KLUNDER, Derk, J. W. (High Tech Campus 44, AE Eindhoven, NL-5656, NL)
1. A sensor comprising:
- a substrate (1) in which an excitation radiation (101,102) having a predetermined wavelength may propagate,
- a wiregrid (2) at a first side of said substrate, having at least one aperture or slit arranged for being filled with a medium (10), said medium comprising at least one particle (51) that can be detected when excited via said excitation radiation, and
- at least one reflective means (3) arranged, at a second side of said substrate which is opposite to the first side, to reflect towards the wiregrid at least a part of excitation radiation that propagate into the substrate and that has been reflected at least once by the wiregrid.
2. A sensor according to claim" 1 1, wherein the at least one aperture or slit has a first dimension below the diffraction limit of the excitation radiation in the medium (10) that fills the apertures. 3. A sensor according to claim 1 , wherein the at least one aperture has a first and second dimension below the diffraction limit of the excitation radiation in the medium (10) that fills the apertures
4. A sensor according to claim 1, or 2 or 3, wherein the reflective means is in contact with the substrate.
5. A sensor according to claim 1, or 2 or 3, wherein the reflective means forms at least two mirrors (31-32) spaced one each other and extending substantially over the surface of the second side of the substrate.
6. A sensor according to claim 1, or 2 or 3, wherein the reflective means forms an array of mirrors (31-32) spaced one each other and extending substantially over the surface of the second side of the substrate.
7. A sensor according to claim 5 or 6, wherein one (300) of the spaces at least, forms an output of the sensor, from where radiations (201) generated by the particle (51) can exit the substrate in order to be detected.
8. A system comprising a sensor according to one of the preceding claims, an excitation source of light (S) and a detection system comprising a detector (13).
IMPROVED SENSORS USING SUB- WAVELENGTH APERTURES OR SLITS
FIELD OF THE INVENTION
The present invention relates to qualitative or quantitative sensors, for example biosensors. The invention applies in particular to luminescence sensors using sub-wavelength aperture or slit structures.
BACKGROUND OF THE INVENTION
Sensors are widely used for measuring a physical attribute or a physical event. They output a functional reading of that measurement as an electrical, optical or digital signal. That signal is data that can be transformed by other devices into information. A particular example of a sensor is a biosensor. Biosensors are devices that detect the presence of (i.e. qualitative) or measure a certain amount (i.e. quantitative) of target molecules such as e.g., but not limited thereto, proteins, viruses, bacteria, cell components, cell membranes, spores, DNA, RNA, etc. in a fluid, such as for example blood, serum, plasma, saliva,.... The target molecules also are called the "analyte". In almost all cases, a biosensor uses a surface that comprises specific recognition elements for capturing the analyte.
Therefore, the surface of the sensor may be modified by attaching specific molecules to it, which are suitable to bind the target molecules which are present in the fluid. For optimal binding efficiency of the analyte to the specific molecules, large surface areas and short diffusion lengths are highly favorable. Therefore, micro- or nano- porous substrates (membranes) have been proposed as biosensor substrates that combine a large area with rapid binding kinetics. Especially, when the analyte
concentration is low (e.g. below 1 nM, or below 1 pM) the diffusion kinetics play an important role in the total performance of a biosensor assay.
The amount of bound analyte may be detected by fluorescence for example. In this case the analyte itself may carry a fluorescent label, or alternatively an additional incubation with a fluorescently labelled second recognition element may be performed.
Detecting the amount of bound analyte can be hampered by several factors, such as scattering, bleaching of the luminophore, background luminescence of the substrate and incomplete removal of excitation light. Moreover, to be able to distinguish between bound labels and labels in solution it is necessary to perform a washing step
(or steps) to remove unbound labels.
A solution to these problems has already been proposed in WO2006/136991 -Al.
In particular, in this document, a luminescent sensor system comprises a luminescence sensor, an excitation radiation source and a detector. The luminescence sensor comprises a substrate provided with at least one aperture or slit having a first dimension and with at least one luminophore in the at least one aperture for being excited by excitation radiation having a wavelength.
The at least one aperture or slit is for being filled with a medium.
The medium comprises at least one luminescent particle to be detected. In use the sensor may be immersed in the medium, e.g. in a liquid medium, or the at least one aperture or slit may be filled with the medium in any other suitable way, e.g. by means of a micropipette in case of a liquid medium, or e.g. by spraying a gas over the sensor and into the at least one aperture or slit.
The first dimension of the at least one aperture or slit is smaller than the wavelength of the excitation radiation in the medium that fills the at least one aperture.
Even though this sensor provides many advantages, a problem may still arise when the surface of detection is large.
