Patent Application
20110188030
The invention relates to a microelectronic sensor device and a method for optical examinations in a sample medium adjacent to the contact surface of a carrier, wherein the examinations comprise the total internal reflection of an input light beam. Moreover, it relates to the use of such device.
The US 2005/0048599 A1 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. A problem of this and similar setups is that the optical effects depend on the refractive index of the sample medium, which may vary from charge to charge. This may severely deteriorate the accuracy of quantitative measurements.
Based on this situation it was an object of the present invention to provide alternative means for making optical examinations with a sample medium that are based on total internal reflection (TIR), wherein it is desirable that the examinations can be made with a high accuracy and robustness with respect to different sample media.
This object is achieved by a microelectronic sensor device according to claim 1, a method according to claim 10, and a use according to claim 15. Preferred embodiments are disclosed in the dependent claims.
The microelectronic sensor device according to the present invention serves for making optical examinations in a sample medium (e.g. blood or saliva) that is provided adjacent to the contact surface of a carrier (wherein the carrier does 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 sample medium. 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 carrier will usually be made from a transparent material, for example glass or poly-styrene, to allow the propagation of light of a given (particularly visible, UV, and/or IR) spectrum. The term “contact surface” is chosen primarily as a unique reference to a particular part of the surface of the carrier, and though target components will in many applications actually contact and bind to said surface, this does not necessarily need to be the case.
The microelectronic sensor device comprises the following components:
The described microelectronic sensor device allows for optical examinations of a sample medium with the help of a total internal reflection at the contact surface to this medium. At the same time, the device provides an independent measurement of the refractive index of the sample medium. This refractive index usually affects significantly the optical processes that are associated to the total internal reflection; taking the independently measured refractive index into account can therefore make the outcome of such processes more robust with respect to variations in the refractive index of the sample medium. The same advantage is achieved if the conditions of TIR are changed based on the measured refractive index. This change can for example compensate the effect of a variation of the refractive index on desired optical processes.
In general, there are many possibilities how the evaluation unit can take the measured refractive index into account. In a practically important example, the evaluation process that is executed by the evaluation unit may be based on a (direct or indirect) estimation of the decay distance of evanescent waves that are generated during the total internal reflection of the input light beam at the contact surface. This approach is based on the fact that many TIR-related optical examinations make use of evanescent waves to exactly localize processes in a small volume adjacent to the TIR-interface, wherein the size of this volume is crucially dependent on the decay distance of the evanescent waves, which in turn depends on the refractive index of the sample medium.
In a typical application of the microelectronic sensor device, the evaluation unit is adapted to determine the amount of target particles—e.g. atoms, ions, (bio-)molecules, cells, viruses, or fractions of cells or viruses, tissue extract, etc., including labels like magnetic, fluorescent, or radioactive particles—that are present in the sample medium at the contact surface of the carrier. This amount can particularly be determined due to the effect that such target particles scatter light of the evanescent waves which are generated during the total internal reflection of the input light beam, thus leading to a so-called frustrated total internal reflection (FTIR). The degree of frustration will then provide information about the amount of target particles at the contact surface. The amount of detected target particles at the contact surface may have a direct (and known) relation to the amount of target particles present in the sample fluid. In case the target particles are labels for other components, e.g. certain biomolecules, their amount is further related to the amount of these components.
It was already mentioned that there is a variety of possibilities to realized the refractive index measurement unit (RIMU). In a first realization, the RIMU comprises the following components:
As will be explained in more detail with reference to the Figures, the described RIMU exploits the fact that the optical path of a (test-) light beam will experience deflections when it passes (oblique) through an interface between two media of different refractive indices. Thus the spatial position of the transmitted test-light beam allows to infer the refractive index of the sample medium. It should be noted that the test-light source and/or the test-light detector may be realized by the light source and/or the light detector, respectively, of the microelectronic sensor device, or that they may alternatively be separate components. Moreover, the estimation module may at least partially be integrated into the evaluation unit of the microelectronic sensor device.
The mentioned test-light detector may comprise a single light-sensitive sensor unit and a scanning mechanism to find the spatial position of the transmitted test-light beam by moving said sensor unit through a search region. In an alternative embodiment, the test-light detector comprises a plurality of sensor units, which may be realized for example by the pixels of a charge coupled device (CCD) or a CMOS chip. This embodiment has the advantage that the test-light detector can remain at a fixed position in space and that the spatial position of the transmitted test-light beam can be inferred from the particular sensor unit(s) it impinges on, or, more generally, from the light distribution over the sensor units. In a similar embodiment, e.g. a split photodiode can be used, consisting of at least two, preferably closely spaced and identical detector parts. When the beam diameter in the detector plane is comparable to the lateral dimension of the detector parts, the beam position on the detector can be inferred from the ratio of the signals from the respective detector parts.
The transparent walls and the intermediate test chamber may in principle have an arbitrary design as long as they allow the transmission of the test-light beam in such a way that the spatial position of the transmitted beam depends on the refractive index of the medium in the test chamber. In a preferred embodiment, the two transparent walls have parallel sides, i.e. all four front- and backsides of the walls are parallel to each other. In this case a test-light beam that is transmitted through the walls at an oblique angle will be displaced in a parallel way depending on the refractive index of the sample medium between the walls.
