparticle etc.) that has some property (e.g. optical density, magnetic susceptibility, electrical charge, fluorescence, radioactivity, etc.) which can be detected, thus indirectly revealing the presence of the associated target component. The "target component" and the "label particle" may optionally also be identical. The method comprises the following steps (which may be executed in the listed or any other order as well, and which may be repeated several times): a) Letting the target components bind to a binding surface, which may particularly be an inner surface of a sample chamber in which the sample to be investigated is provided. The term "letting" shall include both a passive process, for example the admission of a migration of the target components due to diffusion or gravity, as well as an active movement of the target components, for example by an induced hydrodynamic flow. b) Measuring at least one "reference value" for the amount of target components at the binding surface before and/or after the aforementioned step a) is executed. The term "reference" is chosen in this context primarily as a unique name for a particular type of measurement; moreover, this term indicates that this measurement will often serve as a kind of standard for other measurements. c) Removing unbounded target components from the binding surface, wherein this removal can for example comprise an exchange of the sample. d) Measuring a "signal value" for the amount of target components at the binding surface. The term "signal" is again primarily used as a unique name for a particular type of measurement; moreover, this term indicates that the signal value comprises the information one is actually interested in, namely the (exact) amount of bound target components at the binding surface.

The measurement of the reference values and the signal value are preferably done by the same measurement principle and optionally also with the same operating parameters, though this does not necessarily need to be the case. e) Relating the mentioned signal value to the reference value(s).

The described method has the advantage that it does not only consider the signal value, which contains the information about the amount of bound target components at the binding surface, but also at least one reference value which is determined under different conditions at the binding surface. The reference value(s) will therefore comprise information about uncontrollable circumstances of the measurement, e.g. contaminants in the sample, which also affect the signal value. Relating the signal value to the reference value can for example help to compensate these effects, thus improving the accuracy of the method. Moreover, relating the signal value to the reference value can provide information about the reliability of the obtained results.

In a preferred embodiment of the method, the target components are forcibly moved in the sample by magnetic or electric fields acting on their label particles. This requires of course that the label particles are susceptible to electric and/or magnetic fields, i.e. that the particles are for example electrically charged or magnetized. The movement of the target components by said fields may particularly be directed towards the binding surface or away from the binding surface. Magnetic particles serving as labels in the target components may for example actively be pulled to the binding surface during step a) or may be removed from the binding surface during step c) of the method by magnetic fields. The application of magnetic or electric forces can significantly accelerate both the binding process and the separation of unbound components, thus making the method suited for a fast, automatic measurement with high accuracy and reliability.

The amount of target components at the binding surface may be measured by any suited principle, for example by a magnetic sensor of the kind described above. Preferably, the amount of target components at the binding surface is measured optically, for example via an interaction of the target components or their labels with evanescent light waves. An optical measurement has the advantage that it does not interfere with the aforementioned manipulation of the label particles by electrical or magnetic fields. A movement of the target components by these fields can therefore be done simultaneously to optical measurements, allowing for example to actively pull target components to the binding surface or repel them from the binding surface during the measurements.

The amount of target components at the binding surface may optionally be monitored over an extended period of time to determine the point in time when a valid reference value and/or signal value is measured. Such a monitoring is particularly possible in the aforementioned case of a (e.g. optical) measurement principle that does not interfere with a movement of target components (e.g. by electrical or magnetic fields). The right point in time for the measurement of a valid reference value or signal value is typically reached when the monitored value becomes stationary, i.e. if its changes per unit of time fall below a given threshold. This may indicate for example that the removal of all unbound target components from the binding surface is practically completed.

According to another preferred embodiment of the invention, a reference value, which is called "background reference value" in the following, is measured while a minimal amount of target components is present at the binding surface. What a "minimal amount" is depends on the situation of the particular setup.

Thus the aforementioned background reference value may for example be measured while a test or reference sample without any target components and/or without any label particles contacts the binding surface. Besides the absence of target components or label particles, the reference sample shall preferably have a composition that is as similar to the composition of the actual sample as possible.

