The invention relates to a microelectronic sensor device and a method for the detection of target particles that are bound to binding sites at the binding surface of a carrier. Moreover, it relates to the use of such a 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 measurement approaches is that the signal one is interested in is often only a small variation of a large base signal, making accurate and robust measurements difficult, e.g. due to limitations in electronic gain that can be applied on the total signal.
Based on this situation it was an object of the present invention to provide means for an improved detection of bound target particles, wherein it is desired that a higher sensitivity and/or accuracy is achieved.
This object is achieved by a microelectronic sensor device according to claim 1, a method according to claim 2, and a use according to claim 13. Preferred embodiments are disclosed in the dependent claims.
The microelectronic sensor device according to the present invention serves for the qualitative or quantitative detection of target particles that are bound to binding sites at the “binding surface” of a carrier, wherein said binding surface and carrier (and of course the target particles) do not necessarily belong to the device. The “target particles” may particularly comprise a combination of target components (e.g. biological substances like biomolecules, complexes, cell fractions or cells) and “label particles” (e.g. atoms, molecules, complexes, nanoparticles, microparticles etc.) that have some property (e.g. optical density, magnetic susceptibility, electric charge, fluorescence, or radioactivity) which can be detected. The term nanoparticle is used for particles having at least one dimension ranging between 3 nm and 5000 nm, preferably between 10 nm and 3000 nm, more preferred between 50 nm and 1000 nm. The carrier is usually a solid body, for example from a transparent material like glass or a transparent plastic, having one dedicated surface region that is called “binding surface” here and that comprises at least one, typically however a large number of binding sites. The binding sites will usually be realized by capture molecules that are attached to the binding surface and that can specifically bind to target particles (molecules) in a sample fluid. In general, the binding can be based on a chemical binding, an electrostatic attraction, Van-der-Waals forces or the like.
The microelectronic sensor device comprises the following components:
The described microelectronic sensor device has the advantage to allow for a more accurate and robust evaluation of the sensor signals because the measurement of the sensor unit is correlated with the induced movement of the detected target particles. Moving for example all bound target particles out of the sensitive region (or all into the sensitive region) will yield two signals, a measurement with and a reference measurement without target particles, from which the actual effect of the target particles can be inferred with high accuracy.
The invention further relates to a method for the examination of target particles that are bound to binding sites at the binding surface of a carrier, wherein the method comprises 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 the following, various further developments of the invention will be described that relate both to the microelectronic sensor device and the method defined above.
In a first particular embodiment, the target particles are moved by the activity of the actuation unit through zones of the sensitive region in which the sensor unit has different sensitivity. The target particle will therefore evoke different sensor signals of the sensor unit when being in different sensitivity zones. Preferably, the sensitivity of the sensor unit varies continuously throughout its sensitive region; in this case, even the smallest movement of a target particle induces a variation in the sensor signal of the sensor unit. In general, the non-uniform sensitivity of the sensor unit guarantees that the induced movement of the target particle will have an effect on the sensor signal, which can be taken into account during the evaluation of this signal.
It was already mentioned that the movement of the target particles can be induced in several different ways. In a preferred embodiment, the target particles are moved by an interaction with a magnetic and/or an electric field. This is possible if the target particles have a property to which a magnetic or electric field can couple, e.g. if the particles have a magnetic or electric dipole moment or if such a moment can be induced. In this embodiment the movement of the target particles can very well be controlled by the generation of the magnetic or electric field. For the generation of the field, the actuation unit preferably comprises a field generator, for example a permanent magnet, an electromagnet, or an electrode or electrode pair.
