WO2008155716A1

WO2008155716A1 - Microelectronic sensor device for detecting label particles

Info

Publication number: WO2008155716A1
Application number: PCT/IB2008/052378
Authority: WO
Inventors: Coen Adrianus Verschuren
Original assignee: Koninklijke Philips Electronics N. V.
Priority date: 2007-06-21
Filing date: 2008-06-17
Publication date: 2008-12-24
Also published as: EP2181322A1

Abstract

The application 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). At least two parts of a resulting output light beam (L2) which are composed of different fractions of (i) 'nominal light' (Lnom) that is totally internally reflected in the investigation region (13) in a predetermined way and (ii) light (Ls) from another origin are then detected by a light detector (31), wherein the latter part may for example comprise scattered light (Ls) of the input light beam (L1). 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). Moreover, the amount of light (Ls) from other sources, e.g. scatter, can be estimated and used to correct the measurement results.

Description

MICROELECTRONIC SENSOR DEVICE FOR DETECTING LABEL PARTICLES

The invention relates to a microelectronic sensor device and a method for optical examinations in an investigation region at a binding surface of a carrier, particularly for the detection of target components, for example biological molecules, comprising label particles. Moreover, it relates to a light detector that is particularly suited for such a sensor device.

The US 2005/0048599 Al discloses a method for the investigation of microorganisms that are tagged with particles such that a (e.g. magnetic) force can be exerted on them. In one embodiment of this method, a light beam is directed through a transparent material to a surface where it is totally internally reflected. Light of this beam that leaves the transparent material as an evanescent wave is scattered by microorganisms and/or other components at the surface and then detected by a photodetector or used to illuminate the microorganisms for visual observation.

Based on this situation it was an object of the present invention to provide means for an improved detection of target components comprising label particles. In particular, it is desired that the method is simple and that its robustness, sensitivity and/or accuracy is improved with respect to the state of the art.

This object is achieved by a microelectronic sensor device according to claim 1, a light detector according to claim 12, and a method according to claim 14. Preferred embodiments are disclosed in the dependent claims.

The microelectronic sensor device according to the present invention serves for optical examinations in an investigation region at a binding surface of a carrier (wherein the investigation region and the carrier do not necessarily belong to the device). In this context, the term "examination" is to be understood in a broad sense, comprising any kind of manipulation and/or interaction of light with some entity in the investigation region. The investigation region will typically be a small volume at the surface of the (preferably transparent) carrier in which material of a sample to be examined can be provided. The examinations may preferably comprise the qualitative or quantitative detection of target components comprising label particles, wherein the target components may for example be biological substances like biomolecules, 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 carrier usually comprises a binding surface at which target components can collect. The term "binding surface" is chosen primarily as a unique reference to a particular part of the surface of the carrier, and though the target components will in many applications actually bind to said surface, this does not necessarily need to be the case. All that is required is that the target components can reach the binding surface to collect there (typically in concentrations determined by parameters associated to the target components, to their interaction with the binding surface, to their mobility and the like). The microelectronic sensor device comprises the following components: a) A light source for emitting a light beam, called "input light beam" in the following, into the carrier such that it is totally internally reflected in the investigation region at the binding surface of the carrier. The light source may for example be a laser or a light emitting diode (LED), optionally provided with some optics for shaping and directing the input light beam. The "investigation region" may be a sub-region of the binding surface or comprise the complete binding surface; it will typically have the shape of a substantially circular spot that is illuminated by the input light beam. Moreover, it should be noted that the occurrence of total internal reflection requires that the refractive index of the carrier is larger than the refractive index of the material adjacent to the binding surface. This is for example the case if the carrier is made from glass (n = 2) and the adjacent material is water (n = 1.3). It should further be noted that the term "total internal reflection" shall include the case called "frustrated total internal reflection", where some of the incident light is lost (absorbed, scattered etc.) during the reflection process. b) A light detector for determining the amount of light in at least two parts of an output light beam which are composed of different fractions of

(i) light that will be called "nominal light" in the following and that comprises light of the input light beam which was totally internally reflected in a predetermined ("nominal") way in the investigation region, and (ii) light from another origin. The "predetermined way" of total internal reflection typically corresponds to a reflection under ideal conditions, i.e. with all optical components having ideal geometric shapes and mutual position, all surfaces being ideally smooth, all materials being ideally pure etc. When the measurements of the light detector are evaluated, the underlying calculations will usually assume such ideal conditions. It is not necessary that the output light beam comprises all the light that was totally internally reflected in a nominal way (though this will preferably be the case), as some of this light may for example be used for other purposes or simply be lost, or that it completely consists of totally internally reflected light, as it may also comprise e.g. scattered light or fluorescence light. The detector may comprise any suitable sensor or plurality of sensors by which light of a given spectrum can be detected, for example photodiodes, photo resistors, photocells, a CCD chip, or a photo multiplier tube.

