WO2010032157A1

WO2010032157A1 - Wire-grid sensor with continuously controllable height of detection volume

Info

Publication number: WO2010032157A1
Application number: PCT/IB2009/053915
Authority: WO
Inventors: Derk J. W. Klunder
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2008-09-17
Filing date: 2009-09-08
Publication date: 2010-03-25

Abstract

The invention relates to a wire-grid biosensor assembly for detecting e.g. particles, beads, or molecules,where the height of the detection volume in the wire-grid can be adjusted by illuminating the wire-grid at angles above the critical angle and controlling the mixture of TE and TM polarized light. The wire-grid is formed on a substrate and has slits with a short axis being smaller than the diffraction limit of the applied radiation and a long axis being larger than the diffraction limit. The wire-grid is irradiated with excitation radiation through the substrate at an angle of incidence larger than the critical angle of the interface between the substrate and a medium that fills the slits. By adjusting a linear polarization state, θ, of the excitation radiation with a polarizer, the height of the detection volume in the slits, or similarly the penetration depth of the evanescent field, can be controlled.

Description

WIRE-GRID SENSOR WITH CONTINUOUSLY CONTROLLABLE HEIGHT OF

DETECTION VOLUME

FIELD OF THE INVENTION The present invention relates to a wire-grid sensor assembly for detecting e.g. particles, beads, or molecules. More specifically to an assembly and a method for the adjustment of the height of the evanescent detection volume.

BACKGROUND OF THE INVENTION Wire-grid sensors are often applied in relation to luminescent biosensing for detecting labeled target compounds, or in testing the binding affinity of target compounds. However, wire- grid sensors can be used in setups applying other means of sensing.

Typically, capture molecules are deposited in a pattern on a surface (hereafter the sample side of the surface). A solution containing target analytes is flown over the pattern and the target analytes attach to the capture molecules if there is a match. The matched sites are either designed to become luminescent, or are made so by introducing luminescently labeled compounds (luminophores such as fluorophores) that bind to matched sites with a high degree of specificity or by labeling the target molecules with luminescent compounds. Thereafter, excitation light is directed to the surface in a way so that it generates an evanescent field at the sample side of the surface. This evanescent field will be (partially) absorbed by the matched sites and generate luminescent light. The luminescent signal is filtered to remove the excitation wavelengths and is monitored by a CCD imaging system. Detection of multiple target analytes is achieved by spatial multiplexing, where spots with different capture molecules are deposited on the sample side of the surface. The reason for using the evanescent field for excitation is to ensure that the field excites only luminophores bound to biomolecules on or at the vicinity of the surface. Free luminophores in the solution are only excited to a very limited degree, and will therefore essentially not contribute to the background noise.

The evanescent field used for excitation typically results from total internal reflection (TIR) of light incident at the surface at angles larger than the critical angle. Thereby, no light is transmitted, only the evanescent field penetrates into sample side of the surface. In these cases, the surface is typically a waveguide or a microscope slide as is the case for a total internal reflection fluorescence microscope (TIRFM).

A special class of luminescence sensors is wire-grid sensors where sub-diffraction-limited metal wire-grids are formed on substrate surfaces, typically by photolithography, and are used as binding surface for biomolecules. In these setups, the wire-grid is illuminated with polarized light which is reflected by the sub-diffraction- limited metal wire-grid, and a rapidly decaying evanescent field is generated in the volume between the wires. Little or no excitation light is therefore transmitted by the wire grid. By patterning the wire grid with spots of capture molecules that bind at the sample side of the surface of the carrier on which the wires are deposited. By flowing a solution containing target analytes over the wire grid, the target molecules can bind with the capture molecules resulting in matched sides on the sample side of the surface. The matched sites are either designed to become luminescent, or are made so by introducing luminescently labeled compounds (luminophores such as fluorophores) that bind to matched sites with a high degree of specificity or by labeling the target molecules with luminescent compounds. The luminophores inside the slits can be excited by the evanescent field generated by the incident excitation light. This leads to luminescence light signals emitted from a small volume at the bottom, that is close to the sample side of the surface of the carrier, of the slits, and this luminescent light will typically be visible from both sides of the wire-grid..