The induced movement of the target particles may optionally be modulated, preferably in a periodical way with a given modulation frequency (wherein the frequency determines the period of some periodic course which needs not necessarily be sinusoidal). To this end, the actuation unit may comprise a modulator for modulating its activity in a controlled and preferably adjustable way. Actively modulating the movement of the bound target particles has the advantage that this movement can be adjusted to a mode that is optimal for the intended evaluation purposes. Moreover, the information about the controlled activity modulation can be exploited by the evaluation module as it implicitly comprises the desired information about the induced movement of the target particles. Thus the control signal with which a modulator controls the actuation unit may in parallel be supplied to the evaluation module for taking it into account during the evaluation of the sensor signal. Furthermore, by inducing and detecting the movement of the particles in the same frequency domain, e.g. by using synchronous modulation and demodulation techniques, noise sources in other frequency domains can be suppressed extremely efficiently.
It was already said that the sensor unit can apply any suitable measurement principle. In a preferred embodiment, the sensor unit applies an optical measurement in which the sensor signal is derived from an output light beam that comes from the carrier and that comprises light from a frustrated total internal reflection of an input light beam at the binding surface. In this embodiment, the microelectronic sensor device will comprise a light source for emitting the input light beam towards the binding surface in such a way—i.e. under an appropriate angle—that it is totally internally reflected there. 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. Moreover, the sensor device will comprise a light detector for detecting the mentioned output light beam, wherein this detection typically comprises the measurement of the amount of light in the output light beam (e.g. expressed as the intensity of this beam). The light 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.
For a total internal reflection to occur at the binding surface, this surface must be the interface between two media, e.g. glass and water, at which total internal reflection (TIR) can take place if the incident light beam hits the interface at an appropriate angle (larger than the associated critical angle of TIR). Such a setup is often used to examine small volumes of a sample at the TIR-interface that are reached by exponentially decaying evanescent waves of the totally internally reflected beam. Target particles that are present in this volume can then scatter and/or absorb some of the light of the evanescent waves which will accordingly not be coupled out anymore into the reflected light beam. In this scenario of a “frustrated total internal reflection”, the output light beam of the sensor device will comprise the reflected light of the input light beam, wherein the small amount of light missing due to scattering and/or absorption of evanescent waves contains the desired information about the target components in the investigation region. Depending on the concentration of analytes to be measured in the bioassay, the signal one is interested in (missing light) can be very small with respect to a relatively large DC, i.e. constant, background. Furthermore, due to the relatively large background, the signal is prone to disturbances from any source. The proposed application of an induced movement of the target particles helps in this situation to improve the accuracy of the measurements.
According to a further development of the aforementioned embodiment, the input light beam can be modulated, wherein the modulation is preferably done in a periodical way with a given input frequency. Modulating the input light beam provides it with a characteristic fingerprint which allows to distinguish in the sensor signal effects that go back to this input light beam from other effects, e.g. contributions of ambient light.
In the above embodiments in which an output light beam is generated, this beam may optionally be detected with a camera (e.g. a CCD camera) taking exposures with
Thus it is possible to observe with a camera modulation frequencies in the output light beam that are higher than the maximal frame rate of the camera.
The sensor signal that is provided by the sensor unit is preferably demodulated by the evaluation module with respect to one or more given frequencies, particular with respect to a modulation of the induced movement of the target particles and/or with respect to a modulation of an input light beam (if such a modulation and such an input light beam are used). To perform this demodulation, the evaluation unit may comprise a demodulator as it is well known to a person skilled in the art of (analogue or digital) signal processing. With the help of the demodulation, effects that genuinely go back to the target particles and/or the input light beam can be distinguished from other effects, i.e. from disturbances.
In a particular realization of the aforementioned embodiment, the modulation of the induced movement of the target particles and the modulation of an input light beam are adjusted such that the movement-modulation appears in the demodulated sensor signal as a sideband with respect to the light-modulation. This is for example the case if a sinusoidal light-modulation takes place at a much higher frequency than a sinusoidal movement-modulation.