The described microelectronic sensor device allows for example a sensitive and precise quantitative or qualitative detection of target components in an investigation region at the binding surface. This is due to the fact that the totally internally reflected input light beam generates an evanescent wave that extends from the carrier surface a short distance into the adjacent material. If light of this evanescent wave is scattered or absorbed by target components or label particles present at the binding surface, it will be missing in the output light beam. The amount of light in the output light beam (more precisely the amount of light missing in the output light beam when compared to the input light beam) is therefore an indication of the presence and the amount of target components/labels at the binding surface. One advantage of the described optical detection procedure comprises its accuracy as the evanescent waves explore only a small volume of typically 10 to 300 nm thickness next to the binding surface, thus avoiding disturbances from the bulk material behind this volume. A high sensitivity is achieved when the reflected light is measured as all effects are detected that reduce the amount of totally internally reflected light. Moreover, the optical detection can optionally be performed from a distance, i.e. without mechanical contact between the carrier and the light source or light detector.

Besides the "nominal light" stemming from a (frustrated) total internal reflection that takes place in a predetermined way at the investigation region, the output light beam will often also comprise light from other origins, particularly light from the input light beam that was scattered for example at the optical entrance window of the carrier. As such scattered light directly depends on the amount of input light, it cannot be detected by modulations of the light source. To address this problem, the proposed microelectronic sensor device comprises a light detector that is adapted to measure at least two parts of the output light beam which are composed of different fractions of nominal light and light from another origin. Preferably one of the two light parts has known fractions of nominal light and/or "other light", because this may allow to estimate the absolute amount of nominal TIR light in the output light beam, i.e. the value one is interested in.

The "light from the other source" may particularly comprise (or completely consist of) scattered light. Scattered light is in practice always created due to e.g. inaccuracies of the optical components and may severely affect the measurements. The scattered light may possibly also have undergone total internal reflection in the investigation region, but not in a predetermined way (e.g. not at a predetermined angle or position).

The light detector of the microelectronic sensor device may preferably comprise at least two sensitive detection areas that provide individual measurement signals. If these detection areas are simultaneously illuminated by an output light beam, they can be used to separately determine the two light parts of the output light beam that are composed of different fractions of nominal light and light from other sources. Thus the light detector can for example be used for determining the amount of scattered light in the output light beam which in turn allows to find the amount of nominal light one is actually interested in. The two sensitive detection areas of the light detector typically lie within a circle with a diameter of less than 1 mm, preferably less than 10 μm. The aforementioned sensitive detection areas of the light detector may comprise at least one area that is circular, annular or rectangular. A circular sensitive detection area may preferably be used in the centre of the arrangement of sensitive detection areas to cover that cross section of the output light beam that is (inter alia) illuminated by nominal light. Annular or rectangular sensitive detection areas may be positioned around such a central detection area to measure light quantities that only consist of scattered light.

The light detector with several sensitive detection areas may particularly comprise a "central detection area" that corresponds to a region within the cross section of the output light beam which is reached by nominal light, and at least one "peripheral detection area" that corresponds to a region within the cross section of the output light beam which is only reached by light from the other origin(s), e.g. scattered light. Such a decomposition of the whole area illuminated by the output light beam is often possible as the nominal light will typically be restricted to a certain area while e.g. scattered light is spread over a larger range. It should however be noted that the terms "central" and "peripheral" are used here basically as names to distinguish the two kinds of sensitive detection areas, though their arrangement will often (but not necessarily always) actually be central and peripheral with respect to each other. Thus the peripheral detection area(s) is (are) in a preferred embodiment arranged symmetrically with respect to an associated central detection area. This allows to separately detect central and peripheral parts of the output light beam.

