Method of detecting label particles

Abstract

The invention relates to a method for the detection of target components that comprise label particles, for example magnetic particles (1). The method includes (a) collecting the target components at a binding surface (12, 112, 512) of a carrier (11, 111, 211, 311, 411, 511); (b) directing an input light beam (L1, L1a, L1b) into the carrier such that it is totally internally reflected in an investigation region (13, 313a, 313b) at the binding surface (12, 112, 512); and (c) determining the amount of light of an output light beam (L2, L2a, L2b) that comprises at least some of the totally internally reflected light. Evanescent light generated during the total internal reflection is affected (absorbed, scattered) by target components and/or label particles (1) at the binding surface (12) and will therefore be missing in the output light beam (L2). This can be used to determine the amount of target components at the binding surface (12) from the amount of light in the output light beam (L2, L2a, L2b). A magnetic field generator (41) is optionally used to generate a magnetic field (B) at the binding surface (12) by which magnetic label particles (1) can be manipulated, for example attracted or repelled.

Claims

1. A method for detection of target components comprising label particles, comprising: a) collecting the target components comprising label particles at a binding surface of a carrier, said label particles including light scattering and/or light absorbing particles; b) directing an input light beam into the carrier such that it is totally internally reflected in an investigation region at the binding surface, wherein at least a portion of the input light beam that is totally internally reflected leaves the binding surface as an output light beam; c) measuring an amount of light in the output light beam; and d) determining an amount of light of the input light beam that is missing in the output light beam due to scattering and/or absorbing of the input light beam by the label particles based on the measured amount of light in the output light beam to indicate a presence and/or an amount of the target components at the binding surface.

2. The method according to claim 1, wherein the label particles are manipulated by a magnetic or electrical field that attracts or repels the label particles from the investigation region.

3. The method according to claim 1, wherein determining the amount of light of the input light beam that is missing in the output light beam due to scattering and/or absorbing of the input light beam by the label particles includes steps of: measuring the amount of light in the input light beam; measuring the amount of light in the output light beam; and relating the measured amount of light in the input light beam to the measured amount of light in the output light beam.

4. The method according to claim 3, further comprising the step of indicating the presence and/or the amount of the target components at the binding surface based on the amount of light of the input light beam that is missing in the output light beam due to scattering and absorbing of the input light beam by the label particles.

Description

(1) 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:

(2) FIG. 1 schematically shows the general setup of a microelectronic sensor device according to the present invention;

(3) FIG. 2 shows the angles of incidence when the input light beam and the output light beam are oriented under Brewster angle;

(4) FIG. 3 shows a microelectronic sensor device with a well having a spherical bottom;

(5) FIG. 4 shows the design of FIG. 3 with additional means for focusing a light beam;

(6) FIG. 5 shows a well having a plurality of hemispheres at the bottom;

(7) FIG. 6 shows a well having a bottom in the form of a truncated pyramid;

(8) FIG. 7 shows the design of FIG. 6 with a cavity for an electromagnet;

(9) FIG. 8 is a diagram showing a normalized measurement signal s over time t for solutions with different concentrations of morphine labeled with magnetic particles;

(10) FIG. 9 is a diagram showing a normalized measurement signal s over time t for solutions containing morphine labeled with magnetic particles and different concentrations of free morphine;

(11) FIG. 10 illustrates the formation of pillars of magnetic beads in a magnetic field;

(12) FIG. 11 is a diagram showing a normalized measurement signal s over time t for solutions with different concentrations of morphine labeled with magnetic particles when only one step of magnetic attraction is applied;

(13) FIG. 12 is a diagram showing a normalized measurement signal s over time t for solutions containing saliva and morphine labeled with magnetic particles;

(14) FIG. 13 is a diagram showing a normalized measurement signal s over time t for a two-step PTH assay in comparison to a solution containing no PTH;

(15) FIG. 14 shows a dose-response curve for PTH in buffer for optical detection;

(16) FIG. 15 is a diagram like that of FIG. 13 for different concentrations of PTH;

(17) FIG. 16 shows a dose-response curve for PTH in buffer and in blood for optical detection;

(18) FIG. 17 shows a bead-response curve for detection with a GMR sensor;

(19) FIG. 18 shows a bead-response curve for optical detection.