The penetration depth of the evanescent field from the sample side of the surface and into the (typically liquid) sample, also referred to as the height of the detection volume generated by the evanescent field, determines how far away from the sample side of the surface luminophores are excited. US 7,193,711 describes a TIR based luminescence sensors where the height and intensity of the evanescent field is controlled by controlling optical properties of a control layer, e.g. thickness, refractive index, and reflectivity characteristic.

It is a disadvantage that the means of controlling the penetration depth of the evanescent field of the prior art requires controlling either the angle of incidence or optical properties of the materials of the sensor, since such means are difficult to vary during the course of a measurement or require a high-end set-up to provide the necessary accurate control of e.g., the angle of incidence. It is therefore a problem of prior art sensing concepts that these require accurate control of the angle of incidence to adjust the penetration depth.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an adjustment of the height of the detection volume generated by the evanescent field in a wire-grid sensor that overcomes the disadvantages of the prior art. According to the invention the height of the detection volume, H, can be controlled by controlling the distribution of evanescent field from TE and TM components of polarized excitation light. The contributions to the evanescent field from TE and TM components vary with at least the angle of incidence as well as the polarization state of the excitation light. For this purpose, and for linearly polarized light, the polarization state, θ, is defined as the angle of the electric field component relative to the slits of the wire-grid, with θ=0° corresponding to TE polarized light.

In a first embodiment, the invention provides a wire-grid sensor assembly with a continuously controllable height of detection volume, the wire-grid sensor assembly comprising: - a source of excitation radiation having wavelength λ for illuminating a wire-grid; a wire-grid formed by a material that is substantially non-transmissive for the excitation radiation on a surface part of a transparent substrate and defining a plurality of parallel slits with a width W along a short axis being smaller than a diffraction limit of the excitation radiation and a length L along a long axis, perpendicular to the short axis, being larger than the diffraction limit of the excitation radiation; an adjustable polarizer for adjusting a linear polarization state, θ, of the excitation radiation; the wire-grid sensor assembly being arranged so that the excitation radiation illuminates the wire- grid at an angle of incidence on the substrate surface part holding the wire-grid which is larger than a critical angle etc of the interface between the substrate and a medium that fills the slits. In a second embodiment, the invention provides a method for controlling a height of the detection volume of a wire-grid sensor, comprising: irradiating a wire-grid formed on a substrate surface part with excitation radiation at an angle of incidence, α, on said substrate surface part,

- wherein the wire-grid is formed by a material that is substantially non-transmissive for the excitation radiation and that define a plurality of parallel slits with a width W along a short axis being smaller than a diffraction limit of the excitation radiation and a length L along a long axis, perpendicular to the short axis, being larger than the diffraction limit of the excitation radiation; and

- wherein the angle of incidence α is larger than a critical angle etc of the interface between the substrate and a medium that fills the slits; and adjusting a linear polarization state, θ, of the incident excitation radiation to control a height, H, of the detection volume in the slits.

In the following, a number of preferred and/or optional features, elements, examples and implementations will be described in relation to various embodiments of the invention. Features or elements described in relation to one embodiment or aspect may be combined with or applied to the other embodiments or aspects where applicable. Also, a description of underlying mechanisms of the invention as realized by the inventors are explained for explanatory purposes, and should not be used in ex post facto analysis for deducing the invention.

The wire-grid sensor assembly may be applied in e.g. biosensors or chemical sensors. Even though the described examples relates to detecting the presence of luminescently labeled particles, the invention is not limited to luminescent sensing but can rely on other detection mechanisms. The particles to be detected may be organic or inorganic particles, beads, molecules (including macro molecules), micro organisms, etc.

In a preferred embodiment, the wire-grid is a grating formed by a material that is substantially non-transmissive for the excitation radiation with wavelength λ, preferably an electrically conductive material (i.e. the complex refractive index has a non-zero imaginary component). In another preferred embodiment, the wire-grid is formed on a surface of optically transparent solid dielectric substrate. In a typical implementation, the wire-grid is a metal film lattice formed on a glass or plastic substrate (e.g., Polystyrene) substrates by photolithographic processes. The substrate with the wire-grid is also referred to as the substrate wire-grid.

