Patent Application
20100221842
The invention relates to a microelectronic sensor device for the detection of target components.
In an inhomogeneous assay, the concentration of a targeted bio-molecule can be determined by measuring the surface concentration of the targeted bio-molecule or beads [that are representative for the targeted bio molecule] bound at the sensor surface. As an example, one can think of a competitive assay where the binding surface (substrate) is covered with target molecules. The beads may be covered with specific [for the target molecule] antibodies and are dispersed in a fluid that contains the target molecules. The free target molecule in the sample competes with the immobilized target molecule on the sensor surface for binding to the antibody-coated bead. In case of a low concentration, the chance that an antibody binds with a target molecule at the sensor surface is higher than the chance that an antibody binds with a target molecule in the solution. By measuring the surface concentration of beads that are bound at the substrate, one can determine the concentration of the target molecule. Accurate measurement of the concentration however requires a highly surface specific detection scheme that is sufficiently insensitive for beads in the solution.
Furthermore, the detected signal should be independent from the sample matrix, which can be whole-blood, whole-saliva, urine or any other biological fluid. For optical detection schemes high surface specificity can be achieved by reducing the measurement volume. One way to achieve this is by confocal imaging where the measurement volume is reduced to typically a few wavelengths (e.g., 1 micron). 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 desire exists to provide a microelectronic sensor device for the detection of target components wherein the media for containing the target components are not limited to materials having a refractive index smaller than the carrier and the refractive index of the particles attached to the targeted components can be chosen above as well as below the refractive index of the carrier without significantly impacting the sensitivity, for example, to provide the sensor device for biosensing purposes. Accordingly, in one aspect of the invention, there is provided a microelectronic sensor device for the detection of target components, comprising a carrier with a binding surface at which target components can collect; a source for emitting a beam of radiation incident at the binding surface; a detector for determining an amount of said emitted radiation in a reflective mode. In one aspect of the invention, the binding surface is provided by a plurality of aperture defining structures having a smallest in plane aperture dimension W1 smaller than a diffraction limit, the diffraction limit defined by a medium for containing the target components.
In another aspect of the invention there is provided a method of detecting a presence of a target component in a medium, comprising: providing a binding surface at which target components can collect, by a plurality of aperture defining structures having a smallest in plane aperture dimension W1 smaller than a diffraction limit, the diffraction limit defined by a medium for containing the target components; emitting a beam of radiation incident on the binding surface, the binding surface formed by a plurality of aperture defining structures having a smallest in plane aperture dimension W1 smaller than a diffraction limit, the diffraction limit defined by a medium for containing the target components; and detecting an amount of said radiation in a reflective mode. These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter.
The microelectronic sensor device according to the present invention may serve for the qualitative or quantitative detection of target components, wherein the target components may for example be biological substances like biomolecules, complexes, cell fractions or cells. The term “label and/or particle” shall denote a particle (atom, molecule, complex, nanoparticle, microparticle etc.) that has some property (e.g. optical density, magnetic susceptibility, electrical charge.) which can be detected, thus indirectly revealing the presence of the associated target component. A “target component” and a “label particle” may be identical. The microelectronic sensor device, according to an aspect of the invention comprises the following components:
The term “binding surface” is chosen here 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 carrier should have a high transparency for light of a given spectral range, particularly light emitted by the light source that will be defined below. The carrier may for example be produced from glass or some transparent plastic. The carrier may be permeable; it provides a carrying function for aperture defining structures provided on the carrier having a smallest in plane aperture dimension (W1) smaller than a diffraction limit
Wmin=wavelength/(2*nmedium) (1)
with λ the wavelength in vacuum and nmedium the refractive index of the medium in front of the wire grid.
In a preferred embodiment, the aperture defining structure defines a first and a second in-plane vector that are parallel to a slab of material that is not transparent (examples are metals such as gold (Au), silver (Ag), chromium (Cr), aluminium (Al)). The first (smallest) in-plane aperture dimension is parallel to the first in-plane vector and the second (largest) in-plane aperture dimension is parallel to the second in-plane vector.
1 Apertures of the first-type with a first in-plane dimension W1 below the diffraction limit and a second in-plane dimension W2 above the diffraction limit there is a transmission plane that is composed of the first in-plane vector and a third vector that is normal to the first and second in-plane vectors. R-polarized incident light, that is light having an electric field orthogonal to the plane of transmission, is substantially reflected by the aperture defining structure and generates an evanescent field inside the aperture. T-polarized light incident on an aperture defining structure composed of apertures of the first type, that is light having an electric field parallel to the planes of transmission of the one or more apertures, is substantially transmitted by the aperture defining structure and generates a propagating field inside the aperture.
2 For apertures of the second-type with both in-plane dimensions below the diffraction limit we cannot define a plane of transmission. Incident light of any polarization (such as linearly, circularly, elliptically, randomly polarized) is substantially reflected by the aperture defining structure and generates an evanescent field inside the aperture.