In an alternative approach, the background reference value may be measured immediately after the actual sample has contacted the binding surface, wherein the term "immediately" refers in a reasonable way to the kinetics of the binding of target components to the binding surface. Thus the time interval available for an "immediate" measurement will typically comprise the whole time until 5 %, preferably 1 % of the final amount of bound target components (stationary case) is reached. Without an active acceleration of the binding process, this time interval will extend in most cases over a period that is long enough for a secure measurement of the background reference value. In another particular embodiment of the above approach, target components are repelled from the binding surface by magnetic or electric fields acting on their label particles during the measurement of the "background reference value". This has the advantage that no target-component-free sample must be provided. To avoid a negative interaction between the particle repulsion and the measurement, both activities are preferably based on different principles, for example on the use of magnetic fields and optical measurements, respectively. According to another embodiment of the invention, a reference value, which is called "saturation reference value" in the following, is measured before unbound target components are removed from the binding surface. This value therefore represents a situation in which a high amount of both bound and unbound target components is present at the binding surface, thus providing a valuable additional information.

The aforementioned "saturation reference value" may particularly be measured while target components are attracted to the binding surface by magnetic or electric fields acting on their label particles. Again, this is particularly possible if the attraction is executed via e.g. magnetic fields while the measurement is based on e.g. optical principles that do not interfere with the particle manipulation. Actively attracting target components creates a situation in which a maximal amount of both bound and unbound target components is present at the binding surface during the determination of the saturation reference value. There are different ways in which a signal value can be related to the reference value(s). According to one particular embodiment, the signal value is corrected in dependence on the at least one reference value such that it better correlates to the actual amount of bound target components present at the binding surface. The background reference value mentioned above may for example be subtracted from the signal value (and any other reference value) as it represents an offset for all measurements that is present even for a binding surface free of target components. Similarly, the saturation reference value mentioned above may be used as a normalization factor as it represents the maximal amount of target components that can be present at the binding surface. Another possibility to relate the signal value to the reference value(s) is the determination of a reliability measure/index for the signal value from the reference value. If the background and the saturation reference values described above are for example close to each other, i.e. if the spread between minimal and maximal possible measurement signal is small, this may be taken as an indication of a small reliability of the measurements.

A further possibility to relate the signal value to the reference value(s) comprises the comparison of the reference value(s) with a range of predetermined "allowed values" to detect an error state which is indicated by reference values outside said range. In this context, the comparison of the reference value(s) may both be done directly or indirectly (i.e. via a term that is derived from the reference value(s)). If the background reference value mentioned above is for example extraordinarily high for the sample under investigation, this may be taken as an indication of a malfunction of the used instruments, an unusual contamination of the sample or the like.

The invention further relates to a microelectronic sensor device for the detection of target components comprising label particles in a sample, said device comprising the following components: a) A sample chamber with a binding surface. The sample chamber is typically an empty cavity or a cavity filled with some substance like a gel that may absorb a sample; it may be an open cavity, a closed cavity, or a cavity connected to other cavities by fluid connection channels. The binding surface is an inner surface of the sample chamber that is typically covered with binding sites (e.g. antibodies) for the target components. b) A sensor unit for measuring the amount of target components at the binding surface. The sensor unit may for example comprise a light source for emitting an incident light beam and a light detector for measuring the amount of transmitted, reflected (e.g. by total internal reflection (TIR) or frustrated TIR), or absorbed light or of secondary light like fluorescence light that is stimulated at the binding surface. c) A field generator for generating an electrical or magnetic field which can affect the label particles, e.g. exert forces on them. d) A controller for controlling the sensor unit and the field generator such that at least one reference value and a signal value are determined and related to each other in a method of the kind described above.

The sensor device provides a hardware platform for the execution of the method that was explained above. Therefore, reference is made to the preceding description for more information on the details, advantages and improvements of that sensor device. 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:

Figures 1 to 4 show four consecutive stages of a measurement procedure according to the present invention in an associated microelectronic sensor device; Figure 5 shows measurement signals for the three consecutive filling states "wash-liquid/empty/blood" of a sample chamber; Figure 6 shows measurement signals for samples with different concentrations of PTH.