In particular, for a given power of the excitation source, the sensitivity of detection may decrease when the detection surface increases.
SUMMARY OF THE INVENTION
Thus, an aim of the present invention is to alleviate the aforementioned problems.
More particularly, an object of the present invention is to provide improved qualitative or quantitative sensors having a large detection surface, for example biosensors, and more particularly to improved luminescence sensors of this type and using sub- wavelength aperture or slit structures. In this effect, a sensor according to the invention comprises:
- a substrate in which an excitation radiation having a predetermined wavelength may propagate,
- a wiregrid at a first side of said substrate, having at least one aperture or slit arranged for being filled with a medium which comprises at least one luminophore, the latter being to be excited via said excitation radiation, and
- at least on reflective means arranged, at a second side of said substrate which is opposite to the first side, to reflect towards the wiregrid at least a part of excitation radiation that propagate into the substrate and that has been reflected at least once by the wiregrid. Thus, according to the invention, a radiation that propagates into the substrate may encounter at least three reflections.
In particular, an excitation radiation entering into the substrate may be reflected by the wiregrid, and thus deviated towards the second side of the substrate. Then, the excitation radiation in the substrate may be reflected by the reflective means, back towards the wiregrid.
Finally, the excitation radiation may be reflected again by the wiregrid before leaving the substrate via the second side.
Such multiple reflections in the substrate make it possible to recycle the excitation radiation along the detection surface, the intensity being kept substantially constant if desired.
Others aspects of the sensor of the invention are the following: the at least one aperture or slit has a first dimension below the diffraction limit of the excitation radiation in the medium (10) that fills the apertures; the at least one aperture has a first and second dimension below the diffraction limit of the excitation radiation in the medium (10) that fills the apertures; the reflective means is in contact with the substrate;
the reflective means forms at least two mirrors spaced with respect to one another and extending substantially over the surface of the second side of the substrate; the reflective means forms an array of mirrors spaced with respect to one another and extending substantially over the surface of the second side of the substrate; one of the spaces at least, forms an output of the sensor, from where radiations generated by the particle can exit the substrate in order to be detected.
These and other characteristics, features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawing, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference figure quoted below refer to the attached drawing.
BRIEF DESCRIPTION OF THE DRAWING
Fig.l shows a system according to a preferred embodiment of the invention. This system is based on fluorescence detection.
DETAILED DESCRIPTION OF THE EMBODIMENTS
As illustrated in Fig.l, the system comprises a light source S, a bio sensor 1, 2, 3 and a detection system 11, 21, 12, 13.
The biosensor comprises a substrate 1, preferably transparent to light having a predetermined wavelength or range of wavelengths.
The biosensor further comprises, on top of the substrate 1, wires of a wire grid 2.
A medium 10, such as a liquid fills the wiregrid and comprises fluorescent labels as particles to be detected.
The liquid also comprises target molecules such as e.g., but not limited thereto, proteins, viruses, bacteria, cell components, cell membranes, spores, DNA, RNA, etc. in a fluid, such as for example blood, serum, plasma, saliva.
The fluorescent labels are supposed to attach to the target molecules.
Preferably, the wire grids have at least one aperture or slit with a first dimension below the diffraction limit in the medium (10). With the diffraction limit defined as the ratio between the wavelength of the excitation light in medium (10) divided by 2. The apertures or slits are defined in an aperture-plane that is parallel to the substrate and has first and second dimensions parallel to the aperture-plane.
As can be seen in figure 1, a fluorescent label 51 in one aperture or slit of the wiregrid has been shown for illustrative purpose.
The biosensor further comprises an array of mirrors at the bottom side of the substrate. The mirrors may have been deposited or adhered to the top surface of substrate 1 using any kind of method known in the art.
This array is configured such that it can reflect light propagating into the substrate towards the wiregrid, or in other words towards the top surface of the substrate 1.
Accordingly, an excitation light propagating into substrate 1 may encounter multiple reflections therein, and in particular reflections with wires of the wiregrid and with mirrors of the array.
In this way, the excitation light is recycled over a large surface in the substrate, said surface extending parallel to a plane defined by the wiregrid.
In particular, in figure 1, it can be seen that an excitation light 101 generated by the source S enters into substrate 1 via the bottom side according to a predetermined angle.
This light propagates towards the top side of the substrate and undergoes there a first reflection with selected wires of the wiregrid.
The reflected light 102 propagates towards the bottom side of the substrate and undergoes there a second reflection with a mirror 31.
At this stage, the excitation light has travelled into the substrate over a first distance.
After the reflection with mirror 31, the excitation light 103 has been deviated towards the top side of the substrate.