The wire-grid is to be illuminated by excitation light through the substrate. The substrate surface part holding the wire-grid is preferably planar so that the wire-grid is defined in a plane sheet. In the following, the angle of incidence, α, is the angle of the excitation radiation measured at the plane sheet from an axis in the z-direction normal to the plane sheet, with α = 0° corresponding to normal incidence. As there is a transition from higher refractive index material (ni) of the substrate to a lower index medium (n₂) filling the slits of the wire-grid, excitation radiation will be totally reflected (for all polarization states) for angles larger than the critical angle α_c determined by; α_c = arcsin (n₂/ni). The medium filling the slits is typically a liquid such as water-based solutions.

In a preferred embodiment the wire-grid and substrate are back-lit, i.e. illuminated through the substrate from the backside of the substrate (at the substrate surface opposite the wire-grid). In an alternative embodiment, the wire grid is illuminated by waveguiding where the excitation light is coupled in via a side facet of the substrate and the excitation light is transported towards the wire grid by means of multiple total internal reflections. Both these principles for generating an evanescent field at the sample side are well described in the prior art. In the remainder of this description, we will focus on embodiments where the wire-grid and substrate are back-lit, without this being construed as being in any way limiting to the scope of the claims.

As incident radiation will refract into a smaller angle of incidence upon entering the substrate, as the medium (which is typically air) on the backside of the substrate has a refractive index that is in general smaller than the refractive index of medium that fills the space between the wires (which is typically water), it is not possible to couple light that is total internally reflected at the substrate side holding the wire-grid (α > etc) into the substrate. This requires a means to locally frustrate the total internal reflection at the backside of the substrate, such as a diffraction grating or a prism. A prism is preferred over a diffraction grating, as the amount of light that can be coupled into the substrate is typically larger than for a diffraction grating. The prism can either be integrated with the substrate or can be an external prism attached to the backside of the substrate. It is preferred to comprise a prism on a surface part of the substrate opposite the surface part holding the wire-grid for coupling exciting radiation into the substrate. The components of the wire-grid sensor assembly are then preferably arranged so that the excitation radiation is incident on a facet of the prism at an angle of incidence smaller than the critical angle of the interface between the prism and the surrounding medium, even more preferably at normal angle at incidence.

The volume between the wires of the wire-grid are slits with sub-diffraction limit dimensions in only one transverse dimension. The diffraction limited dimension is defined as: d= λ/2, where λ is the wavelength of the excitation light in the relevant medium. It follows that slits are elongated or oblong with a long axis and a short axis, and will be referred to as "slits" regardless of their aspect ratio (width W along short axis/length L along long axis). The ratio between the width of the space between the wires (size of slit along the short axis) and the pitch between the wires (width of space between the wires + wire width) is referred to as duty cycle.

It should be noted that, in an optional embodiment, the invention may also be utilized with wire-grids where the volume between the wires define holes with sub-diffraction limit dimensions in all transverse dimensions, where the width in a first transverse direction is different than in the second transverse direction . In this case the holes support two modes, a first mode with an electric field in said first transverse direction and a second mode with an electric field in said second transverse direction, where the two modes have different heights of the evanescent detection volume. By controlling the polarization state, one can vary the height of the detection volume between the heights of the detection volumes for the two modes. Advantage of this embodiment is that it is more or less independent of the angle of incidence, and does not require an angle of incidence that would is total internally reflected at the interface between the substrate and the medium that fills the space between the wires; one could also use normal incident light. Disadvantage is that the control of the height of the detection volume by the angle of incidence is limited.

The evanescent intensity in the z-direction into the sample side of the surface may be described as

/ =Io e^z/H, (1) where Io is the evanescent intensity at the substrate surface part holding the wire-grid and H is the evanescent decay length. The height of the evanescent detection volume, follows from integration of this intensity along the z-direction and equals the evanescent decay length H for a wire grid with a height (H_wg) of the wires substantially larger than the evanescent decay length. .

When the substrate surface part holding the wire-grid is illuminated with polarized excitation light, the polarization states are defined as:

- TE polarized light is incident radiation where the projection of electric field on the substrate surface part holding the wire-grid is parallel to the long axis of the slits. If the wire-grid is formed in a conducting material, TE polarized light results in a rapidly decaying evanescent wave in the space between the wires, with a decay length that does not depend on the angle of incidence, i.e. also for angles of incidence smaller than the critical angle.