In a further development of the aforementioned embodiment, there is a plurality of central detection areas that are arranged in line in a first direction. Typically, each of these central detection areas is associated to a different investigation region of the microelectronic sensor device which may for example be scanned by one input light beam or simultaneously be processed by parallel input light beams and output light beams. Moreover, there may optionally be at least one peripheral detection area associated to each of the aforementioned lined-up central detection areas, wherein all these peripheral detection areas are disposed

(i) in the same line as the central detection areas, which yields a compact, line-wise design of the light detector;

(ii) perpendicular to the line of central detection areas, which allows to analyze the spatial light distribution with minimal interference between neighboring output light beams; and/or (iii) diagonal to the line of central detection areas, which allows to analyze the spatial light distribution in two dimensions with little interference between neighboring output light beams.

The light detector may optionally comprise an array with a plurality of light sensitive elements which are called "pixels" in the following. Such a light detector may for example be realized by a CMOS circuit or a charge coupled device (CCD) as it is well known from e.g. digital photography. This makes a spatially (and optionally also spectrally) resolved measurement of the output light beam possible as well as simultaneous measurements of several output light beams.

The microelectronic sensor device optionally further comprises a "scatter correction module" for estimating the actual amount of nominal light in the output light beam. As explained above, the output light beam will in practice usually have unknown additional components of scattered light spread over its cross section. The scatter correction module helps to separate this overlay (or optionally an overlay of light from other origins) from the nominal light one is actually interested in. This may for example be achieved by first estimating the amount of scattered light from measurements in the above mentioned "peripheral detection areas" which are only hit by scattered light, and by then subtracting this amount from the total light amount measured in the "central detection area". To this end the intensity profile of the scattered light may optionally be determined by fitting some modeling curve to the measurements in the peripheral detection areas. The microelectronic sensor device may optionally further comprise an

"analyzer module" for analyzing the spatial distribution of the light in the output light beam and for setting different detection areas on the sensitive area of the light detector that are reached by at least two light parts of the output light beam which are composed of different fractions of nominal light and light from other sources, e.g. scattered light. If for example a single output light beam is measured by a CCD chip, the peak of the measured intensity profile may be located by the analyzer module, and a circular central detection area with a predetermined or an individually adapted diameter may be set at this peak. Similarly, appropriate detection areas for a plurality of simultaneously measured output light beams may automatically be determined by the analyzer module. In a preferred embodiment of the invention, the microelectronic sensor device comprises a field generator for generating a magnetic and/or an electrical field that can affect the label particles. The field generator may for example be realized by a permanent magnet, a wire, a pair of electrodes, or a coil. The generated field may affect the label particles for instance by inducing a magnetization or a polarization and/or by exerting forces on them. Such a microelectronic sensor device allows a versatile manipulation of target components via fields, which may for example be used to accelerate the collection of target components at the binding surface and/or to remove undesired (unbound or, in a stringency test, weakly bound) components from the binding surface.

In the general case, the space next to the carrier at the side of the binding surface may be arbitrarily designed. It is for example possible that this space is exterior to the microelectronic sensor device and that target components are applied to the binding surface by spraying or painting; the space may also be open to the surroundings for detecting target components in e.g. the ambient atmosphere. Moreover, it is possible that the target components reach the binding surface through the carrier, e.g. by diffusion. In preferred embodiments of the invention, the microelectronic sensor device comprises however a sample chamber which is located adjacent to the binding surface and in which a sample with target components can be provided. The sample chamber is typically an empty cavity or a cavity filled with some substance like a gel that may absorb a sample substance; it may be an open cavity, a closed cavity, or a cavity connected to other cavities by fluid connection channels. As was already mentioned, the microelectronic sensor device may be used for a qualitative detection of target components, yielding for example a simple binary response with respect to a particular target molecule ("present" or "not-present"). Preferably the sensor device comprises however an evaluation module for quantitatively determining the amount of target components in the investigation region from the detected output light beam. This can for example be based on the fact that the amount of light in an evanescent light wave, that is absorbed or scattered by target components, is proportional to the concentration of these target components in the investigation region. The amount of target components in the investigation region may in turn be indicative of the concentration of these components in an adjacent sample fluid according to the kinetics of the related binding processes.