A new sequence of reflections will then occur, with other wires and with another mirror 32 of the array.
The excitation light will then have travelled into the substrate over a second distance larger than the first one and, preferably, without any loss of intensity.
As the person skilled in the art will then have recognized, the biosensor of the invention may be adapted easily to create as many sequences as necessary for recycling the excitation light in a substrate having a large area.
It is to be noted that, in case the aperture or slit has a second dimension above the diffraction limit in the medium (10) that fills the aperture, typically the excitation light
(101) generated by the source S has a polarization commonly referred to as R- polarized light. R-polarized light is characterized in that the projection of the electric field on the aperture-plane is parallel to the second dimension of the aperture.
As known in the art, R-polarized excitation light creates an evanescent excitation field in the space between the wires (2) of the wire grid.
The evanescent excitation field results in the generation of fluorescence light 201 in case that a fluorescent label 51 is present in the space between the wires where the evanescent field is.
According to the invention, it thus possible to create such an evanescent field over a large area of the wiregrid, without significantly compromising for the intensity of the excitation light.
Returning to figure 1, in the preferred embodiment the mirrors of the array 3 are spaced with respect to each other by a predetermined distance.
One of these distances, at least, is chosen such that a sufficient amount of fluorescent light which has been generated by fluorophores after being excited by the evanescent field, can escape the substrate via the bottom side.
In this embodiment, the minimum- without compromising in the efficiency with which the fluorescence can be detected- distance (d) separating two mirrors is determined by the numerical aperture of the first lens (11). For a thickness of the substrate t=700 μm and a numerical aperture of the first lens (11) of NA=O.3 results in a minimum for the distance (d) separating two mirrors of d>W(NA "2 -l), which results in d>221 μm. The width (w2) of the mirror sections must be equal to or larger than the area (wl) on the wire grid that one wants to illuminate.
Of course, other widths and distances may be contemplated and the widths and distances may not be constant over the whole array.
In the example given by figure 1 , the fluorescent label 51 generates a fluorescent light that at least partially propagates into substrate 1.
In particular, this light can exit the substrate through an aperture 300 defined between mirrors 31 and 32.
This fluorescent light then propagates in the system, outside of the biosensor, and enters the detection system. The detection system comprises successively a first lens 11 for collecting fluorescence, a filter 21 for wavelength (emission filter) and polarization filtering of the fluorescent light 201, a second lens 12 and a detector 13 (CCD typically) the second lens being for imaging the filtered fluorescent light onto said detector.
More precisely, a function of the filter 21 is to filter out parasitic excitation light by means of properly configured emission filters. Typical parameters for an emission filter are a reasonably high transmission for the fluorescent light and a strong (by 5 orders or more) rejection of the excitation light. An example of such a filter, is a commercially available emission filter by Omega Optical, Inc.: XF3076 (695 AF 55) having a rejection by at least 5 orders of magnitude for excitation wavelengths around 635 run.
An additional function of the filter is polarization filtering of the excitation light.
In this regard, it may be noted that this filter together with the wiregrid constitute a crossed polarizer to the fluorescent light which may be generated in the medium above the detection surface, and more generally above the wiregrid. Using such a crossed polarizer with a transmission axis (polarization direction) orthogonal to the wire grid, the fluorescence generated on top of the wires of the wire grid may substantially be suppressed.
Of course, the invention is not limited to the preferred embodiment described herein above. In particular, the reflective means may be a single mirror instead of an array of mirrors, this single mirror having at least a hole through which fluorescent light can escape from the substrate and can be detected.
In an alternative embodiment, the reflective means may be a reflective polarizer with a transmission axis parallel to the transmission axis of the wire grid, resulting in reflection of R-polarized excitation light.
In an alternative embodiment, one may also utilize total internal reflection on the second side of the substrate.
In this case the excitation light (102) that has been reflected at least once by the wire grid is directed back towards the wire grid by total internal reflection at the second side of the substrate.
Thus, a requirement for this embodiment is that the angle of the reflected excitation light (102) may be larger than the critical angle on the second side of the substrate. Further, even though the systems dscribed above use the biosensor in reflection mode (where detector and source are the same side of the substrate) , the principles of the invention may also be applied when using transmission mode (where the detector and source are on opposite sides of the substrate). In this regard, the previously mentioned single mirror may not necessarily comprise a hole in this case, as the light may be detected at the top side of the system instead of the bottom one.
Further, in transmission mode, the filter may consist of an emission filter only, a polarisation filter being optional.
Finally, the invention is not limited to fluorescence detection. In particular, the skilled person in the art will adapt easily the principles of the invention to the detection of non- luminescent labels, such as magnetic labels or beads for example.