WO2013102661A1

WO2013102661A1 - Spectroscopic sensor for bio-sensing

Info

Publication number: WO2013102661A1
Application number: PCT/EP2013/050103
Authority: WO
Inventors: Carsten Thirstrup
Original assignee: Carsten Thirstrup
Priority date: 2012-01-04
Filing date: 2013-01-04
Publication date: 2013-07-11

Abstract

The present invention relates to a spectroscopic sensor comprising an optical sensor unit comprising a solid transparent member (1), an inclined- side- surface (2) disposed on the solid transparent member, a reflective diffractive optical element disposed on the solid transparent member, and a sensing portion (5). The reflective diffractive optical element is adapted to diffract and further direct light beams to an output coupling surface ( 9 ) disposed on the solid transparent member. Moreover, the reflective diffractive optical element and the output coupling surface are arranged to direct, with spatial separation of different wavelengths, light beams onto the external detector surface (11).

Description

SPECTROSCOPIC SENSOR FOR BIO-SENSING FIELD OF THE INVENTION

The present invention relates to spectroscopic sensors for bio-sensing, in particular wavelength interrogation surface plasmon resonance sensors and localized surface plasmon resonance sensors.

BACKGROUND OF THE INVENTION

There is a growing need for analyses of biological and chemical substances within health care, environmental protection, drug development, food safety, forensics, and industrial quality control. In the art, there are pure spectroscopic analytical methods including ultraviolet-visible, near- infra red, infrared and Raman spectroscopy combined with advanced data processing techniques such as multivariate modelling. Other methods including fluorescence spectroscopy and surface plasmon resonance spectroscopy combine spectroscopic detection of substances with biosensor elements based on biochemical interactions with or biomolecular recognition of molecules and/or particles. The surface plasmon resonance (SPR) sensor is an optical sensor making use of biosensor elements, and it is being used extensively for analysis of bio-chemical and chemical substances. There are two main types of surface plasmon resonance sensors. The traditional type of SPR sensor is based on optical excitation of surface plasmons in a thin film of a noble metal such as gold or silver on a transparent dielectric substrate having a refractive index higher than the substance above the metal film. Surface plasmons are excited optically by coupling p-polarized light through the dielectric substrate to the metal film at the resonant wavelength and angle of incidence of light. The light totally internally reflected from the metal film can be monitored by an optical detector system, where a strong reduction of the light intensity is detected at the resonant wavelength and angle of incidence of light. The change in resonance conditions can be monitored by the detector system as function of changes in the refractive index within a few hundred nanometres from the metal surface. This type of SPR sensor can be operated in two different modes [M. Piliarik and J. Homola, Optics Express 17, 16505 (2009)] . One mode of operation is angular interrogation, where the wavelength is fixed and the SPR response is monitored as function of angle of incidence of light to the metal film. Another mode of operation is wavelength interrogation, where the angle of incidence of light to the metal film is fixed, and the SPR response is monitored as function of wavelength.

The localized-surface-plasmon resonance sensor (LSPR) is another type of optical sensor, which is based on optical excitation of surface plasmons in nanoscale particles of noble metals [B. Sepiilveda, P.C. Angelome, L. M. Lechuga, and L.M. Liz-Marzan, Nano Today 4, 244 (2009)], and it combines bio-sensing with spectroscopy. Usually gold nano-particles of a few tens of nanometres are used. Localized surface plasmons are collective oscillations of electrons in the metal nano-particles surrounded by the dielectric. Because of the increased confinement in a nano-particle, the electric field is enhanced close to the surface of the nano-particle compared to the traditional type of SPR, where surface plasmons are excited on a metal film. The light transmitted through a substance comprising noble metal nano-particles is absorbed in a particular

wavelength range, and the absorption peak wavelength changes with changes in the refractive index surrounding the nano-particles. The LSPR involves increased absorption and scattering and it is a more sensitive measurement technique than traditional SPR. The LSPR is usually detected as light absorption by light transmission through a substance comprising the nano-particles. Measurements of scattering by total internal reflection spectroscopy is also possible, but this technique usually exhibits poorer signal- to-noise ratio [B. Sepiilveda, P.C. Angelome, L. M. Lechuga, and L.M. Liz-Marzan, Nano Today 4, 244 (2009)] .