Depending on the particular task the microelectronic sensor device or the method are applied for, the sensor signal may be evaluated with respect to different aspects. Preferably, the sensor signal is evaluated with respect to the presence and/or the amount of target particles in the sensitive region of the sensor unit, thus allowing to determine for example the concentration of particular biomolecules in a sample fluid. Alternatively or additionally, the sensor signal may be evaluated with respect to the binding characteristics of the binding between the target particles and the binding surface. In this case it is exploited that the reaction of the target particles to certain actuation forces, e.g. induced by an electric or magnetic field, depend on the strength with which these target particles are bound, i.e. on the properties of the associated binding sites (capture molecules). Certain aspects of the induced movement of the target particles—like damping factor, resonance frequency, amplitude, phase shift etc.—will therefore carry valuable information about the binding site and/or the operating conditions at the binding surface (e.g. the viscosity of the surrounding fluid).
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 in the Figures refer to identical or similar components.
The interface between the carrier 11 and the sample chamber 2 is formed by a surface called “binding surface” 12. This binding surface 12 is coated with capture elements 3, e.g. antibodies, which can specifically bind to the target particles.
The sensor device comprises a magnetic field generator 51, 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 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 binding surface 12 in order to accelerate the binding of the associated target particle to said surface. A second electromagnet 51′ at the top of the sample chamber 2 can optionally be used to ‘wash’ away the particles 1 which have not been bound (e.g. because all binding sites 3 are occupied). This ‘washing’ can also be accomplished by applying magnetic fields using the first electromagnet 51 in such a way that all unbound target particles are removed from the measurement area/volume, as can be done using a horse-shoe electromagnet configuration. In this case, the binding forces between the target particles 1 and the binding sites 3 should be larger than the applied magnetic forces, therefore the bonds remain intact during the washing procedure (assuming that the bond between the binding sites 3 and the surface 12 is strong enough as well). It should further be noted that also electrostatic forces may be utilized, driving (non-magnetic) label particles using an alternating electric field.
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 through an “entrance window”. The input light beam L1 arrives at the binding surface 12 at an angle θ0 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”) 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 32 that is coupled to the detector 31.
As light source 21, e.g. a commercial CD (λ=780 nm), DVD (λ=658 nm), or BD (λ=405 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.
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 evanescent wave of 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. This principle is based on the fact that an evanescent wave penetrates (exponentially dropping in intensity) 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. It should be noted that this “near region” is defined by the penetration depth ζ of the evanescent wave in the sample chamber, which depends on wavelength λ, entrance angle θ of the input light beam L1, and on the refractive indices nA of the substrate 11 and nB of the medium directly above the interface 12 (e.g. blood or water) and is given by the following formula:
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 typical interaction distance of the evanescent wave of about 100 to 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 optical measurement 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 nA=1.52. Medium B in the sample chamber 2 will be water-based and have a refractive index nB close to 1.3. This corresponds to a critical angle θc of 60°. An angle of incidence of θ=70° is therefore a practical choice to allow fluid media 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:
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).
A problem of the described measurement approach may arise from the fact that the starting signal, i.e. the sensor signal S when no target particles 1 are attached to the binding surface 12, is high. Binding of target particles to the binding surface will decrease this high signal. Thus, the signal ‘x’ corresponding to the amount of target particles bound to the binding surface is measured in an (1−x) way, as this is the optical signal S. This has a disadvantage, because one is interested in the signal ‘x’ which is rather small compared to the measured optical signal (1−x). This may cause so-called “gain-problems”, as the starting signal is large with respect to the ‘x’ signal. Therefore it is difficult to amplify the ‘x’ signal, as the background signal is amplified as well, which can for example result in “overflow” in amplifiers and ADC's, and an amplification of noise contributions e.g. due to laser intensity fluctuations or detector noise etc. Also, the measured ‘x’ signal is very sensitive to gain variations because gain variations cannot be distinguished from x-changes. Furthermore, if the background signal changes, e.g. because some light from an external source hits the detector or cartridge, the outcome of the measurement is influenced, which is highly undesirable.
It would therefore be highly desirable from the point of view of circuit design and signal processing to convert the (1-x) measurement into a measurement that only measures ‘x’, i.e. the amount of target particles 1 bound to the binding surface 12.