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

(21) FIG. 1 shows the general setup of a microelectronic sensor device according to the present invention. A central component of this device is the 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 superparamagnetic 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).

(22) 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.

(23) 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.

(24) The sensor device further comprises a light source 21, for example a laser or an LED, that generates an input light beam L1 which is transmitted into the carrier 11. The input light beam L1 arrives at the binding surface 12 at an angle larger than the critical angle θ.sub.c 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 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 output 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.

(25) In the light source 21, a commercial DVD (λ=658 nm) laser-diode can be used. A collimator lens may be used to make the input light beam L1 parallel, and a pinhole 23 of e.g. 0.5 mm may be used to reduce the beam diameter. For accurate measurements, a highly stable light source is required. However, even with a perfectly stable power source, temperature changes in the laser can cause drifting and random changes in the output.

(26) To address this issue, the light source may optionally have an integrated input light monitoring diode 22 for measuring the output level of the laser. The (low-pass filtered) output of the monitoring sensor 22 can then be coupled to the evaluation module 32, which can divide the (low-pass filtered) optical signal from the detector 31 by the output of the monitoring sensor 22. For an improved signal-to-noise ratio, the resulting signal may be time-averaged. The division eliminates the effect of laser output fluctuations due to power variations (no stabilized power source needed) as well as temperature drift (no precautions like Peltier elements needed).

(27) A further improvement can be achieved if not (or not only) the laser output itself is measured, but the final output of the light source 21. As FIG. 1 coarsely illustrates, only a fraction of the laser output exits the pinhole 23. Only this fraction will be used for the actual measurement in the carrier 11, and is therefore the most direct source signal. Obviously, this fraction is related to the output of the laser, as determined by e.g. the integrated monitor diode 22, but will be affected by any mechanical change or instability in the light path (a laser beam profile is approximately elliptical with a Gaussian profile, i.e. quite non-uniform). Thus, it is advantageous to measure the amount of light of the input light beam L1 after the pinhole 23 and/or after eventual other optical components of the light source 21. This can be done in a number of ways, for example: a parallel glass plate 24 can be placed under 45° or a beam splitter cube (e.g. 90% transmission, 10% reflection) can be inserted into the light path behind the pinhole 23 to deflect a small fraction of the light beam towards a separate input-light monitoring sensor 22′; a small mirror at the edge of the pinhole 23 or the input light beam L1 can be used to deflect a small part of the beam towards a detector.

(28) The Figure shows a “second 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 input light beam L1. As this fluorescence light is usually emitted isotropically to all sides, the second 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.

(29) The described microelectronic sensor device applies optical means for the detection of magnetic particles 1 and the target components for which detection is actually of interest. 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.

(30) According to Snell's law of refraction, the angles θ.sub.A and θ.sub.B with respect to the normal of an interface between two media A and B satisfy the equation
n.sub.A sin θ.sub.A=n.sub.B sin θ.sub.B

(31) with n.sub.A, n.sub.B 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 n.sub.A=2) will for example refract away from the normal under an angle θ.sub.B at the interface with a medium B with lower refractive index such as air (n.sub.B=1) or water (n.sub.B≈1.3). A part of the incident light will be reflected at the interface, with the same angle as the angle θ.sub.A of incidence. When the angle θ.sub.A of incidence is gradually increased, the angle θ.sub.B of refraction will increase until it reaches 90°. The corresponding angle of incidence is called the critical angle, θ.sub.c, and is given by sin θ.sub.c=n.sub.B/n.sub.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:

(32) $\exp \left(-k\sqrt{n_A^2{\sin }^2\left({\theta }_A\right)-n_B^2}.Math.z\right)$

(33) with k=2π/λ, θ.sub.A being the incident angle of the totally reflected beam, and n.sub.A and n.sub.B the refractive indices of the respective associated media.