Both traditional SPR sensors and LSPR sensors require detector systems, which are either tailored to the application or they require spectroscopic systems like optical fibre based spectrometers. Commercial spectroscopic systems are available, but they usually have a large size, are costly, not flexible in use, and they can normally only measure one substance at a time. On the other hand, there is a large variety of commercial image sensors and cameras, which may be used as optical detectors for SPR/LSPR sensors based on wavelength interrogation, but in the art there is a lack of simple and flexible optical interfaces between the sensors and the optical detectors.

There have been attempts to make miniature and low cost SPR sensors. US pat. no. 6,183,696 has miniaturized an SPR sensor by integrating the light source (a light- emitting-diode), a photodiode detector array and a metal film comprising a sensing layer into one unit. However, the device requires a large surface to be covered by the sensing layer and it is therefore not suited for measuring multiple substances. Since it is based on angular interrogation, it cannot be used for measuring LSPR. US pat. appl. no. 2007/0285666 describes an information-acquiring device, which is adapted to measure a change of wavelength characteristics on an optical transmittance in a substance to be detected. The invention is designed for miniaturizing both the sensor unit and the detection device, and it is suitable for using localized surface plasmon resonance. The miniaturization is made integrating the light source and the light-receiving means onto the same substrate, such as silicon or gallium arsenide. However, the sensing element and the wavelength-varying means comprising elements such as a diffraction grating and a concave mirror are separate components and they need to be aligned with each other and with the light source and the detection device on the substrate. In addition, the integration of the light source and the detector system onto a semiconductor substrate means that the sensor system lacks flexibility.

US pat. no. 7,262,845 describes an invention of a miniaturized diffractive imaging spectrometer, which comprises a highly compact device, which can receive multiple optical input signals from input channel waveguides and produces a spectrally resolved image, covering a wide spectral range with high resolution. A diffractive optical element collimates the input signal from a plurality of waveguides and focuses the light onto an image sensor via an aberration correction prism. The image sensor is attached to the prism having an optimized angle with respect to the diffractive optical element of approximately 53°. This invention has solved the problem of miniaturizing the spectrometer by integrating the optical elements including the wavelength-varying means and the detector into the same unit. The invention is adapted for a multitude of inputs, and the miniaturized spectrometer may be designed for a wavelength range between 400 and 700 nm. However, the image sensor needs to be in proximity with the aberration correcting prism and at an angle, which limits its flexibility. It may therefore be difficult to interface the angled prism-coupling to commercially available cameras or image sensor systems. In addition, the waveguide coupling described by this invention is tailored towards specific applications with one waveguide for each sensor element and it does not provide a solution for optical coupling to surface plasmons.

There is therefore a need in the art of spectroscopic sensors for bio-sensing and chemical sensing of a multitude of substances, in particular for using surface plasmon resonance and localized surface plasmon resonance, where the sensor elements, the wavelength-varying means and the imaging optics are integrated into the same unit enabling an easy and flexible interface to commercially available light sources and detector systems. It may be seen as an object of embodiments of the present invention to provide a spectroscopic sensor being a small size and compact system for analyzing a multitude of bio-sensor and chemical sensor elements.

It may be seen as a further object of embodiments of the present invention to provide a spectroscopic sensor, which is suitable for using surface plasmon resonance and/or localized surface plasmon resonance.

It may be seen as an even further object of embodiments of the present invention to provide a spectroscopic sensor comprising a separate optical sensor unit and a separate reader unit, where the optical sensor unit comprises wavelength varying means and imaging optics integrated in a solid transparent member and where the reader unit comprises light sources and a detector system.