In a further development of the aforementioned embodiment, the microelectronic sensor device comprises a recording module for monitoring the determined amount of light in the output light beam over an observation period. Thus it will be possible to monitor the kinetics with which target components collect at or depart from the binding surface. This may reveal valuable information about the target components and/or the prevailing ambient conditions. The evaluation module and/or the recording module are typically coupled to the light detector and may be realized by some data processing hardware, e.g. a microcomputer, together with associated software. In the following, several embodiments of the microelectronic sensor device will be considered in which the carrier comprises a plurality of investigation regions at which different input light beams can be totally internally reflected. One carrier then allows the processing of several investigation regions and thus for example the search for different target components, the observation of the same target components under different conditions and/or the sampling of several measurements for statistical or reference purposes. The "different input light beams" may optionally be components of one broad light beam that is homogeneously generated by the light source.

The different input light beams that are used in the aforementioned embodiment may be different with respect to time. This is for example the case if the microelectronic sensor device comprises a scanning module for sequentially coupling the light source to different investigation regions. Alternatively or additionally, it may comprise a scanning module for optically coupling the light detector to different investigation regions on the binding surface. The scanning modules may for example comprise optical components like lenses or mirrors for directing the incident or the output light beam in a suitable way. The scanning modules may also comprise means for moving the carrier with respect to the light source and/or light detector.

In another embodiment of the microelectronic sensor device with a plurality of investigation regions, a plurality of light sources and/or a plurality of light detectors is present that are directed to different investigation regions at the binding surface. In this case it is possible to process a plurality of investigation regions simultaneously, thus speeding-up the associated measurement process accordingly. This embodiment can of course be combined with the previous one, i.e. there may for example be a scanning module for scanning the input light beams of a plurality of light sources over different arrays of investigation regions and/or a scanning module for directing the output light beams from different arrays of investigation regions to a plurality of light detectors. By using scanning modules, the number of light sources/detectors can be kept smaller than the number of investigation regions.

In another embodiment with a plurality of investigation regions, the microelectronic sensor device comprises a plurality of individually controllable (magnetic or electrical) field generators that are associated to different investigation regions. In this case it is possible to manipulate the label particles in each investigation region individually according to the requirements of the particular tests that shall be performed there.

The microelectronic sensor device may in principle be used with any kind of label particles. It is however preferably provided with label particles that specifically fit to the other components of the device. The sensor device may especially comprise label particles with a mantle of a transparent material, wherein this mantle typically covers (completely or partially) one or more kernels of another material, e.g. iron-oxide grains. In this case light of an evanescent light wave at the binding surface can readily enter the label particles where it is absorbed and/or scattered and thus lost for the output light beam. The transparent material of the mantle may particularly be a material with a similar refractive index as the material of the carrier, because this optimizes the transition of light from the carrier to the label particles. The mantle may for example consist of the same material as the carrier.

The microelectronic sensor device may optionally comprise a "second light detector" for determining (qualitatively or quantitatively) fluorescence light emitted by target components at the binding surface. The fluorescence can be stimulated by the evanescent wave of the input light beam in a small volume adjacent to the binding surface and then be detected, thus indicating the presence (and amount) of fluorescent target components.

In another embodiment of the invention, the microelectronic sensor device comprises an "input light monitoring sensor" for determining the amount of light in the input light beam. The result of this measurement may then be used during the evaluation of the measurements of the light detector, for example during the estimation of the amount of target components or label particles at the binding surface. Taking into account the measured amount of light in the input light beam allows to compensate for unpredictable light source variations due to e.g. current fluctuations or aging effects.

While it is in principle possible that the carrier has some dedicated structure with multiple components of different materials, it is preferred that the carrier is homogenously fabricated from a transparent material, for example a transparent plastic. The carrier can thus readily be produced for example by injection moulding. The investigation region of the carrier has preferably a high smoothness in order to minimize unwanted influences on the (frustrated) total internal reflection. With λ being a characteristic (e.g. peak or average) wavelength of the light constituting the input light beam, the smoothness of the investigation region is preferably better than 0.5 λ, most preferably better than 0.1 λ (which means that the height difference between microscopic "valleys" and "tips" of the carrier surface in the investigation region is smaller than these values).