(34) For a typical value of the wavelength λ, e.g. λ=650 nm, and n.sub.A=1.53 and n.sub.B=1.33, the field amplitude has declined to exp(−1)≈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 FIG. 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.

(35) 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.

(36) 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.sub.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 n.sub.A=1.52, n.sub.B is allowed up to a maximum of 1.43). Higher values of n.sub.B would require a larger n.sub.A and/or larger angles of incidence.

(37) 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.

(38) FIG. 2 illustrates in more detail the angles of incidence of the input light beam L1 and the output light beam L2 at the entrance window 14, the binding surface 12, and the exit window 15 of a carrier 11. When the entrance and exit windows 14, 15 are orthogonal to the incoming beam, normally a part of the light (typically around 4%) is reflected back, causing e.g. in the light source 21 non-desirable output fluctuations of a laser (called “laser feedback”). This distorts the measurement. Furthermore, interference effects can occur in the light detector at the detection side as well, as typically a perpendicular orientation is used here, too, and a coherent light source (laser) is used.

(39) Due to unwanted heating of the measurement cartridge, which occurs e.g. due to the heating of the actuation magnets 41 during operation or due to other external factors, a slight shift of the positions of the carrier's facets can lead to slow variations of the intensity, on both the light source and the detection branch of the setup, that are difficult to eliminate from the measurement. By placing the light detector at an angle (rather than perpendicular) with respect to the incoming output light beam L2, some of the problems occurring at the detection side of the setup can already be eliminated effectively. At the light source side, however, this is not possible.

(40) It is therefore desirable to make the entrance and exit windows 14, 15 of the carrier 11 such that reflections are eliminated without the use of expensive optical anti-reflex coatings.

(41) To solve this problem, it is proposed to place the entrance and exit windows 14, 15 under Brewster angle with respect to the incoming light beam, and to provide this beam with a (linear) polarization in the plane of incidence (called “p-polarization”). As is known from optics (cf. e.g. Pedrotti & Pedrotti, Introduction to Optics, Prentice Hall), a reflected beam vanishes if the p-polarized incident beam hits the surface of a (transparent) medium under Brewster angle.

(42) A p-polarized input light beam L1 can be achieved by choosing the right orientation of a semiconductor laser in the light source, or by using a half wave plate to rotate the polarization to the correct orientation.

(43) The propagation of the input light beam L1 inside the carrier 11 is fixed due to the fact that it should impinge on the binding surface 12 with an angle θ.sub.3 larger than the critical angle θ.sub.c of TIR. This fixes also the orientation of the entrance and exit windows or facets of the carrier 11, if their angle θ.sub.2 or θ.sub.6, respectively, with the refracted beam shall correspond to the Brewster angle. This in turn fixes the direction of the input light beam L1 and the output light beam L2.

(44) The angle of incidence θ.sub.1 of the input light beam L1 is equal to Brewster angle when the sum of this angle and the angle θ.sub.2 of refraction is 90°. This condition in combination with Snell's law leads to the following formula for the angle of incidence at Brewster angle:
tan(θ.sub.1)=n.sub.2/n.sub.1,

(45) where n.sub.1 is the refractive index of the medium in which the input light beam L1 propagates before refraction, normally air, and n.sub.2 of the medium where the refracted ray propagates, normally the plastic of the carrier (e.g. polycarbonate, zeonex or polystyrene).

(46) For the angle θ.sub.2 of the refracted beam one finds
tan(θ.sub.2)=n.sub.1/n.sub.2.

(47) Another condition that needs to be satisfied is that the input light beam L1 should be incident at an angle θ.sub.3 close to but beyond the critical angle θ.sub.c of total internal reflection at the binding surface 12, i.e.
θ.sub.3>θ.sub.c with sin(θ.sub.c)=n.sub.3/n.sub.2,

(48) where n.sub.3 is the refractive index of the medium above the binding surface 12. Furthermore, the angles at the side of the output light beam L2 are mirrored with respect to the input side, i.e.
θ.sub.4=θ.sub.3, θ.sub.5=θ.sub.2, and θ.sub.6=θ.sub.1.