It may be seen as an even still further object of embodiments of the present invention to provide a spectroscopic sensor, which has an easy and flexible interface to commercially available light sources such as white light emitting diodes or white light emitting diode arrays and commercially available cameras or image sensors such as complementary- metal-oxide-semiconductor (CMOS) image sensors.

DESCRIPTION OF THE INVENTION

The above-mentioned objects are complied with by providing, in a first aspect, a spectroscopic sensor comprising - an optical sensor unit comprising a solid transparent member, an inclined-side-surface disposed on the solid transparent member, said inclined- side-surface being adapted to receive incoming light beams of a plurality of wavelengths and being adapted to direct first light beams as essentially collimated light beams towards a reflective diffractive optical element disposed on the solid transparent member, and a sensing portion adapted to comprise a sample to be analysed, said sensing portion being positioned in an optical path between the incoming light beams and an external detector surface, wherein the reflective diffractive optical element is adapted to diffract and further direct second light beams to an output coupling surface disposed on the solid transparent member, said output coupling surface being adapted to direct third light beams onto the external detector surface, and wherein the reflective diffractive optical element and the output coupling surface are arranged to direct, with spatial separation of different wavelengths, the third light beams of the plurality of wavelengths onto the external detector surface.

The sensor of the present invention is advantageous in that it offers a simple, a robust and a cost effective layout. In fact, one may choose to use the sensor as a disposable device.

The term "external detector surface" is to be understood as a detector surface being external relative to the optical sensor unit. The external detector surface may form part of an external detector unit which may be attachable to/detachable from the optical sensor unit. Optionally the external detector unit may house one or more light sources for providing one or more light beams of a plurality of wavelengths.

The reflective diffractive optical element may comprise a variation in a grating spacing, wherein said variation in the grating structure and a shape of the output coupling surface in combination may focus, with spatial separation of different wavelengths, the third light beams onto the external detector surface. The spectroscopic sensor may further comprise collimating means. The collimating means may be integrated into the solid transparent member such that the incoming light beams are directed as essentially collimated light beams towards the inclined-side- surface. The collimating means may comprise one or more collimating lens' or one or more collimating lens arrays. The sensing portion may be disposed on a surface of the solid transparent member. This surface may involve an outer surface, or part of an outer surface, of the solid

transparent member. In fact the surface may form an integral part of a surface portion of the solid transparent member.

The sensing portion may comprise an electrically conducting material, such as gold, silver or aluminium for supporting surface plasmons. The sensing portion may be disposed in front of the collimating means. In this configuration the sensing portion may comprise nano-particles comprising a conducting material, such as gold, silver or aluminium for supporting localized surface plasmons.

The plurality of wavelengths may cover a spectral range from 400 nm to 700 nm, or from 550 nm to 800 nm.

The solid transparent member may comprises a polymer material, such as acrylics, polycarbonate, polyetherimide or polyolefin, such as cyclic olefin copolymer, whereas the inclined-side-surface may comprise an externally arranged reflective material, such as gold, silver, aluminium, or a dielectric reflective coating. In a second aspect the present invention relates to a spectroscopic sensor assembly comprising a spectroscopic sensor according to the first aspect in combination with a light source emitting essentially white light. The essentially white light source may comprise one or more white light emitting diodes or one or more white light emitting diode arrays, such as one or two-dimensional light emitting diode displays. The assembly may further comprise an external sensor comprising the external sensor surface, said external sensor comprising a one or a two-dimensional CMOS image sensor or CCD image sensor.

In a third aspect the present invention relates to a method of determining biochemical and/or chemical substances within a sample using a spectroscopic sensor or sensor assembly according to the first and second aspects.