The described microelectronic sensor device allows 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 light beam, which is preferably R-polarized for apertures of the first type and may have any polarization for apertures of the second type, that is incident on the aperture defining structure generates an evanescent wave that extends from the end of the aperture adjacent to the carrier a short distance into the aperture. If light of this evanescent wave is scattered or absorbed by target components or label particles present at the binding surface, it will result in a reduction of the power/energy in specularly reflected light beam. The power/energy in the reflected light beam (more precisely the reduction of the power/energy in the reflected light beam due to the presence of target components or label particles present at the binding surface) 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 that extends typically 10 to 30 nm into the aperture from the end of the aperture adjacent to the carrier, thus avoiding disturbances (such as scattering, reflection) from the bulk material behind this volume. As the reduction of the specularly reflected light is caused by essentially only the target components or label particles present at the binding surface, a high sensitivity is achieved. 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.
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 reflected light. 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 a sample fluid that is in communication with the aperture according to the kinetics of the related binding processes.
Turning to
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 functionalized or coated with capture elements, e.g. antibodies, ligands, which can specifically bind the target components.
It is reminded here that a functionalized surface or particle is referred to as a surface or particle whereon capture elements, e.g. antibodies, ligands, which can specifically bind the target components are immobilized.
The sensor device may optionally comprise 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 10 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 10 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 a LED, that generates an incident light beam 101 which is transmitted into the carrier 11. The incident light beam 101 arrives at the binding surface 12 and is reflected as a “reflected light beam” 102. The reflected light beam 102 leaves the carrier 11 and is detected by a light detector 31, e.g. a photodiode. The light detector 31 determines the power/energy of the reflected light beam 102 (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. On the carrier surface 12, a slab of material that is not transparent, preferably metal (for example gold (Au), silver (Ag), chromium (Cr), aluminium (Al)) is provided in the form of strips 20, defining a wire grid having a smallest in plane aperture dimension (W1) smaller than a diffraction limit, the diffraction limit defined by the ratio between wavelength and twice the refractive index of the medium 2 containing the target components 10. The angle of incidence θ can in principle vary from 0 to 90°. Due to the diffraction limited nature of the aperture, in investigation area 13 an evanescent field is created that may be selectively disturbed due to the presence of particles that are bound by carrier surface 12 or at least within reach of the evanescent field generated by the aperture defining structures 20.
The described microelectronic sensor device applies optical means for the detection of particles 10 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. The use of magnetic labels in a wiregrid biosensor has the advantage (compared to the use of non-magnetic labels) that magnetic actuation can be applied for various reasons:
upconcentration of target molecules near the surface (catch assay) to improve assay speed and detection limit.
magnetic washing for stringency (instead of more complex and less-reproducible fluid washing).
In
In
Typical bead sizes are in the order of 10-1000 nm. Typical parameters for a wire grid made of Aluminium used for red excitation light (e.g., HeNe laser having a wavelength of 632.8 nm) are a period of 140 nm (59% of the diffraction limit in water for this wavelength); duty cycle of 50% and a height of 160 nm. For these parameters, the (1/e) intensity decay length in an aperture filled with water is only 17 nm. The maximum bead size (i.e., beads that ‘just’ fit in the space between the wires) is limited to somewhat smaller than 70 nm for these parameters. For an aperture of the first type, a preferred value for the first in-plane dimension W1 is less than 50% of the diffraction limit or less than 119 nm (for a wavelength of 632.8 nm and an aperture filled with water), more preferred the first in-plane dimension W1 is less than 40% of the diffraction limit or less than 95 nm (for a wavelength of 632.8 nm and an aperture filled with water), and most preferred the first in plane dimension W1 is less than 30% of the diffraction limit or less than 71 nm (for a wavelength of 632.8 nm and an aperture filled with water). A preferred value for the second in plane dimension W2 is at least the diffraction limit or at least 238 nm (for a wavelength of 632.8 nm and an aperture filled with water), more preferred the second in plane dimension W2 is 20 to 200 times the diffraction limit or 4.8 to 48 μm (for a wavelength of 632.8 nm and an aperture filled with water), even more preferred the second in plane dimension W2 is 200 to 2000 times the diffraction limit or 48 to 480 μm (for a wavelength of 632.8 nm and an aperture filled with water), and most preferred the second in plane dimension W2 is at least 200 times the diffraction limit or 480 μm (for a wavelength of 632.8 nm and an aperture filled with water).
As an example consider the case of beads with a diameter of 200 nm. For this diameter, a period of 580 nm and a duty cycle of ⅔ is a reasonable choice; opening between the wires of 387 nm. In order to avoid propagating diffraction orders for the transmitted light, the grating period should be below the diffraction limit in water (index of refraction of 1.33): for a period of 580 nm, this implies that the wavelength of the incident light is at least 1540 nm. For a wavelength of 1600 nm and a thickness of 600 nm, this results in an (1/e) intensity decay length of 109 nm and a background suppression (for the bulk on top of the wire grid) of 250.