(49) For typical values of n.sub.1=1 (air), n.sub.2=1.5 (transparent plastic), and n.sub.3=1.3 (water like), the following figures can be derived: θ.sub.3=θ.sub.4>60°, θ.sub.1=θ.sub.6=56°, θ.sub.2=θ.sub.5=34°.

(50) By placing the entrance and exit windows of the carrier at Brewster angle, unwanted reflections back into the laser are prevented without the need of an expensive anti-reflection coating. Furthermore, by placing the detector at an angle, rather than perpendicular, interference effects on the detector side can be prevented as well. By doing so, expansion or shrinkage of the carrier/cartridge during a measurement, e.g. due to thermal effects, will not influence the measurement result.

(51) In the environment of a laboratory, well-plates are typically used that comprise an array of many sample chambers (“wells”) in which different tests can take place in parallel. FIGS. 3-7 show different possible embodiments of one well of such a well-plate that are particularly suited for an application of the explained measurement principle. The production of these (disposable) wells is very simple and cheap as a single injection-moulding step is sufficient.

(52) The light source 121 shown in FIG. 3 is arranged to produce a parallel light beam L1, incident at the well bottom surface at an angle larger than the critical angle θ.sub.c. To prevent excess reflection of this input light beam L1 at the first interface from air to the carrier 111 (e.g. glass or plastic material), the bottom of the well comprises a hemispherical shape 114 of radius R, with its centre coinciding with the detection surface 112. The input light beam L1 is directed towards this same centre. At the reflection side, a photodetector such as a photodiode 131 is positioned to detect the intensity of the output light beam L2. A typical diameter D of the well 102 ranges from 1 to 8 mm. The Figure further indicates a magnet 141 for generating magnetic actuation fields inside the well 102 (this magnet is not shown in the following Figures for simplicity).

(53) FIG. 4 shows an alternative embodiment in which the light source comprises some optical element like a lens 222 to produce an input light beam L1 which is substantially focused to the centre of the hemisphere 214. At the detection side, a similar optical element 232 can be used to collect and detect the light intensity of the output light beam L2.

(54) In a further development of the measuring procedure, multiple input light beams and output light beams can be used to simultaneously detect the presence of different target molecules at different locations in the same well. FIG. 5 shows in this respect a well with multiple hemispheres 314a, 314b on the well bottom that can be used to couple the light from multiple input light beams L1a, L1b to respective investigation regions 313a, 313b on the bottom of the well. Multiple photodetectors (not shown) may be used in this case to measure the multiple output light beams L2a, L2b.

(55) FIG. 6 shows an alternative embodiment in which a prism or truncated pyramidal structure 414 is used to couple the light of the input light beam L1 and the output light beam L2. The sloped edges of the pyramid should be substantially perpendicular to these light rays. Advantages of this design are that it is simple to produce and does not block beams from neighboring areas. Neighboring wells are indicated in this Figure by dashed lines.

(56) As indicated in FIG. 6, it is possible to use a single, parallel input light beam L1 with a diameter covering all detection areas on the well bottom. As a detector, multiple photodiodes can be used, aligned with each individual detection area. Alternatively, a CCD or CMOS chip (not shown) such as used in a digital camera can be used to image the reflected intensity response of the entire well bottom, including all detection areas. Using appropriate signal processing, all signals can be derived as with the separate detectors, but without the need for prior alignment.

(57) FIG. 7 shows a further embodiment in which the well bottom 511 comprises an open cavity 515 with its center outside the optical path of the input light beam(s) L1 and the output light beam(s) L2. This allows the following advantageous features: A (T-shaped) ferrite core 542 of a magnetic coil 541 for improved field intensity and concentration can be placed close to the binding surface 512, allowing a compact and low-power design. A self-aligning structure is achieved: if the optics and the magnetic field generator 541 are fixed, an auto-alignment of the well on the ferrite core 542 takes place.

(58) The magnetic beads 1 that are used in the described embodiments of the invention are typically poly-styrene spheres filled with small magnetic grains (e.g. of iron-oxide). This causes the beads to be super-paramagnetic. The refractive index of poly-styrene is nicely matched to the refractive index of a typical substrate material of well-plates. In this way optical outcoupling of light is enhanced.