In a fourth and final aspect the present invention relates to use of a spectroscopic sensor or sensor assembly according to the first and second aspects for determining

biochemical and/or chemical substances within a sample.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will now be explained in further details with reference to the accompanying figures, wherein

Fig. 1 illustrates (a) an embodiment of the present invention of a spectroscopic sensor comprising a solid transparent member (1) with integrated mirror-coated inclined-side- surface (2) having an angle (10) less than 45° to a bottom surface (3) for input coupling of one or more light beams comprising a plurality of wavelengths (4), a sensing portion (5) being adapted to totally internally reflect the light beams (4), a reflective diffractive optical element (8) on the top surface (14) of the solid transparent member (1) and an output coupling surface (9) for coupling of the light beams (4a, 4b) to an external detector surface (11) with spatial separation of different wavelengths; (b) An

embodiment of the present invention of a spectroscopic sensor comprising a solid transparent member (21) with an integrated mirror-coated inclined-side-surface (22) having an angle (30) larger than 45° to a bottom surface (23) for input coupling of one or more light beams (24), a sensing portion (25) being adapted to totally internally reflect the light beams (24), a reflective diffractive optical element (28) on the top surface of the solid transparent member (21) and an output coupling surface (29) for coupling of the light beams (24a; 24b) to an external detector surface (31) with spatial separation of different wavelengths,

Fig. 2 illustrates (a) an embodiment of the present invention of a spectroscopic sensor comprising a solid transparent member (41) with a sensing portion (45) being adapted to transmit the incoming light beams with a plurality of wavelengths (44), an air gap (56), a collimation lens or collimation lens array (53), which is integrated into the solid transparent member (41), an integrated mirror-coated inclined-side-surface (42) having an angle (50) less than 45° to a bottom surface (43) for input coupling of one or more light beams (44), a reflective diffractive optical element (48) on the top surface (54) of the solid transparent member (41), and an output coupling surface (49) for coupling of the light beams (44a; 44b) onto an external detector surface (51) with spatial separation of different wavelengths; (b) An embodiment of the present invention of a spectroscopic sensor comprising a solid transparent member (61) with a first integrated mirror-coated inclined-side-surface (62) having an angle (70) essentially equal to 45° to a bottom surface (63) for input coupling of light beams (64), a sensing portion (65) being adapted to transmit the light beams, a second side-surface (76) having an angle (80) less than 45° to the bottom surface (63), a reflective diffractive optical element (68) on the second side-surface of the solid member (76), and an output coupling surface (69) for coupling of the light beams (64a; 64b) onto an external detector surface (71) with spatial separation of different wavelengths,

Fig. 3 is a schematic cross-sectional view of one embodiment of the output coupling surface having a cylindrical shaped surface with a radius (R) and a centre line of the cylinder (O), Fig. 4 illustrates (a) an example of a calculation of ray tracing in one embodiment of the present invention for two wavelengths of light, at 600 nm (illustrated with three solid rays) and at 750 nm (illustrated with three dashed rays), (b) a plot of a calculation of the beam position (solid curve) and the beam width (dashed curve) on the external detector surface (y=0 in (a)) as function of wavelength for the ray tracing in (a),

Fig. 5 illustrates (a) an embodiment of the present invention of a spectroscopic sensor comprising a solid transparent member (101) with integrated mirror-coated inclined- side- surface (102) having an angle (110) less than 45° to a bottom surface (103) for input coupling of one or more timely separated light beams comprising a plurality of wavelengths (104a, 104b, 104c), a sensing portion (105) being adapted to totally internally reflect the light beams (104a, 104b, 104c), a reflective diffractive optical element (108) on the top surface (114) of the solid transparent member (101) and an output coupling surface (109) for coupling of the timely separated light beams

(104a', 104b', 104c') to an external detector surface (111); (b) a plot of a calculation of the spectral distribution of light beams at the input to the bottom surface (103) along this surface for a predetermined position of a light ray on the external detector surface as function of wavelength, and

Fig.6 illustrates an example of an application of the present invention of a spectroscopic sensor where (a) illustrates components of the application including a spectroscopic sensor (94), a clamp component (95) and a smartphone (90) and (b) illustrates the components assembled.