The investigation region of the carrier may optionally be covered with at least one type of capture element that can bind one or more target components. A typical example of such a capture element is an antibody to which corresponding antigens can specifically bind. By providing the investigation region with capture elements that are specific to certain target components, it is possible to selectively enrich these target components in the investigation region. Moreover, undesired target components can be removed from the binding surface by suitable (e.g. magnetic) repelling forces (that do not break the bindings between desired target components and capture elements). The binding surface may preferably be provided with several types of capture elements that are specific for different target components. In a microelectronic sensor device with a plurality of investigation regions, there are preferably at least two investigation regions having different capture elements such that these regions are specific for different target components.

According to another embodiment of the invention, the surface of the carrier is substantially perpendicular to the incident light beam and/or to the reflected light beam in the region where this beam enters or leaves the carrier, i.e. the angle of incidence lies in a range of about ±5° around 90°. In this case the direction of the incident light beam and/or the reflected light beam will not or only minimally change during the transition from a surrounding medium into the carrier or vice versa. Moreover, reflection will be minimized. Additionally or alternatively, the corresponding regions may also have an anti-reflection coating. To prevent feedback into the light source (e.g. a laser), it may be preferable to have the incident beam (at most) a few degrees off-perpendicular.

The carrier may particularly comprise at least one surface with a form similar or identical to a hemisphere or a truncated pyramid. These forms function like lenses and/or prisms and thus provide a favorable guidance of the incident and the output light beam.

The carrier may further optionally comprise a cavity in which a (magnetic or electrical) field generator can at least partially be disposed. The source of the field can thus be positioned as close as possible to the binding surface, allowing to generate high field strengths in the investigation region with minimal effort (e.g. electrical currents) and with minimal disturbances for other regions (e.g. neighboring investigation regions). Moreover, such a cavity can be used to center the carrier with respect to the field generator, the light source and the light detector. While the microelectronic sensor device may in principle be constructed as a "one-piece" unit of solidly mounted components, it is preferred that the carrier is designed as an exchangeable component of the device, for example a well-plate. Thus it may be used as a low-cost disposable part, which is particularly useful if it comes into contact with biological samples and/or if its coating (e.g. with antibodies) is used up during one measurement process. The invention further relates to a light detector for a microelectronic sensor device of the kind described above, wherein said light detector comprises at least two sensitive detection areas that provide individual measurement signals. These detection areas can thus simultaneously be illuminated by an output light beam during the operation of the light detector. The two sensitive detection areas can therefore be used to separately determine two light parts of the output light beam, particularly light parts that are composed of different fractions of totally internally reflected nominal light and light from another origin. Thus the light detector can for example be used for determining the amount of scattered light in the output light beam which in turn allows to find the amount of nominal light one is actually interested in. The two sensitive detection areas of the light detector typically lie within a circle with a diameter of less than 1 mm, preferably less than 10 μm.

The invention further relates to a method for optical examinations in an investigation region at a binding surface of a carrier, particularly for the detection of target components comprising label particles, wherein said method comprises the following steps: a) Directing an input light beam into the carrier such that it is totally internally reflected in the investigation region at the binding surface. b) Determining the amount of light in at least two parts of an output light beam which are composed of different fractions of (i) "nominal light" that was totally internally reflected in the investigation region in a predetermined way and (ii) light from another origin, e.g. scattered light.

The method comprises in general form the steps that can be executed with a microelectronic sensor device of the kind described above. Therefore, reference is made to the preceding description for more information on the details, advantages and improvements of that method. In a preferred embodiment of the method, the amount of nominal light in the output light beam is estimated. Thus an undesired overlay due to e.g. scattered light can be corrected for.

According to a further development of the method, the spatial distribution of the light in the output light beam is analyzed, and detection areas are set according to this analysis such that each of them is reached by one of at least two light parts of the kind described above (i.e. parts with a different relative composition of nominal light and other light). By such an analysis it is possible to adjust the detection areas to the conditions of a particular measurement situation. In a preferred embodiment of the method, the label particles are manipulated by a magnetic and/or an electrical field, wherein this manipulation may particularly comprise the attraction of the particles to or their repulsion from the investigation region.