(59) Experimental Results A

(60) In the following, some experimental results will be described that were obtained in a setup with a well-plate like that of FIG. 3. Standard 96 wells polystyrene titerplates were used with a flat bottom (6 mm in diameter, about 1 mm bottom thickness). To get the hemispherical bottom, glass lenses were attached to the bottom using refractive index matched immersion oil (n=1.55). The glass lenses were polished down from a hemispherical shape (6 mm diameter) to a thickness of 2 mm. The model assay chosen for the set of experiments is drugs of abuse in saliva. Drugs of abuse are generally small molecules that only possess one epitope and for this reason cannot be detected by a sandwich assay. A competitive or inhibition assay is the method to detect these molecules. A well-known competitive assay setup is to couple the target molecules of interest onto a surface, and link antibodies to a detection tag (e.g. enzyme, fluorophore, or magnetic particle). This system was used to perform a competitive assay between the target molecules from the sample and the target molecules on the surface, using the tagged antibodies. The tag in these experiments was a magnetic particle. Upon actuation, a permanent magnet was placed under the well by mechanical movement. The distance between the bottom of the well and the magnet was about 2 mm. A permanent magnet in the well was used for magnetic washing.

(61) FIG. 8 shows the normalized measurement signal s over time t for a first sensitivity test. For that, the bottom of a well was prepared for detection of the target molecules. The target under investigation was morphine. Morphine is a small molecule, with only one epitope, so a competitive assay has to be performed to indicate the amount of morphine in a sample. A clear polystyrene surface (96 wells titerplate) was coated for 2 hrs with a range of concentrations of BSA-morphine from 1 pg/ml to 1 μg/ml. Then functionalized superparamagnetic nanoparticles “MP” (300 nm Carboxyl-Adembeads functionalized with monoclonal anti morphine antibodies) solved in PBS+10 mg/ml BSA+0.65% Tween-20 were inserted into the wells (1:20 dilution of MPs, total amount of solution was 50 μl). The MPs were attracted to the surface by alternated application of magnetic forces (in the order of 10 fN) as indicated by symbol A in FIG. 8. In the end, unbound particles were removed from the surface by a washing step, indicated by symbol W in FIG. 8. The Figure shows that the lowest concentration of BSA-morphine (10 pg/ml) yields the largest dynamic measurement range. Also, the steepness of the curve after actuation is the largest, enabling fast response/short measuring time and the highest sensitivity.

(62) To test the sensitivity of the assay, the ability of free morphine to compete for functionalized MP binding to the surface was tested. FIG. 9 shows the resulting normalized signal s collected by the detector as a function of time t. A clear polystyrene surface (96 wells titerplate) was coated for 2 hrs with 10 pg/ml BSA-morphine. MPs functionalized with anti-morphine antibodies premixed with a defined amount of free morphine solved in PBS+10 mg/ml BSA+0.65% Tween-20 were inserted into the wells (1:20 dilution of MPs, total amount of solution was 40 μl). As described above and indicated in the Figure, the MPs were actuated four times at t=30 s, t=140 s, t=210 s, t=290 s during 15 sec (cf. symbol A). At t=390 s the non-bound MPs have been removed from the well by means of magnetic washing W, i.e. the non-bound MPs are removed by applying a magnetic force using a permanent magnet in the fluid above the binding surface.

(63) It can be seen from the Figure that for the highest concentrations of free morphine, the signal reduction (after magnetic washing W) is low, while for a low concentration of free morphine the signal reduction is high (a high concentration of MPs on the surface leading to a clear reduction in signal after magnetic washing W).