- TM polarized light is incident radiation where the projection of electric field on the substrate surface part holding the wire-grid is perpendicular to the long axis of the slits. TM polarized light can be transmitted for angles of incidence smaller than the critical angle and and results in an evanescent field with a decay length given by equation (2) for angles of incidence larger than the critical angle.

For TE polarized light, a decaying evanescent field is generated in the volume between the wires, and the height of the detection volume generated by the evanescent field from TE polarized light, H_TE, is determined by the dimensions of the wire-grid.

For TM polarized light, a decaying evanescent field is generated in the slits, and the height of the detection volume generated by the evanescent field from TM polarized light, H_TM, is determined by the angle of incidence α:

where λ is the wavelength, and n_sub_strate and n_medmm are the refractive indices on the substrate holding the wire-grid and the medium filling the slits, respectively.

The height of the detection volume as a function of the polarization state of the excitation light and for fixed angle of incidence is:

_H(₀ ) _ ^w Sm²Ie )H ^T,_TM + ^X _TE COS² (θ )H_rT_jE

(3)

X_TM sin ² (θ ) + X_TE cos ² (θ )

where X_TE and X_TM are the efficiencies with which TE and TM polarized light are coupled into the wire-grid. The coupling of the incident light into the wire-grid depends on the angle of incidence. For normal incident light the coupling efficiency into the TE polarized light X_TE is approximately equal to the duty cycle of the wire-grid, so that for a typical wire-grid with a duty cycle of 50% is X_TE ~ 0.5. For TM polarized light the coupling efficiency is typically larger than for TE polarized light, for the example of a wire-grid with aluminum wires and a duty cycle of 50%, X_TM ~ 0,8. X_TE and X_TM may be determined by numerical simulation. Thus, controlling the polarization state allows continuous adjustment of the height of the detection volume between Η(0) = H_TE and H(90) = H_TM, which of these are the largest will depend on the given dimensions of the wire-grid and the angle of incidence. The control of the height of the detection volume H(θ) by adjustment of the polarization state of light incident on an exemplary wire-grid is illustrated in Figure 1. Here, the exemplary wire-grid is formed on a glass substrate and illuminated under an angle of incidence larger than the critical angle.

It is preferred that the adjustable polarizer can vary the polarization continuously between a TE polarization state and a TM polarization state. This will allow the height, H, of the detection volume in the slits to be continuously controlled between a minimum value, H_min, and a maximum value, H_max. It is further preferred to vary the height, H, of the detection volume in the slits between a minimum value, H_min, and a maximum value, H_max, without changing the angle of incidence. The endpoints of the range in which the height of the detection volume can be adjusted can be controlled by proper selecting of wire-grid parameters, that control H_min, and angle of incidence, that controls H_max. It is therefore preferred to adjust the angle of incidence α > α_c to control an extremum value, H_TM, of the height of the detection volume corresponding to the incident excitation radiation having a TM polarizations state. Similarly, it is preferred to select the material and/or dimensional parameters of the wire-grid to control an extremum value, H_TE, of the height of the detection volume corresponding to the incident excitation radiation having a TE polarizations state. Typical material parameters may be the complex index of refraction of the wires, typical dimensional parameters may be the width (W) along the short axis, i.e. the space between the wires and the duty cycle.

In the present description, several angles and orientations are determined by the terms 'parallel', 'perpendicular', or 'normal to' . It is to be understood, as will also be recognized by the person skilled in the art, that angles and orientations that are not exactly parallel or normal to (i.e. 0 ° or 90°) will result in the same effect, albeit possibly with a lower efficiency. The invention can therefore be embodied, implemented and applied using small deviations from exactly parallel or normal for the relevant angles and orientations, and it is stressed that the claims are intended to cover also such small deviations. It may be preferred that the term 'parallel' means an angle of 0 ° ± 3 ° such as 0^o ± 5 ° or 0^o ± 10°. Similarly, it may be preferred that the terms 'perpendicular' , or 'normal to' mean an angle of 90 ° ± 3 ° such as 90 ° ± 5 ° or 90 ° ± 10 °.