While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of examples in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The present description mainly concerns spectroscopic sensors based on wavelength interrogation based SPR sensors and LSPR sensors, but it also applies to other types of biosensors and chemical sensors employing light spectroscopy. Surface plasmon resonance sensors include traditional SPR sensing with totally internally reflected light from a sensing portion comprising an electrically conducting film (hereafter named SPR sensors), and localized-surface-plasmon-resonance sensors with light being transmitted through a sensing portion comprising electrically conducting nano-particles (hereafter named LSPR sensors). The electrically conducting film in SPR sensors is primarily made of noble metals such as gold or silver with a thickness of a few tens of nanometres, typically 50 nm, but it can also be aluminium films. The electrically conducting nano-particles in LSPR sensors are primarily gold or silver particles, but aluminium nano-particles may also be used.

Fig. 1 illustrates two embodiments of the present invention comprising a sensing portion (5; 25) where the light beams (4; 24) are totally internally reflected. In Fig. 1(a), the angle (10) between the inclined-side-surface (2) and the bottom surface (3) of the solid transparent member (1) is smaller than 45°, and in Fig. 1(b), the angle between the inclined-side-surface (22) and the bottom surface (23) of the solid transparent member (21) is larger than 45°. In Fig. 1(a), one or more incoming light beams comprising a plurality of wavelengths (4) originating from a light source (12) and collimated with a lens, lens system or lens array (13) enter the solid transparent member (1) of a spectroscopic sensor from the bottom surface (3) at an angle of incidence, which is essentially perpendicular, impinges onto a mirror-coated inclined-side-surface (2) having an angle (10) to the bottom surface of less than 45°. The light beams are reflected from the mirror-coated inclined-side-surface

(2) , further reflected from the bottom surface (3) and illuminates the backside surface of an electrically conducting film (7) disposed on the top surface (14) of the solid transparent member (1). The light beams are further reflected from the bottom surface

(3) , diffracted from a reflective diffractive optical element (RDOE) (8) and directed towards an output coupling surface (9). The light beams are further refracted from the output coupling surface (9) of the solid transparent member and focussed onto an external detector surface (11) with spatial separation of different wavelengths. The output coupling surface (9) joins the plane bottom surface (3) at a position (9')-

In Fig. 1(b), one or more incoming light beams comprising a plurality of wavelengths (24) originate from a light source (32) and are collimated by a lens, a lens system or a lens array (33) and enter the solid transparent member (21) of a spectroscopic sensor from the bottom surface (23) at an angle of incidence which is essentially perpendicular, impinges onto a mirror-coated inclined-side-surface (22) having an angle (30) to the bottom surface (23) larger than 45°. The light beams reflected from the mirror-coated inclined-side-surface (22) illuminates the backside surface of an electrically conducting film (27) disposed on the top surface (34) of the solid transparent member (21). The light beams are further reflected from the bottom surface (23), diffracted from a reflective diffractive optical element (RDOE) (28) and directed towards an output coupling surface (29). The light beams are further refracted from the output coupling surface (29) of the solid transparent member (21) and focussed onto an external detector surface (31) with spatial separation of different wavelengths. The output coupling surface joins the plane bottom surface at a position (29')- Fig. 1(a) and (b) illustrates schematically the first order diffraction of a light beam at two different wavelengths, a short wavelength within the plurality of wavelengths illustrated with solid rays (4a; 24a) and a long wavelength within the plurality of wavelengths illustrated with dashed rays (4b; 24b). The output coupling surface (9; 29) has a shape which in combination with the spatial variation of the grating spacing of the reflective diffractive optical element (RDOE) (8; 28) enables the light beam (4; 24) to be focussed onto the external detector surface (11 ; 31) for a wavelength range within the plurality of wavelengths.