(64) The signal reduction during actuation and magnetic relaxation as found in these experiments, together with information already collected from microscopic investigations proposes the following interpretation of the results: Upon magnetic actuation, MPs are concentrated to the surface, without showing an increase in binding to the surface (no signal decrease). Upon removing of the magnetic field, the signal drops indicating MPs binding to the surface. Application of a magnetic field then induces pillar formation: MPs become magnetized and those freely movable (a-specifically bound MPs and MPs freely in solution) will bind to the specific bound MPs in the direction of the magnetic field lines, which are perpendicular to the binding surface. This state is illustrated in FIG. 10, which also indicates the evanescent field EF. Since the evanescence detection system will only detect MPs at the surface, pillar formation during magnetic actuation will result in a reduction in signal change. Upon removal of the magnetic field, the MPs will lose their magnetic property, and fall to the surface again where binding can take place.

(65) To obtain a fast assay, the actuation scheme can be optimized using the above results. FIG. 11 shows a dose-response curve on polystyrene wells coated with 10 pg/ml BSA-morphine. MPs functionalized with anti-morphine antibodies premixed with a defined amount of free morphine solved in PBS+10 mg/ml BSA+0.65% Tween-20 were inserted into the wells (1:20 dilution of MPs, total amount of solution was 40 μl, final morphine concentrations between 1 and 1000 ng/ml). MPs were actuated at A using a permanent magnet below the well for 15 seconds, to up-concentrate the MPs near the surface. Next, the MPs were allowed to bind to the surface for 60 seconds. The data show that already after 20 seconds the binding rate of magnetic particles to the surface is a direct measure for the concentration of free morphine in solution. This means that the measurement procedure can be simplified and more rapid, since no washing step is needed. For this to occur rapidly, the magnetic upconcentration step A is necessary.

(66) Next, the background signal from saliva was tested. Filtered saliva was introduced in a well and the signal was followed for 120 seconds. The background is negligible as can be seen in FIG. 12. As a comparison, the signal of MPs in PBS+10 mg/ml BSA+0.65% Tween20 mixed with 0.1 ng/ml morphine is included as well. At t=13 s, both the saliva (SL) and the morphine solution+MPs were injected. It can be seen that the background signal from the saliva is <1% and can be neglected.

(67) Experimental Results B

(68) To verify the sensitivity of the detection method a two-step PTH (PTH=parathyroid hormone) assay was carried out in the described well-plates on an optical substrate. In FIG. 13 the signal transients s (arbitrary units) are plotted as function of time t for both a blanc (0 nM, upper curve) and a relatively high (4 nM) concentration. A clear difference in the kinetic binding regime is observed and also a clear signal difference after washing W remains.

(69) In order to compare magnetic read-out (via Giant Magneto-Resistance (GMR) sensors as they are for example described in the WO 2005/010543 A1 or WO 2005/010542 A2) with optical read-out (via the principle of frustrated total internal reflection explained above), the PTH dose response curve is plotted in FIG. 14.

(70) The corresponding transient curves for the optically detected PTH assay are given in FIG. 15. The dose-response curve in FIG. 14 is measured in a buffer matrix. The curve shows the optical signal s as percentage of the signal caused by reflection from an empty substrate. It is interesting to note that the curve is linear on a log-log scale (similar to the magnetically detected curve). Furthermore, detection limits can be calculated according to ‘blanc+2*standard deviation of the blanc’ (wherein “blanc” indicates the signal level when testing a sample with zero target concentration).

(71) For the magnetic read-out this value is equal to 3 pM. For the optical experiment, which was done with a very basic experimental set-up, this value was equal to 13 pM. It can be concluded that both detection techniques seem to have the same sensitivity.

(72) Next it is very important to verify the background signal for the optical detection method when measuring in complex matrices. For this reason the same PTH assay was carried out in a blood matrix. From the results shown in FIG. 16 (Bld.=blood, Buf.=buffer) it is clear that the resulting dose-response very well matches the curve that was measured in buffer. Also the blank signal is very low. This nice property is attributed to the fact that the total internal reflection is caused by the refractive index difference between the optical substrate material and the matrix. The matrix can consist of different components such as plasma, (red blood) cells, etc. However, all these components have a significantly lower refractive index than the substrate material. Therefore, total internal reflection is not influenced by the matrix. Only when beads are bound (e.g. high-index polystyrene with magnetic grains) the total internal reflection is frustrated and a drop in reflected intensity can be measured.