It may be of interest to know the value of H or a parameter indicative of H for different polarization states. Hence, the wire-grid sensor assembly may comprise means for correlating a chosen polarization state with a height of the evanescent detection volume in the wire-grid. Such means for correlating may be in digital or analogue form, and may be embodied by storage holding e.g. a look-up table, a curve, a formula, a program, or an algorithm from which H can be determined from θ. The means for correlating may be determined using theoretic calculations, numeric simulations or by measurements of corresponding θ and H for a given wire-grid sensor assembly. It can be seen as the basic idea of the invention that the evanescent penetration depth and thus the height of the detection volume in the wire-grid can be adjusted by illuminating the wire- grid at angles above the critical angle and controlling the mixture of TE and TM polarized light.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will be described, by way of example only, with reference to the drawings, in which

Figure 1 is a graph showing the control of the height of the detection volume H(θ) by adjusting the polarization state of light incident on a wire-grid.

Figure 2 is a side-view of an illustration of a substrate wire-grid to be used in a wire-grid sensor assembly in accordance with an embodiment of the invention.

Figure 3 is a top-view of an illustration of a substrate wire-grid to be used in a wire-grid sensor assembly in accordance with an embodiment of the invention.

Figure 4 illustrates a wire-grid sensor assembly according to an embodiment of the invention.

Figure 5 is a graph with a curve describing H_TE from a numerical simulation for a wire-grid sensor assembly in accordance with an embodiment of the invention. Figure 6 is a graph with curves X_TE (α) and X_TM (α) from numerical simulations for a wire- grid sensor assembly in accordance with an embodiment of the invention.

DETAILED DESCRITION OF THE INVENTION

As mentioned previously, Figure 1 shows the dependence of the height of the detection volume H(θ) on the polarization state θ for an exemplary wire-grid. The wire-grid is formed on a glass substrate (n_non__ss = n_gi_ass = 1.5) and the sample side covered with water (n_ss = n_water= 1.33) and is illuminated with light with a wavelength of 633 nm at an angle of incidence just above the critical angle; α = 64° > etc = 62.5°. The wire-grid had H_TE = 20 nm.

Figure 2 illustrates a side-view of a substrate wire-grid 1 to be used in a wire-grid sensor assembly in accordance with an embodiment of the invention. A substrate 2 holds a wire-grid 3 with wires 4 of height H_wg defining slits 5 having a short axis (SA) of width W and a long axis (LA) (not shown). The substrate has a front surface part 6 holding the wire-grid and an opposite back surface part 7 where the excitation radiation is coupled in. The substrate is formed in a dielectric material that is substantially transmissive for the excitation radiation, e.g. glass (silica), or plastic (polycarbonate, polystyrene). The wires 4 are formed in a material that is substantially non-transmissive (preferably has an imaginary part of the refractive index larger than 1 ; even more preferably larger than 3) for the excitation radiation, e.g. metal such as Aluminum, Gold, Silver, or Copper. Exemplary sizes for the width W of the slits along the short axis should be below the diffraction limit. Here some examples are given with in between brackets the values for a wire grid with water in the space between the wires and a wavelength in vacuum of 633 nm: preferably smaller than λ/2 (119 nm); more preferably between 0.15 * λ and λ/4 (71-119 nm); most preferably between 0.1* λ and 0.2* λ (48-95 nm). With λ as the wavelength in the medium that fills the space between the wires. The length L along the long axis is typically substantially larger than the pitch of the wire grid, ranging from typically 10 μm up to a few cm. The figure is no to scale. The figure also shows the exciting radiation 8 illuminating the front surface part 6 at an angle of incidence α.

Figure 3 is a top view of the substrate wire-grid 1 of Figure 2. Here, the TE and the TM polarization states of the incident exciting radiation 8 are shown in relation to the wire-grid 3. Firstly, it can be seen that the substrate wire-grid 1 is arranged so that the excitation radiation 8 illuminates the wire-grid 3 from a direction which, when projected onto the substrate surface part holding the wire-grid, is perpendicular to the long axis LA of the slits, which is a preferred arrangement. Secondly, it can be seen that for the TE polarized light, the projection of electric field E on the substrate surface part holding the wire-grid is parallel to the long axis LA of the slits. Thirdly, for the TM polarized light, the projection of electric field E on the substrate surface part holding the wire-grid is perpendicular to the long axis LA of the slits.