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

Figure 1 shows the general setup of a microelectronic sensor device according to the present invention. One component of this device is a carrier 11 that may for example be made from glass or transparent plastic like poly-styrene. The carrier 11 is located next to a sample chamber 2 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 1, for example super paramagnetic beads, wherein these particles 1 are usually bound as labels to the aforementioned target components (for simplicity only the magnetic particles 1 are shown in the Figure). It should be noted that instead of magnetic particles other label particles, for example electrically charged of fluorescent particles, could be used as well.

The interface between the carrier 11 and the sample chamber 2 is formed by a surface called "binding surface" 12. This binding surface 12 may optionally be coated with capture elements, e.g. antibodies, which can specifically bind the target components. The sensor device comprises a magnetic field generator 41, for example an electromagnet with a coil and a core, for controllably generating a magnetic field B at the binding surface 12 and in the adjacent space of the sample chamber 2. With the help of this magnetic field B, the magnetic particles 1 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 1 to the binding surface 12 in order to accelerate the binding of the associated target component to said surface.

The sensor device further comprises a light source 21, for example a laser or an LED, that generates an incident light beam Ll which is transmitted into the carrier 11. The incident light beam Ll arrives at the binding surface 12 at an angle larger than the critical angle θ_c of total internal reflection (TIR) and is therefore totally internally reflected as a "reflected light beam" L2. The reflected light beam L2 leaves the carrier 11 through another surface and is detected by a light detector 31, e.g. a photodiode. The light detector 31 determines the amount of light of the reflected light beam L2 (e.g. expressed by the light intensity of this light beam in the whole spectrum or a certain part of the spectrum). The measurement results are evaluated and optionally monitored over an observation period by an evaluation and recording module 32 that is coupled to the detector 31.

The Figure shows a further light detector 31' that can alternatively or additionally be used to detect fluorescence light emitted by fluorescent particles 1 which were stimulated by the evanescent wave of the incident light beam Ll. As this fluorescence light is usually emitted isotropically to all sides, the detector 31' can in principle be disposed anywhere, e.g. also above the binding surface 12. Moreover, it is of course possible to use the detector 31, too, for the sampling of fluorescence light, wherein the latter may for example spectrally be discriminated from reflected light L2. Though the following description concentrates on the measurement of reflected light, the principles discussed here can mutatis mutandis be applied to the detection of fluorescence, too.

The described microelectronic sensor device applies optical means for the detection of magnetic particles 1 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. This is achieved by using the principle of frustrated total internal reflection which is explained in the following.

According to Snell's law of refraction, the angles Θ_A and Θ_B with respect to the normal of an interface between two media A and B satisfy the equation

ΠA sinθA = nβ sinθβ with Π_A, nβ being the refractive indices in medium A and B, respectively. A ray of light in a medium A with high refractive index (e.g. glass with Π_A = 2) will for example refract away from the normal under an angle Θ_B at the interface with a medium B with lower refractive index such as air (nβ = 1) or water (nβ « 1.3). A part of the incident light will be reflected at the interface, with the same angle as the angle Θ_A of incidence. When the angle Θ_A of incidence is gradually increased, the angle Θ_B of refraction will increase until it reaches 90°. The corresponding angle of incidence is called the critical angle, θ_c, and is given by sinθ_c = n_B/n_A. At larger angles of incidence, all light will be reflected inside medium A (glass), hence the name "total internal reflection". However, very close to the interface between medium A (glass) and medium B (air or water), an evanescent wave is formed in medium B, which decays exponentially away from the surface. The field amplitude as function of the distance z from the surface can be expressed as: exp(-k^n_A ² sm²(0_A) - n_B ² ■ z) with k = 2π/λ, Θ_A being the incident angle of the totally reflected beam, and Π_A and nβ the refractive indices of the respective associated media.