(73) Experimental Results C

(74) An important proof for the proposed technology is the so-called bead-response curve. It gives an indication of the signal change per bead attached to the sensor surface. Ideally, detection of a single bead is possible (in presence of noise, disturbances). In this situation further improving the detection technology is not needed anymore. The biological detection limit can then only be improved by methods such as upconcentration of beads (in a catch-assay), etc. FIG. 17 shows the bead-response curve in case of detection with a GMR-type of sensor and 300 nm beads (Δs=signal change; BD=bead density; NB=number of beads). For these beads the detection limit was 3 beads on 40 μm.sup.2 for a sampling frequency of 1 Hz.

(75) In order to estimate the bead-response for optical detection, a number of samples (glass-slides) were prepared with various bead concentrations. The resulting surface coverage was determined using an optical microscope, followed by a measurement of the optical signal (change) s compared to a clean reference sample without beads. The experimental data as obtained with a simple set-up are plotted in FIG. 18. In this set-up, the noise level corresponds to a signal change at a surface coverage SC of 0.01%. These data show that the sensitivity of this technique is at least similar to state-of-the-art results using GMR sensors at the same bead concentrations.

(76) In summary, the invention relates to a microelectronic sensor device for the detection of target components that comprise label particles, for example magnetic particles 1. The sensor device comprises a carrier 11 with a binding surface 12 at which target components can collect and optionally bind to specific capture elements. An input light beam L1 is transmitted into the carrier and totally internally reflected at the binding surface 12. The amount of light in the output light beam L2 is then detected by a light detector 31. Evanescent light generated during the total internal reflection interacts with the label particles 1 bound to target particles at the binding surface 12 leading to absorption and/or scattering and will therefore be missing in the output light beam L2. This can be used to determine the amount of target components at the binding surface 12 from the amount of light in the output light beam L2, L2a, L2b. A magnetic field generator 41 is optionally used to generate a magnetic field B at the binding surface 12 by which the magnetic label particles 1 can be manipulated, for example attracted or repelled.

(77) The label particles 1 are for instance magnetic beads, which means magnetic particles MP, in an example paramagnetic, ferromagnetic, or super-paramagnetic particles or beads. These label particles 1 are subject to reflection of the light beam L1 impinging at the binding surface 12 of the carrier. The more label particles 1 are bound to the binding surface 12 the more the light beam L1 will not be totally internal reflected at the binding surface 12 but an evanescent wave will be generated. The light beam L2 reflected by the binding surface 12 is so called frustrated by the effect of scattering of the incoming light beam L1 by the label particles 1. The more label particles 1 bound to the binding surface the more the reflected light beam L2 is frustrated. The light detector 31, 131 measures the light beam L2 coming from the binding surface 12 and uses the reflected light beam L2 for measuring the amount of label particles 1 bound at the binding surface 12. The more label particles 1 bound to the binding surface 12 the more scattering of the light beam L1 due to the label particles 1 takes place with the generation of the evanescent wave. The label particles 1 enabling the effect described have for instance the following features. Magnetic beads of a width of roughly 300 nm of uniform superparamagnetic particles containing a polymer core shell structure. These label particles 1 or magnetic beads show a scatter of light beam L1 which is sufficient for detection of the reflected light beam L2 to determine the label particles 1. Label particles 1 of similar materials and widths are also feasible, for instance label particles 1 of a width of 200 nm. Nevertheless, the scatter of light from only the target components to which the label particles 1 bind is found not to be suitable for detection. This means the direct measurement of target components by frustrated internal reflection to measure the amount of these target components is not possible. The detection and subsequent calculation of the amount of target particles 1 is made possible by the scattering of light at the label particles 1. Herewith, no detection of fluorescence of fluorescent material is needed, as used in state of the art with other optical detection systems. Further in this context it is to be stressed that detection of fluorescence light emitted by the target components is an additional feature which is combinable with the described method and sensor device.

(78) While the invention was described above with reference to particular embodiments, various modifications and extensions are possible, for example: 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 jetprinting 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.

(79) 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.