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:

Figure 1 schematically shows the general setup of a microelectronic sensor device according to the present invention;

Figure 2 shows intensity profiles of output light beams (top) and corresponding detection areas (bottom) with (a) only one central detection area, (b) one additional annular detection area, and (c) two additional annular detection areas; Figure 3 shows a CCD sensor surface with three neighboring central detection areas and associated, overlapping peripheral detection areas;

Figure 4 shows different designs for the combination of central circular detection areas with rectangular peripheral detection areas;

Figure 5 shows an enlarged view of an alternative optical structure of the carrier; Like reference numbers or numbers differing by integer multiples of 100 refer in the Figures to identical or similar components.

Figure 1 shows a general setup with a microelectronic sensor device according to the present invention. A central component of this setup 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). 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 input light beam Ll which is transmitted into the carrier 11 through an "entrance window". The input 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 "nominal light" part L_nom of an "output light beam" L2. This output light beam L2 will typically comprise additional light components leaving the carrier, e.g. light L_s of the input light beam that was scattered by the carrier and that therefore propagates in other paths than the predetermined or nominal ones. The output light beam L2 leaves the carrier 11 through another surface ("exit window") and is detected in the sensitive areas C, R (e.g. realized by photodiodes or a CCD or CMOS imaging chip) of a light detector 31. 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.

As 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 Ll parallel, and a pinhole of e.g. 0.5 mm may be used to reduce the beam diameter.

Figure 1 further 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 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. 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. 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 2 when the incident light beam Ll is totally internally reflected. If this evanescent wave then interacts with another medium like the magnetic particles 1, 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). Depending on the amount of disturbance, i.e. the amount of magnetic beads on or very near (within about 200 nm) to the TIR surface (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 n_A = 1.52, n_B is allowed up to a maximum of 1.43). Higher values of n_B would require a larger n_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 11 can consist of a relatively simple, injection-molded piece of polymer material.

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.

Figures 2 to 4 refer in more detail to the problem of measurement disturbances by scattered light L_s. For a reliable measurement, the detected drop in the intensity of the output light beam should accurately correspond to the amount of light which is scattered and/or absorbed at the TIR interface. However, source light Ll being scattered due to non-perfected smoothness, contamination or damage of the optical windows (in particular the entrance window) can reach the detector without being reflected from the TIR interface 12 in a nominal way. This part of the light therefore gives a constant, but a priori unknown offset to the detector signal, affecting the accuracy of the measurement.

A solution to the above problem is based on the observation that in a preferred embodiment of the sensor device, a well-defined (preferably substantially parallel) input light beam is used to illuminate the TIR interface, which is then reflected onto the detector. In the ideal case, the beam diameter on the detector is the same as in the source light path. In other cases, it is at least directly related to the beam diameter in the source light path. Light that is scattered e.g. from the entrance window will generally be distributed over a larger area in the plane of the detector in a regular, but not necessarily uniform way. It is therefore proposed to use a first, "central detection area" C corresponding to the position and diameter of the ideally reflected beam of "nominal light". A second and preferably additional "peripheral detection areas" R, Rl, R2, ... are placed around this central detection area to determine the amount and preferably the distribution of light due to scattering. Based on the above observation, this information is then used to correct the signal from the central detection area C by subtracting an appropriate amount calculated from this information.

In a first particular embodiment of this approach depicted in Figure 2, the detector 131 comprises ring-shaped peripheral detection areas Rl, R2 located around a circular central detection area C. Figure 2a) shows the ideal case, i.e. the light distribution (line RL) from only totally internally reflected nominal light that fits the central detection area C.

Figure 2b) additionally shows a background due to scattered light (line SL), increasing the total intensity (line TL), also in the central detection area C. The ring-shaped peripheral detection area Rl measures the average intensity in this area, providing a reasonable estimate of the background in the central area. Correction of the detector signal from the first, central detection area C is achieved by subtracting a value equal to or derived from the signal from the peripheral detection area Rl . Such a correction may be done by a "scatter correction module" 32. In Figure 2c), a second peripheral detection area R2 has been added.

Within each ring Rl, R2, the average intensity is measured. By interpolation of these values or by fitting these values to a e.g. a Gaussian profile (taking the ring widths into account), a more accurate estimate of the background baseline due to scattering is obtained. This estimate can be subtracted from the central detector signal to yield a significantly improved signal.