For a typical value of the wavelength λ, e.g. λ = 650 nm, and n_A=1.53 and nβ=1.33, the field amplitude has declined to exp(-l) « 0.37 of its original value after a distance z of about 228 nm. When this evanescent wave interacts with another medium like the magnetic particles 1 in the setup of Figure 1, part of the incident 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). Depending on the amount of disturbance, i.e. the amount of magnetic beads on or very near (within about 200 nm) to the binding surface 12 (not in the rest of the sample chamber 2), the reflected intensity will drop accordingly. This intensity drop is a direct measure for the amount of bonded magnetic beads 1, and therefore for the concentration of target molecules. When the mentioned interaction distance of the evanescent wave of about 200 nm is compared with the typical dimensions of anti-bodies, target molecules and magnetic beads, it is clear that the influence of the background will be minimal. Larger wavelengths λ will increase the interaction distance, but the influence of the background liquid will still be very small.

The described procedure is independent of applied magnetic fields. This allows real-time optical monitoring of preparation, measurement and washing steps. The monitored signals can also be used to control the measurement or the individual process steps.

For the materials of a typical application, medium A of the carrier 11 can be glass and/or some transparent plastic with a typical refractive index of 1.52. Medium B in the sample chamber 2 will be water-based and have a refractive index close to 1.3. This corresponds to a critical angle θ_c of 60°. An angle of incidence of 70° is therefore a practical choice to allow fluid media with a somewhat larger refractive index (assuming U_A = 1.52, nβ is allowed up to a maximum of 1.43). Higher values of nβ would require a larger U_A and/or larger angles of incidence. Advantages of the described optical read-out combined with magnetic labels for actuation are the following:

Cheap cartridge: The carrier cartridge 11 can consist of a relatively simple, injection-molded piece of polymer material that may also contain fluidic channels. - Large multiplexing possibilities for multi-analyte testing: The binding surface 12 in a disposable cartridge can be optically scanned over a large area. Alternatively, large-area imaging is possible allowing a large detection array. Such an array (located on an optical transparent surface) can be made by e.g. ink-jet printing of different binding molecules on the optical surface. The method also enables high-throughput testing in well-plates by using multiple beams and multiple detectors and multiple actuation magnets (either mechanically moved or electro-magnetically actuated).

Actuation and sensing are orthogonal: Magnetic actuation of the magnetic particles (by large magnetic fields and magnetic field gradients) does not influence the sensing process. The optical method therefore allows a continuous monitoring of the signal during actuation. This provides a lot of insights into the assay process and it allows easy kinetic detection methods based on signal slopes.

The system is really surface sensitive due to the exponentially decreasing evanescent field. - Easy interface: No electrical interconnect between cartridge and reader is necessary. An optical window is the only requirement to probe the cartridge. A contact-less read-out can therefore be performed. Low-noise read-out is possible.

In the following, a typical measurement that can be executed with the described device will be explained in more detail. Figure 1 shows in this respect the time point ti of the beginning of the whole measurement procedure, when the sample with magnetic particles 1 as labels of the target components is introduced in the initially empty sample chamber 2. The measurements may already be running, yielding a "dry- well-signal" S_dr that is indicated in the diagram on the right-hand side of the Figure. It is assumed in this context that the signal s depends in a monotonically increasing way on the amount of magnetic particles 1 at the binding surface 12 (while the latter is inversely related to the amount of totally internally reflected light L2). As there are no particles at the binding surface at time ti, the "dry- well — signal" sa_r has a minimal value. The aforementioned "dry- well- signal" sa_r could be used as a reference to eliminate well-to-well variations such as optical alignment, lens reflection and transmission differences. In an ideal situation, a normalization with respect to this "dry- well-signal" should be preferable: due to the choice of incident angle, the signal should be exactly the same for the dry sample chamber and for a sample chamber with liquid (but no beads near the surface). However, experiments show that there is a small but significant signal change upon injection of the sample liquid into the empty well. This is exaggeratedly illustrated in Figure 2 which shows the apparatus at a time t₂ after the sample fluid has filled the sample chamber 2. The electromagnet 41 has been positioned above the sample chamber to attract with its gradient field B the magnetic particles 1 to the top surface and keep them away from the binding surface 12. Of course also a second magnet could be used for this purpose, which would avoid the need for moving one magnet 41 around the sample chamber. Due to the presence of the sample fluid at the binding surface 12, some disturbance of the total internal reflection may take place, leading to the increase of the signals to a "background reference value" S_bg as shown in the diagram. The increase of the signal from the zero- signal to the background reference value S_bg is caused by small disturbances and imperfect smoothness of the detection surface. This leads to some amount of scattering and influences the intensity of the reflected beam. Depending on the refractive index difference with the surrounding medium (liquid or air), the scattering and the total internal reflection will be affected, leading to a slightly different optical signal. One solution to this problem is to require a perfectly smooth detection surface. In practice, a high-grade optical quality (λ/10 or better smoothness) can be achieved also in injection-molded plastic products. An alternative and more attractive solution to this problem requires that normalization should be done with a reference equal to the optical signal obtained when a medium with preferably the same refractive index as the liquid under test (and no beads) is present on the detection surface, i.e. with the background reference signal S_bg defined above.