Figure 4 illustrates a wire-grid sensor assembly 10 according to an embodiment of the invention, and is used to embodying a method for controlling the height of the detection volume according to another embodiment of the invention. In Figure 4, the substrate wire-grid 1 of Figure 2 is shown with a source of excitation radiation 11 , which is preferably a laser as the light generated by a laser can easily be collimated, for illuminating a wire-grid and an adjustable polarizer 12, which is preferably a polarizer on a rotation stage, for adjusting a linear polarization state, θ, of the excitation radiation. These components form the basic wire-grid sensor assembly 10 according to an embodiment of the invention.

Referring to the arrangements illustrated in Figures 2 and 4, the wire-grid 3 is irradiated with excitation radiation 8 through the substrate 2 at an angle of incidence α that is larger than a critical angle etc of the interface between the substrate 2 and a medium that fills the slits 5. By adjusting a linear polarization state, θ, of the incident excitation radiation 8 using the polarizer 12, a height, H, of the detection volume (not shown) in the slits 5 can be controlled according to Equation 3 described previously.

Figure 4 also shows a prism 13 for coupling the excitation radiation into the substrate under an angle smaller than the critical angle of the air-prism interface. For this purpose, the prism is arranged with a facet 14 where the excitation radiation is incident with a substantially normal angle of incidence. To ensure that the excitation radiation is not refracted between the prism and the substrate, the prism should preferably be formed in the same material as the substrate and secured with an index-matched paste. Alternatively, the prism can be formed as part of the substrate, e.g. by injection molding.

The wire-grid sensor assembly according to an embodiment of the invention can be used in a sensor setup. Here, setup can include a signal detector 15, e.g. a luminescence detector for detecting luminescent radiation 17 from labeled particles that are excited by the evanescent field in the slits of the wire-grid 3. This has the advantage that the excitation light will not couple to this side of the wire-grid and thus not disturb the measurements. Still, in order to sort out stray exciting radiation a wavelength filter 16 may be used. In other set-ups, the sensor and filter are placed on the backside of the substrate, to detect the luminescent radiation 17 through the substrate. In this case, a filter should be used to block scattered excitation radiation. This set-up, however, may be advantageous when transmission of luminescent radiation 17 on the sample side is blocked by e.g. fluid handling infrastructure as described next.

The wire-grid sensor can comprise a fluid handling infrastructure (not shown), e.g. a cover with flow channels, canals or compartments, for adapting the substrate surface part holding the wire-grid to receive a sample solution containing particles to be detected in the slits.

The detection volume generated by the evanescent field from TE polarized light, H_TE, is set by the dimensions of the wire-grid, and the variation and/or specific values of H_TE, can be determined by empirical measurement on a given wire-grid sensor assembly. Alternatively, H_TE, can be determined by numerical simulations for a given wire-grid geometry and excitation radiation wavelength. Figure 5 shows a calculated curve of H_TE and H_TM (for angles of incidence below the critical angle) as a function of the width between the space of the wires for a wire grid with aluminum wires and a wavelength in vacuum of 630 nm, which clearly shows that the decay length for TE polarized light is substantially smaller than for TM polarized light. .

X_TE and X_TM are the efficiencies with which TE and TM polarized light are coupled into the wire-grid, and these are dependent on the angle of incidence. For normal incident light, and as a rule-of-thumb, X_TE is approximately equal to the duty cycle of the wire-grid and X_TM is in the range XTE < XTM < 1 • The variation and/or specific values of XTE and XTM can be determined by empirical measurement on a given wire-grid sensor assembly under the variance of α. X_TE and X_TM can also be determined by numerical simulations for a given wire-grid geometry and excitation radiation wavelength. Figure 6 shows curves X_TE (ct) and X_TM (ct) from such numerical simulations for the same wire grid used in the calculations in Fig. 5. Figure 6 clearly shows that the excitation efficiency for TE polarized light is indeed lower than for TM polarized light.