In
Preferably, beads are used with a polymer matrix containing small superparamagnetic grains (e.g. Iron oxide). The index of the beads should be different from the index of the fluid that fills the wires (which is typically water).
A rough estimate for the impact of a bead between the wires on the transmission and reflection of the wire grid samples can be obtained from calculating the impact of filling the space between the wires with a higher index material on the intensity decay. The (1/e) intensity decay length increases from 125 nm for a wire grid filled with SiO2 (index of 1.45) up to 1550 nm for a wire grid filled with Si3N4 (index of 2). If we assume that beads with a diameter of 200 nm can be represented by a uniform layer having a thickness of 100 nm, we find an increase in the transmission by the wire grid—assuming no additional reflections due to index mismatch between the bead and its environment—of 12% and 235% respectively.
The wiregrids 20 have a period (Λ) and define an aperture W1 and thickness T.
For good reflection the opening between the sections of material is preferably below 80% of the diffraction limited opening. The diffraction limited wavelength for an aperture may typically be defined as a wavelength in the medium inside the aperture equal to twice the smallest aperture dimension W1. Typically, the efficiency varies between 0.98 for zero degree incidence, to almost 1 for 90 degree incidence (relative to a normal of a plane of incidence). As an alternative the wiregrids 20 may be replaced by an array of 2D sub-diffraction limited apertures, also referenced as a pin-hole structure. In this case the aperture defining structures is composed of apertures of the second-type mentioned here above. Accordingly these arrays have a high reflection (and evanescent fields inside the apertures) for any polarization.
A disadvantage of the arrangement of embodiment of
Advantages of the described optical read-out combined with magnetic labels for actuation are the following:
In the environment of a laboratory, well-plates are typically used that comprise an array of many sample chambers (“wells”) in which different tests can take place in parallel. The production of these (disposable) wells is very simple and cheap as a single injection-moulding step is sufficient.
While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments.
In one example, other adjacent media are used, in particular, of a refractive index smaller than the carrier medium 12. It is noted that the generation of an evanescent field is also possible using total internal reflection. Depending on the index of refraction nglass for the glass prism, the angle of incidence θA in the carrier, and the wavelength λ of the used light, the magnitude of evanescent field can be described as:
exp(−k√{square root over (nglass2sin2(θA)−nfluid2)}·z)
with z the distance from the interface and k the wavenumber (2π/λ). The penetration depth into water ((1/e) intensity) ranges from 100 nm for silica (index of refraction 1.45) down to 35 nm for a high index glass (index of refraction 2) at a beam angle of 80 degrees with respect to the normal of the detection surface. Here it is assumed that the sample matrix has refractive index nfluid=1.33 (similar to water) and that the wavelength of the used light is 650 nm (DVD laser). However, in the context of total internal reflection, the desired reduction in the specular reflection due to the presence of a bead at the interface between the carrier and the sample matrix sets a minimum for refractive index of bead:
n
bead
≧n
glass·sin(α) (1)
which implies that there is a minimum value for the refractive index of the bead. In particular, for most practical applications the penetration of the evanescent field into the sample matrix (1003) on top of the carrier is preferably limited to particles bound to the substrate. The penetration depth tdecay (1/e intensity of the evanescent field) depends on the refractive index of the prism (nglass) and the sample matrix (nfluid) and the angle of incidence (α):
t
decay=λ/(4·π·√[(nglass·sin(α))2−nfluid2]) (2)
By combining (1) and (2), one finds a criterion for the refractive index of the bead for a given penetration depth of the evanescent field:
In addition, in the context of total internal reflection, the penetration depth into the medium is limited by choice of the carrier material and the medium for containing the target components.
A suitable decay length of for instance 30 nm requires an index of the prism of at least 1.87. Preferably the prisms for total internal reflection are made of low-cost material such as Polystyrene and Polycarbonate, with typical refractive indexes of 1.55 and 1.58 respectively. These materials limit the penetration depth in water to a minimum of 65 nm and 60 nm respectively.
In addition, total internal reflection requires grazing incidence. Also, the decay length depends on the angle of incidence. For a Polycarbonate prism, an angle of incidence of 60 degrees results in a penetration depth of 504 nm. The present invention, using the generation of evanescent fields by a plurality of aperture defining structures having a smallest in plane aperture dimension W1 smaller than a diffraction limit mitigates the limitations of the total internal reflection arrangement.
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.
Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. 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 |
|---|---|---|---|
| 07301408.6 | Sep 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2008/053886 | 9/24/2008 | WO | 00 | 3/22/2010 |