A background reference value of the aforementioned kind could alternatively be obtained by first measuring a reference sample, e.g. water or buffer liquid, without target components and/or magnetic particles 1, and then introducing the actual sample (with target components and magnetic particles 1).

The background reference signal S_bg should be taken immediately after injection of the liquid under test. Due to the finite diffusion time of the beads, there will initially be no beads on the binding surface. With respect to the aforementioned use of a reference sample, this has the advantage that exactly the same liquid (refractive index) is used. A further improvement is achieved by the additional precaution shown in Figure 2, i.e. by using a magnet for magnetic washing during fluid injection and reference signal acquisition.

Experimental tests with a wash liquid as reference sample and human blood as actual sample have shown that the signal change from an empty well to the level directly after blood injection ranges from 0.5 to over 2 %. Although the wash liquid was not tuned to have the same refractive index as the blood, the signal change compared to this level was much smaller, the largest value being 0.7 %. Experiments with reference measurements during magnetic washing will be discussed in connection with Figures 5 and 6. At a time t₃ shown in Figure 3, the magnet 41 has been repositioned below the sample chamber and therefore attracts all magnetic particles 1 to the binding surface 12, thus increasing the diffusion velocity. When the detected signal s is saturated, i.e. its changes are smaller than a predetermined value as approximately all particles 1 are at the binding surface 12, then the corresponding "saturation reference value" s_sat is stored. Due to the high density of the particles 1 at the binding surface, the saturation reference value s_sat represents the upper threshold of the measurement values.

Figure 4 shows the final time point U- The magnet 41 is again disposed above the sample chamber, pulling the magnetic particles 1 away from the binding surface 12. The strength of the magnetic field B is however adjusted in such a way that only unbound particles 1 move to the top while magnetic particles 1' that are bound to the binding sites of the binding surface 12 are left unaffected. This process can again be monitored, i.e. when the change in the signal s becomes smaller than a predetermined threshold, the signal s is stored as the "signal value" s_slg. This signal value will lie somewhere between the background reference value S_bg and the saturation reference value s_sat.

For a water-like sample liquid, the background level S_bg will usually be much lower than the saturated signal level s_sat, with the detection signal s_slg in between, depending on the concentration of target molecules. A normalized and background- corrected value f reflecting the concentration as a fraction of the maximum (saturated) value, is given for example by

I ⁼ (Ssig ^~ Sbg) / (S_sat — Sb_g), i.e. signal s_slg minus background S_bg divided by total signal range (s_sat - S_bg). To obtain a quantitative value for the concentration of target molecules, some external calibration factor or formula can be applied to this corrected signal f.

Depending on the type of sample liquid and the presence of other contaminants (e.g. blood cells or other constituents), it could be possible that the background level S_bg is equal to or even higher than the saturation level s_sat. In the latter case, detection is still possible: the detection level will still be in between the background and the saturation level. Only when the background and saturation levels are very close or equal, it is clear that a reliable detection is not possible. In that case, an error message can be generated by the evaluation and recording module 32, and alternative measurements are necessary. More generally, a reliability measure can be generated from the absolute difference D between background and saturation values, i.e. from

D = (S_sat - S_bg). Thus a larger value of D typically corresponds to a larger reliability, while a value close to zero is very unreliable.