In the embodiment shown in Figure 3, a CCD or other multi-element detector 231 with an array of pixels 233 is used. This is in particular useful for multiplexing (multiple measurements in parallel). First, central detection areas C correspond to a predetermined first set of detector units and investigation regions on the associated carrier. For each independent measurement, a different central detection area C is used. The one or more peripheral detection areas Rl are located around the central detection areas C and correspond to different sets of detector units, generally not comprising pixels in the central detection areas.

The first set of detector units C can be predetermined during manufacturing, similar to the case depicted in Figure 2a) (determined by design and alignment). However, to allow for more tolerance during manufacturing, this set can also be determined by analyzing the light distribution over the detector. For example, finding the intensity peaks will give the centre spots of the central detection areas C, while a fixed radius can be applied for them. Alternatively, the central detection areas C can be determined by observing the distribution of light over a line through the maximum light intensities (e.g. the horizontal line X in Figure 3). This will result in a signal similar to that shown at the top of Figure 2a). The central detection areas C can be distinguished from the background by looking at e.g. the slope or (absolute) derivative of the signal: the maxima of this slope indicate the boundary. The described analysis of beam profiles and the setting of detection areas C, Rl, ... may be done by an "analyzer module" 32. The additional peripheral detection areas Rl can comprise a subset of all pixels not included in the central detection areas C. Figure 4 shows some examples of basic geometries for this case with rectangular peripheral detection areas R. These peripheral detection areas can be defined relative to the positions of the central detection areas C, or predefined during manufacturing. For correction purposes, the peripheral detection areas R indicated in

Figure 4 may be used as single detector units (predefined photodetectors such as the annular ones in Figure 2), allowing a straightforward correction. A more advanced correction is possible by exploiting a pixelated nature of the peripheral detection areas: by measuring the intensity as a function of position on the detector, an accurate intensity profile can be obtained. From this profile, a background baseline correction can be calculated (interpolation or extrapolation, curve fitting to a Gaussian or multiple Gaussian when including the central detection area, etc.).

An intensity profile from top to bottom (assuming a horizontal distribution of the multiplexed central detection areas C) through the centre of a first central detection area C is preferable, because of its simplicity and because potential overlap between the scattered background signal is minimized. Horizontal scans can also be used, but need to take such overlap into account to prevent over-correction. Diagonal scans eliminate the overlap problems to a large extent, and allow a two-dimensional fit of the intensity profile. This has the advantage that such a configuration can adequately deal with non-symmetric background distributions. An exemplary design of the optical structure on the surface of the transparent carrier 11 is shown in more detail in Figure 5. This optical structure consists of wedges 51 with a triangular cross section which extend in y-direction, i.e. perpendicular to the drawing plane. The wedges 51 are repeated in a regular pattern in x-direction and encompass between them triangular grooves 52. When the input light beam Ll (or, more precisely, a sub-beam of the whole input light beam Ll) impinges from the carrier side onto an "excitation facet" 53 of a wedge 51, it will be refracted into the adjacent groove 52 of the sample chamber 2. Within the groove 52, the light propagates until it impinges onto an oppositely slanted "collection facet" 54 of the neighboring wedge. Here the input light that was not absorbed, scattered, or otherwise lost on its way through the sample chamber 2 is recollected into the output light beam L2. Obviously the amount of light in the output light beam L2 is inversely correlated to the concentration of label particles 1 in the grooves 52 of the sample chamber.

As a result a thin sheet of light is propagating along the contact surface, wherein the thickness of this sheet is determined by the wedge geometry and the pitch p (distance in x-direction) of the wedges. A further advantage of the design is that illumination and detection can both be performed at the non-fluidics side of the carrier.

Given the refractive index ni of the carrier (e.g. made of plastic), the refractive index n₂ of the (bio-)fluid in the sample chamber, and the entrance angle i of the input light beam Ll , the wedge geometry can be optimized such that (i) a maximum amount of light is refracted back towards the light detector; and (ii) a maximum surface area is probed by the "reflected" light beam in order to have optimum binding statistics (biochemistry).

In case of a symmetrical wedge structure the refracted ray in the groove 52 between two wedges 51 , sensing refractive index n₂, should be parallel to the optical interface. With respect to the variables defined in Figure 5, this means that O = OL .

Furthermore, in order to have a maximum "clear" aperture for the incoming input light beam, the angle α of the wedge structure should be equal to the entrance angle i of the input light beam:

i = a .