While it is in principle possible that the RIMU with the two transparent walls and the test chamber is a separate entity independent of the carrier, it is a preferred embodiment of the invention that the two transparent walls belong to the carrier. Thus it can be guaranteed that the test chamber between the two walls is automatically filled with the same sample medium that is present adjacent to the contact surface. In this context it should be noted that the invention also refers to a particular carrier design comprising two such transparent walls with an intermediate test chamber between them.
Other approaches for measuring the refractive index of a sample medium are based on the reflection of a test-light beam. Thus another type of refractive index measurement unit (RIMU) may comprise the following components:
The described RIMU exploits the fact that the reflection of a (test-) light beam at an interface to the sample medium depends on the refractive index of said sample medium. A particular advantage of this approach is that the necessary optical instruments (test-light source, test-light detector) can be arranged on the same side of the sample medium. Moreover, the reflection-based test requires no extensive light propagation within the sample medium and can therefore be executed with minimal amounts of a sample. Finally, it should be noted that the test-light source and/or the test-light detector can be identical to the light source and/or the light detector of the microelectronic sensor device, possibly with some necessary adaptations.
In a first particular realization of the aforementioned reflection-based approach, the estimation module is adapted to determine the critical angle of total internal reflection (TIR) at the test surface. As this critical angle depends on the refractive index of the sample medium that contacts the test surface, it is possible to infer the refractive index of a particular sample medium from the measured critical angle of TIR.
The aforementioned RIMU may particularly comprise a scanning unit for varying the angle of incidence of the test-light beam over a predetermined range, wherein this range preferably covers the (expected) critical angle of TIR. Thus the angle of incidence can be swept over a range of angles, and the critical angle of TIR can be found from the observed amount of light in the reflected light beam.
In another embodiment, the RIMU may comprise an optical system for directing simultaneously a plurality of test-light beams under different angles of incidence onto the test surface. In this case a range of angles of incidence can be examined in parallel.
According to still another embodiment, the RIMU may comprise an optical system for directing reflected test-light beams of different angles of incidence to the test-light detector. The test-light detector can then remain at a fixed position in space, which simplifies the mechanical design of the apparatus.
In a second particular realization of the reflection-based approach, the estimation module of the RIMU is adapted to determine the reflectivity of the test surface provided that the test-light beam has an angle of incidence smaller than the critical angle of TIR. This realization is based on the fact that the reflectivity depends (for angles of incidence smaller than the critical angle of TIR) on the refraction index of the sample medium that is adjacent to the test surface. The relation between the refractive index of a sample medium and the reflectivity can for example be determined for given angles of incidence from experiments. It can then be stored in a look-up table that can be used by the estimation module. An advantage of this design is that the hardware requirements are minimal, as a measurement at one given angle of incidence suffices.
It should further be noted that the transmission based approach and at least one of the reflection based approaches (with TIR-angle or reflectivity determination) can be applied in parallel to increase the accuracy and reliability of the determined results.
The invention further relates to a method for optical examinations in a sample medium adjacent to the contact surface of a carrier, comprising the following steps:
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 first preferred embodiment of the method, a test-light beam is transmitted at an oblique angle through a test volume of the sample medium, and the displacement of this test-light beam after its transmission is measured.
In another embodiment of the method, the critical angle of total internal reflection between the sample medium and a test material is determined. The test material may in particular be the same material as that of the carrier.
Moreover, it is possible to measure for a given angle of incidence (smaller than the critical angle of TIR) the reflectivity of a test interface with the sample medium on one side.
The invention further relates to the use of the microelectronic device described above for molecular diagnostics, biological sample analysis, or chemical sample analysis, food analysis, and/or forensic analysis. Molecular diagnostics may for example be accomplished with the help of magnetic beads or fluorescent particles that are directly or indirectly attached to target molecules.
These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. These embodiments will be described by way of example with the help of the accompanying drawings in which:
Like reference numbers or numbers differing by integer multiples of 100 refer in the Figures to identical or similar components.
Though the present invention will in the following be described with respect to a particular setup (using magnetic particles and frustrated total internal reflection as measurement principle), it is not limited to such an approach and can favorably be used in many different applications.
The interface between the carrier 11 and the sample chamber 2 is formed by a surface called “contact surface” 12. This contact surface 12 is coated with capture elements, e.g. antibodies, which can specifically bind the target particles.
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 at the contact surface 12 and in the adjacent space of the sample chamber 2. With the help of this magnetic field, the target 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 target particles 1 to the contact surface 12 in order to accelerate their binding to said surface, or to wash unbound target particles away from the contact surface before a measurement.