Further information can be obtained by comparing the difference D with typical values for the type of liquid, as specified by the user or inherently from the type of test. When the measured value D deviates significantly from the standard value, stored in a local memory, a warning signal can be given. As an example, when the sensor module expects to deal with saliva, and quite different values are found for sa_r, S_bg, s_sat, and/or their differences, then it is clear that something could be wrong (e.g. different or modified liquid, different amount of beads, system malfunction, ...).

In summary, an important aspect of the presented approach is that the sensor device applies an orthogonal detection method, i.e. a method that is independent of applied magnetic fields. This allows real-time monitoring of preparation, measurement and washing steps, which is not possible using the prior art method employing a magnetic sensor. The monitor signals can be used to control the measurement or the individual process steps, but also to calibrate and check the signal levels. For example, to exclude influences from incident light intensity (light power variations in the source), external contamination (e.g. fingerprints) or manufacturing inaccuracies of the sample chamber, etc., independent calibration is necessary. A method for such a calibration and further checks, which are essential steps to ensure a reliable measurement, were explained in detail above.

Figure 5 shows normalized optical signals S (vertical axis) measured over time t (horizontal axis, in arbitrary units) in an experimental setup like that of Figures 1 to 4. The optical signal S may e.g. represent the light intensity of the output light beam L2, and it should be noted that it is inversely related to the signals s in Figures 1 to 4 (i.e. S decreases if s increases and vice versa). The five different curves of the diagram correspond to measurements in different sample chambers ("wells") of a well plate and comprise the phases of presence of a wash liquid in the sample chamber (state (W)); removal of the wash liquid and filling of the sample chamber with air to yield the state of an "empty" sample chamber (state (E)); removal of the air and filling of the sample chamber with human blood (yielding state (B)).

The Figure shows that the optical signal S can change by more than 1% upon injection of e.g. water into an empty well, and that the reverse effect occurs when a filled well is emptied. The amplitude of the signal change varies from well to well, but it is reproducible for the same well. It is supposed that this effect is caused by small disturbances and an imperfect smoothness of the contact surface, which leads to some amount of scattering and influences the intensity of the reflected beam. Depending on the refractive index difference with the surrounding medium (liquid or air), the scattering and the total internal reflection will be affected, leading to a slightly different optical signal. Figure 5 shows experimental results in relation to the above described processes. The diagram refers to a two-step PTH (PTH = parathyroid hormone) assay, wherein the optically detected, normalized signals S are plotted as function of time t. In this experiment, measurements were started with a dry well prepared with various concentrations of PTH. At t = 12 s, a buffer liquid with 300 nm beads was injected into the well, followed by magnetic attraction (permanent magnet) from t = 32 to t = 62 s, then binding of the beads, and finally magnetic washing (dipping a permanent magnet in the liquid) at t = 240 s. Upon injection of the liquid, a signal drop is observed ranging from about 0.25 % to more than 2 %, independent of concentration. Signals are normalized to the signal level directly after liquid injection (here t = 12 s). The experiment shows that diffusion of beads is rather slow: without magnetic actuation, the signal only drops very little in a period of almost 20 seconds (from t = 12 to t = 32 s). This means that the background reference signal taken at t = 12 s is quite reliable (no beads on the surface). Without proper normalization, it would not be possible to reliably detect the lowest concentrations of 0.04 ng/ml and even 0.4 ng/ml (because the signal change with respect to zero is less than the well-to- well variation upon injection). While the invention was described with reference to particular embodiments, various modifications and extensions are possible, for example:

The sensor can be any suitable sensor to detect the presence of magnetic particles on or near to a sensor surface, based on any property of the particles, e.g. it can detect via magnetic methods, optical methods (e.g. imaging, fluorescence, chemiluminescence, absorption, scattering, surface plasmon resonance, Raman, etc.), sonic detection (e.g. surface acoustic wave, bulk acoustic wave, cantilever, quartz crystal etc), electrical detection (e.g. conduction, impedance, amperometric, redox cycling), etc.

In addition to molecular assays, also larger moieties can be detected with sensor devices according to the invention, e.g. cells, viruses, or fractions of cells or viruses, tissue extract, etc.