To determine the height H of the detection volume for a given polarization state θ, means for correlating these can be provided. In one example, a look-up table such as Table 1 below can be used. Figure 1 represents another example of determined simulated correlation curve. Other embodiments for such correlation have been described previously. Table 1

Although the present invention has been described in connection with the specified embodiments, it should not be construed as being in any way limited to the presented examples. The scope of the present invention is to be interpreted in the light of the accompanying claim set.

In the context of the claims, the terms "comprising" or "comprises" do not exclude other possible elements or steps. Also, the mentioning of references such as "a" or "an" etc. should not be construed as excluding a plurality. The use of reference signs in the claims with respect to elements indicated in the figures shall also not be construed as limiting the scope of the invention. Furthermore, individual features mentioned in different claims, may possibly be advantageously combined, and the mentioning of these features in different claims does not exclude that a combination of features is not possible and advantageous.

Claims

1. A wire-grid sensor assembly (10) with a continuously controllable height of detection volume, the wire-grid sensor assembly comprising: - a source (11) of excitation radiation (8) having wavelength λ for illuminating a wire-grid; a wire-grid (3) formed by a material that is substantially non-transmissive for the excitation radiation on a surface part (6) of a transparent substrate (2) and defining a plurality of parallel slits (5) with a width W along a short axis (SA) being smaller than a diffraction limit of the excitation radiation and a length L along a long axis (LA), perpendicular to the short axis, being larger than the diffraction limit of the excitation radiation; an adjustable polarizer (12) for adjusting a linear polarization state, θ, of the excitation radiation; the wire-grid sensor assembly being arranged so that the excitation radiation illuminates the wire- grid at an angle of incidence on the substrate surface part holding the wire-grid which is larger than a critical angle etc of the interface between the substrate and a medium that fills the slits.

2. The wire-grid sensor assembly according to claim 1, further comprising a prism (13) on a surface part (7) of the substrate opposite the surface part holding the wire-grid, the wire-grid sensor assembly being arranged so that said exciting radiation is incident on a facet (14) of the prism at an angle of incidence smaller than the critical angle of the interface between the prism and the surrounding medium.

3. The wire-grid sensor assembly according to claim 1 , further comprising means for correlating a chosen polarization state with a height of the evanescent detection volume in the wire-grid.

4. The wire-grid sensor assembly according to claim 1 , wherein the substrate surface part holding the wire-grid is adapted to receive a sample solution.

5. The wire-grid sensor assembly according to claim 1, wherein the wire-grid sensor assembly is arranged so that the excitation radiation illuminates the wire-grid from a direction which, when projected onto the substrate surface part holding the wire-grid, is perpendicular to the long axis of the slits.

6. The wire-grid sensor assembly according to claim 1 , wherein the adjustable polarizer can vary the polarization continuously between a TE polarization state and a TM polarization state.

7. A method for controlling a height of the detection volume of a wire-grid sensor, comprising: irradiating a wire-grid (3) formed on a substrate surface part (6) with excitation radiation (8) at an angle of incidence, α, on said substrate surface part,

- wherein the wire-grid is formed by a material that is substantially non-transmissive for the excitation radiation and that define a plurality of parallel slits (5) with a width W along a short axis (SA) being smaller than a diffraction limit of the excitation radiation and a length L along a long axis (LA), perpendicular to the short axis, being larger than the diffraction limit of the excitation radiation; and

- wherein the angle of incidence α is larger than a critical angle etc of the interface between the substrate and a medium that fills the slits; and adjusting a linear polarization state, θ, of the incident excitation radiation to control a height, H, of the detection volume in the slits

8. The method according to claim 7, further comprising adjusting the angle of incidence α to control an extremum value, H_TM, of the height of the detection volume corresponding to the incident excitation radiation having a TM polarizations state.

9. The method according to claim 7, further comprising selecting material and/or dimensional parameters of the wire-grid to control an extremum value, H_TE, of the height of the detection volume corresponding to the incident excitation radiation having a TE polarizations state.

10. The method according to claim 7, wherein the adjustment of the polarization state, θ, comprises selecting a polarization state in a continuous range between a TE polarization state and a TM polarization state to continuously control the height, H, of the detection volume between a minimum value, H_min, and a maximum value, H_max.

11. The method according to claim 7, wherein the height, H, of the detection volume in the slits is continuously controlled between a minimum value, H_min, and a maximum value, H_max, without changing the angle of incidence.