The sensor device further comprises a light source 21 that generates an input light beam L1 which is transmitted into the carrier 11 through an “entrance window” 14. As light source 21, a laser or an LED, particularly 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 of e.g. 0.5 mm may be used to reduce the beam diameter. The input light beam L1 arrives at the contact surface 12 at an angle θ=θA larger than the critical angle θc of total internal reflection (TIR) and is therefore totally internally reflected in an “output light beam” L2. The output light beam L2 leaves the carrier 11 through another surface (“exit window” 16) and is detected by 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 measured sensor signals S are evaluated and optionally monitored over an observation period by an evaluation and recording module 50 that is coupled to the detector 31.
It is possible to use the detector 31 also for the sampling of fluorescence light emitted by fluorescent particles 1 which were stimulated by the input light beam L1, wherein this fluorescence may for example spectrally be discriminated from reflected light L2. Though the following description concentrates on the measurement of reflected light, the principles discussed here can mutatis mutandis be applied to the detection of fluorescence, too.
The described microelectronic sensor device applies optical means for the detection of target particles 1. 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 (FTIR). This principle is based on the fact that an evanescent wave propagates (exponentially dropping) into the sample 2 when the incident light beam L1 is totally internally reflected. If this evanescent wave then interacts with another medium like the bound target 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 target particles 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 bound target particles 1, and therefore for the concentration of target particles in the sample. 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 θc of 60°. An angle of incidence of 70° is therefore a practical choice to allow fluid medium with a somewhat larger refractive index (assuming nA=1.52, nB is allowed up to a maximum of 1.43). Higher values of nB would require a larger nA and/or larger angles of incidence.
Advantages of the described optical read-out combined with magnetic labels for actuation are the following:
In the described optical biosensor the refractive index nB of the unknown sample liquid has an influence on the sensor signal S, i.e. the signal per target particle 1. For accurate, quantitative measurements with low coefficient of variation CV this may be a problem (note: The CV is reported as a percentage and calculated from the average or mean and standard deviation as follows: 100*Standard Deviation/Average). For example, the evanescent decay distance z (and thus the strength of the interaction with the target particles) depends on the ratio of refractive indices of carrier material and the sample liquid according to:
with k=2π/λ being the wavenumber of the input light beam, nA and nB the indices of refraction of carrier material and sample liquid, respectively, and θA the angle of incidence of the input light beam L1. Moreover, the amount of scattering depends on the refractive index difference between the sample liquid and the target particles 1.
To address this problem it is therefore proposed here to accurately measure the refractive index nB of the sample liquid. The measurement can then be used to correct the sensor signal for differences in the refractive index, leading to a more accurate, quantitative measurement with low CV.
There are several methods to determine a refractive index. Three attractive methods are particularly suitable for practical implementation in a biosensor device: The first is based on a determination of the displacement of a refracted beam (incident at an angle below the critical angle of TIR) after transmission through the liquid. In the second method, the critical angle is determined from a reflected light beam. A third method involves a reflection measurement at a fixed, incident angle below the critical angle. These methods will below be described in more detail. The correction of the sensor signal S may be done “virtually” in a separate calculating element, embedded in hardware or in software code (or by some other means), or “physically” by adapting the evanescent decay length for example by changing the incident angle of the input light beam L1.
In
Δx=cos θe·(tan θe−tan θ2)/h,
with θ2=arcsin(sin θe/nB). This displacement can be detected using a position sensitive test-light detector 102 or e.g. a pixelated detector such as a CCD. Alternatively, a scanning detector e.g. with a pinhole can be used to determine Δx.
To give an indication of the beam displacements, Table 1 of
Returning to
The RIMU 200 further comprises a particular adaptation of the evaluation module 50 to incorporate also an “estimation module” which can determine the critical angle of TIR, θc. This determination is achieved from measurements which will now be explained in more detail with reference to
In this configuration, the detector output S is monitored while scanning the source. At angles below the critical angle θc, the reflected intensity will be low due to partial reflection and refracted transmission. At angles equal to and larger than the critical angle θc, the intensity will be high and constant due to TIR. For a specific example of nA=1.53, nB=1.33, and λ=650 nm, the measured normalized detector output S* (vertical axis) is shown in the diagram of
For relevant material parameters, an example of the angles involved is shown in Table 2 of
Translating the aforementioned requirements to a position results in Table 3 of
An attractive alternative configuration is shown in
The embodiment of
Returning again to
The RIMU 300 is based on the observation that the reflected intensity at an incident angle θR below the critical angle of TIR, θc, depends on the refractive index nB of the liquid (and the carrier material). Only a single test-light beam is needed, as well as a single, fixed test-light detector 31. This detector can be, but does not need to be, the same as the one used for detecting the target particles 1.
It should further be noted that
While the invention was described above with reference to particular embodiments, various modifications and extensions are possible, for example:
Finally it is pointed out that in the present application the term “comprising” does not exclude other elements or steps, that “a” or “an” does not exclude a plurality, and that a single processor or other unit may fulfill the functions of several means. The invention resides in each and every novel characteristic feature and each and every combination of characteristic features. Moreover, reference signs in the claims shall not be construed as emitting their scope.
| Number | Date | Country | Kind |
|---|---|---|---|
| 07114101.4 | Aug 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB08/52870 | 7/17/2008 | WO | 00 | 2/2/2010 |