The detection can occur with or without scanning of the sensor element with respect to the sensor surface.

Measurement data can be derived as an end-point measurement, as well as by recording signals kinetically or intermittently.

The particles serving as labels can be detected directly by the sensing method. As well, the particles can be further processed prior to detection. An example of further processing is that materials are added or that the (bio)chemical or physical properties of the label are modified to facilitate detection.

The device and method can be used with several biochemical assay types, e.g. binding/unbinding assay, sandwich assay, competition assay, displacement assay, enzymatic assay, etc. It is especially suitable for DNA detection because large scale multiplexing is easily possible and different oligos can be spotted via ink-jet printing on the optical substrate. - The device and method are suited for sensor multiplexing (i.e. the parallel use of different sensors and sensor surfaces), label multiplexing (i.e. the parallel use of different types of labels) and chamber multiplexing (i.e. the parallel use of different reaction chambers).

The device and method can be used as rapid, robust, and easy to use point-of-care biosensors for small sample volumes. The reaction chamber can be a disposable item to be used with a compact reader, containing the one or more field generating means and one or more detection means. Also, the device, methods and systems of the present invention can be used in automated high- throughput testing. In this case, the reaction chamber is e.g. a well-plate or cuvette, fitting into an automated instrument.

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.

Claims

CLAIMS:

1. A method for the detection of target components with label particles (1) in a sample, comprising the following steps: a) letting the target components bind to a binding surface (12); b) measuring at least one "reference value" (s_bg, s_sat) for the amount of target components at the binding surface (12) before and/or after step a); c) removing unbound target components from the binding surface (12); d) measuring a "signal value" (s_slg) for the amount of target components at the binding surface (12); e) relating the signal value (s_slg) to the reference value (s_bg, s_sat).

2. The method according to claim 1, characterized in that target components are forcibly moved in the sample, particularly towards or away from the binding surface (12), by electrical or magnetic fields (B) acting on their label particles (1).

3. The method according to claim 1, characterized in that the amount of target components at the binding surface (12) is measured optically, particularly via an interaction with evanescent light waves.

4. The method according to claim 1, characterized in that the amount of target components at the binding surface (12) is monitored to determine the point in time when the reference value (s_bg, s_sat) and/or the signal value (s_slg) is measured.

5. The method according to claim 1, characterized in that a "background reference value" (s_bg) is measured while a minimal amount of target components is present at the binding surface (12).

6. The method according to claim 5, characterized in that the background reference value (s_bg) is measured while a reference sample without target components and/or label particles contacts the binding surface.

7. The method according to claim 5, characterized in that the background reference value (s_bg) is measured immediately after the sample has contacted the binding surface (12).

8. The method according to claim 5, characterized in that target components are repelled from the binding surface (12) by electrical or magnetic fields (B) acting on their label particles (1) during the measurement of the background reference value (s_bg).

9. The method according to claim 1, characterized in that a "saturation reference value" (s_sat) is measured before unbound target components are removed from the binding surface (12).

10. The method according to claim 9, characterized in that the saturation reference value (s_sat) is measured while target components are attracted to the binding surface (12) by electrical or magnetic fields (B) acting on their label particles (1).

11. The method according to claim 1 , characterized in that the signal value (s_slg) is corrected by the at least one reference value (s_bg, s_sat).

12. The method according to claim 1, characterized in that a reliability measure is determined from the at least one reference value (s_bg, s_sat).

13. The method according to claim 1, characterized in that the reference value (s_bg, s_sat) is compared with a range of allowed values to detect an error state.

14. A microelectronic sensor device for the detection of target components with label particles (1) in a sample, comprising: a) a sample chamber (2) with a binding surface (12); b) a sensor unit (21, 31) for measuring the amount of target components at the binding surface (12); c) a field generator (41) for generating a electrical or magnetic field(B) that can affect the label particles (1); d) a controller (32) for controlling the sensor unit and the field generator such that at least one "reference value" (s_bg, s_sat) and a "signal value" (s_slg) are determined and related to each other in a method according to claim 1.