Introducing these two demands into the law of refraction, ni-sin(i-90°+α) = n₂-sin(o) implies after some calculations that

For a plastic substrate with a refractive index ni = 1.6, and a water-like liquid with a refractive index of n₂ somewhere between 1.3 and 1.4, the optimum wedge angle CC ranges between about 70° and 74°. An appropriate value for the pitch p of the wedges 51 is about 10 μm, giving a sample volume height of about 1.5 μm

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-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 microelectronic sensor device for optical examinations in an investigation region (13) at a binding surface of a carrier (11), particularly for the detection of target components comprising label-particles (1), comprising a) a light source (21) for emitting an input light beam (Ll) into the carrier such that it is totally internally reflected in the investigation region (13); b) a light detector (31, 131 , 231 ) for determining the amount of light in at least two parts of an output light beam (L2) which are composed of different fractions of (i) "nominal light" (L_nom) that was totally internally reflected in the investigation region (13) in a predetermined way and (ii) light (L_s) from another origin.

2. The microelectronic sensor device according to claim 1, characterized in that the light from the other origin comprises scattered light (L_s) of the input light beam (Ll).

3. The microelectronic sensor device according to claim 1, characterized in that the light detector (31, 131, 231) comprises at least two sensitive detection areas (C, Rl, R2, R) that provide individual measurement signals.

4. The microelectronic sensor device according to claim 3, characterized in that at least one of the sensitive detection areas is circular (C), annular (Rl, R2), or rectangular (R).

5. The microelectronic sensor device according to claim 3, characterized in that the light detector (131, 231) comprises a "central detection area" (C) that is reached by nominal light and at least one "peripheral detection area" (Rl, R2, R)) that is only reached by light from the other origin, e.g. scattered light.

6. The microelectronic sensor device according to claim 5, characterized in that the at least one peripheral detection area (Rl, R2, R) is arranged symmetrically with respect to the central detection area (C).

7. The microelectronic sensor device according to claim 5, characterized in that there is a plurality of central detection areas (C) that are arranged in line in a first direction.

8. The microelectronic sensor device according to claim 7, characterized in that at least one peripheral detection area (Rl, R2, R) is associated to each central detection area (C), wherein these peripheral detection areas (Rl, R2, R) are disposed in the same line as the central detection areas (C), perpendicular to the line of central detection areas (C), and/or diagonal to the line of central detection areas (C).

9. The microelectronic sensor device according to claim 1, characterized in that the light detector (231) comprises an array with a plurality of light sensitive elements (233) called "pixels".

10. The microelectronic sensor device according to claim 1, characterized in that it comprises a "scatter correction module" (32) for estimating the amount of nominal light in the output light beam (L2).

11. The microelectronic sensor device according to claim 1, characterized in that it comprises an "analyzer module" (32) for analyzing the spatial configuration of the output light beam (L2) and for setting detection areas (C, Rl, R2, R), wherein each of these detection areas is reached by one of the at least two parts of the output light beam.

12. A light detector (31, 131, 231) for a microelectronic sensor device according to claim 1, comprising at least two sensitive detection areas (C, Rl, R2, R) that provide individual measurement signals.

13. The microelectronic sensor device according to claim 1, characterized in that the carrier (11) comprises at least one hole or groove (52) in the surface of the carrier (11), whereby the hole or groove (52) has a cross section with two oppositely slanted opposing facets (53, 54), particularly a triangular cross section.

14. A method for optical examinations in an investigation region (13) at a binding surface of a carrier (11), particularly for the detection of target components comprising label particles (1), comprising a) directing an input light beam (Ll) into the carrier such that it is totally internally reflected in the investigation region (13) at the binding surface (12); b) determining the amount of light in at least two parts of an output light beam (L2) which are composed of different fractions of (i) "nominal light" (L_nom) that was totally internally reflected in the investigation region (13) in a predetermined way and (ii) light from another origin, e.g. scattered light (L_s).

15. The method according to claim 14, characterized in that the amount of nominal light (L_nom) in the output light beam (L2) is estimated.

16. The method according to claim 14, characterized in that the spatial distribution of the light in the output light beam (L2) is analyzed and that detection areas (C, Rl, R2) are set according to this analysis, wherein each of these detection areas is reached by one of the at least two light parts.