The solution to the above issues that is proposed here starts with the bioassay as usual, i.e. injection of magnetic beads and sample with target molecules, binding of magnetic beads and target molecules to “target particles” 1, binding of target particles 1 to binding sites 3, and washing away of non-bound target particles. As a result there is a binding surface 12 with target particles 1 that are attached to the surface via capture elements 3, e.g. the protein BSA-opi. The amount of light that is coupled out of the input light beam L1 is proportional to the amount of target particles 1 bound to the binding surface 12. However, the amount of light coupled out is also dependent on the distance d between the target particles 1 and the binding surface 12, i.e. the amount of ‘target particle’ present in the evanescent field (sensitive region 13) just above the surface.
This is illustrated in
Changing the distance d between the binding surface 12 and the target particle 1, while stretching and compressing the protein 3 in between, can be done in several ways:
The microelectronic sensor device of
By using the above-mentioned modulation method, the sensor signal S can be appropriately demodulated in the evaluation module 32 to achieve a signal that is directly proportional to the amount of target particles 1 that are bound to the binding surface 12. However, the approach also enables measurement of certain properties of the capture elements 3 (e.g. a protein) between the binding surface and the target particles. Thus structural information on the protein can be derived from the observed Q-factor and resonance frequency, and the size of the protein can be derived from the observed amplitude of the target particle oscillation. Moreover, the viscosity of the fluid (e.g. saliva) in which the measurement takes place can also be inferred.
Furthermore, because of the non-linear relation between the detected signal and the z-position of the particles above the sensor surface, harmonics in the modulated signal gives information about the average of said z-position. This can be used to measure the length of the binding with respect to the optical surface, as well as to characterize the binding probes.
In the described method, the value of ‘x’ which is derived from the sensor signal S by demodulation is independent of the background signal. Furthermore ‘x’ cannot be influenced by any disturbances that occur at a different frequency than the frequency used for modulation/demodulation, e.g. external light, electronic interference etc.
A dashed line shown in
Another structure of the binding surface 12 is shown in
A problem of a modulation of the target particle movement with a low frequency ωm may be that the detection of the resulting low frequent signals takes place in a range where 1/f noise (electronics) may have a predominant contribution to the demodulated signal noise. To address this issue, an additional intensity modulation of the light source 21 and an associated demodulation technique can be used.
By using this double modulation scheme, the (moderate frequency) oscillating target particle signal can be measured conveniently at high frequencies. In order to eliminate erroneous cross-talk from the signal due to laser intensity variations and stray reflections (present at frequency ω1), the sidebands should be sufficiently separated from the main band occurring at ω1. This requires a stable oscillation circuitry driving the laser. Using a laser modulation frequency ω1 of for example 100 kHz and a magnetic or electric field modulation frequency ωm of 1 kHz, the stability of the laser driver should be well below 1 kHz when operated at 100 kHz, which can be readily achieved in practice.
Furthermore, because of the non-linear relation between the detected signal and the z-position of the particles above the sensor surface, higher inter-modulation terms in the signal give information about the average of said z-position. This can be used to measure the length of the binding with respect to the optical surface, as well as to characterize the binding probes.
When a (CCD) camera is used to observe the binding surface 12, said camera maybe too slow to follow the modulation of a magnetic or electric field (frequency ωm) and/or of a laser input light beam (frequency ω1). This problem can be solved by
By shifting phase of the illumination moment with respect to the modulation period, the total period can be scanned. Using a camera has advantages in multi-spots processing compared to single-spot approaches.
While the invention was described above with reference to particular embodiments, various modifications and extensions are possible, for example:
The detection can occur with or without scanning of the sensor element with respect to the sensor surface.
Finally it is pointed out that in the present application the term “comprising” does not exclude other elements or steps, that “a” or “an” does not exclude a plurality, and that a single processor or other unit may fulfill the functions of several means. The invention resides in each and every novel characteristic feature and each and every combination of characteristic features. Moreover, reference signs in the claims shall not be construed as limiting their scope.
Number | Date | Country | Kind |
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07123741.6 | Dec 2007 | EP | regional |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/IB08/54541 | 10/31/2008 | WO | 00 | 6/11/2010 |