As illustrated in schematically in Fig. 1(a) and (b), portions of the light beams with different wavelengths are focussed onto different positions on the external detector surface (11; 31), and the response from a detector system positioned at the external detector surface can be resolved spectrally. The zero order diffracted light beams being diffracted from the RDOE (8; 28) should preferably be suppressed in order not to cause too much stray light on the detector surface. The second side-surface ( 15; 35) may be designed to absorb the zero order diffraction beams, or they may be perpendicular to the zero order diffracted light beam or anti-reflection coated in order to guide the zero order diffracted light beams out of the transparent member or a combination. The inclined-side-surface (2; 22) may be coated externally with a highly reflective material such as a metal including gold, silver or aluminium. The RDOE may also be coated externally with a highly reflective material such as a metal including gold, silver or aluminium. In the two embodiments of Fig. 1, the inclined-side-surface is illustrated as a plane surface, but the scope of the present invention also covers other surfaces of the inclined-side-surface and depending on the shapes of the incoming light beams, the surfaces may be concave or convex. In addition, the scope of the present invention also covers embodiments, where the inclined-side-surface is uncoated and the incoming beams are transmitted through or refracted by the inclined-side-surface. For an SPR sensor, the angle of incidence to the sensing portion (5; 25) should match the surface plasmon resonance. For a solid transparent member with a refractive index of ~ 1.5, a metal surface of gold and a sensing portion with an aqueous solution, the resonance angle (9_res) is typically in the range from 60 to 75°. The corresponding wavelength range is typically in the range from 600 to 800 nm. In Fig. 1(a), the angle a (10) of the inclined-side-surface should be approximately equal to half the value of e_res, i.e. a should preferably be in the range from 30° to 38°, when 6_res is in the range from 60° to 75°. In Fig. 1(b), the angle a (30) of the inclined-side- surface should be approximately equal to 9O° - 0_res/2, i.e. a should preferably be in the range from 52° to 60°, when 6_res is in the range from 60 ⁰ to 75°.

The sensing portions (5; 25) comprising the electrically conducting film (7; 27) and the bio-sensing area comprising sensor elements (6; 26) may be part of a microfluidic system attached to the top surface of the solid body of the spectroscopic sensor. The microfluidic system is not shown in Fig. 1. Since only p-polarized light excites surface plasmons in electrically conducting films, it is preferable that only p-polarized light is incident onto the backside of the electrically conducting film (7; 27). This can be ensured by providing a polarization filter either integrated in the solid transparent member (1; 21), such as onto the backside surface (3; 23) or in front of the incoming light beams (4; 24). Fig. 2 illustrates two other embodiments of the present invention employing a sensing portion being adapted to transmit the light beams and it is particularly suitable for using the localized surface plasmon resonance.

In Fig. 2(a), one or more light beams originating from a light source or an array of light sources (52) are coupled into a sensing portion (45) comprising a bio-sensing area (46) comprising metal nano-particles and being adapted to transmit the light beams. A collimation lens or collimation lens array (53), which is integrated into the solid transparent member (41), collimates and directs the light beams (44) towards a mirror- coated inclined-side-surface (42) having an angle (50) less than 45° to a bottom surface (43). An air gap (56) is disposed between the sensing portion (45) and the collimation lens or lens array (53). The light beams are reflected from the mirror-coated inclined- side-surface (42) and further reflected from the bottom surface (43), the top surface (54), and the bottom surface (43) again and illuminates a reflective diffractive optical element (RDOE) (48), which diffracts and directs the light beams towards an output coupling surface (49) of the solid transparent member (41). The light beams are further refracted from the output coupling surface (49) and focussed onto an external detector surface (51) with spatial separation of different wavelengths. The output coupling surface (49) joins the bottom surface (43) at a position (49')- Fig. 2(b) illustrates another embodiment of the present invention adapted to localized surface plasmon sensing with the sensing portion (65) comprising a bio-sensing area (66) comprising metal nano-particles, but with a different position of the sensing portion and the reflective diffractive optical element (RDOE). One or more light beams (64) originating from a light source or an array of light sources (72) and collimated by a lens, a lens system or a lens array (73) enter the solid transparent member (61) of a spectroscopic sensor from the bottom surface (63) at an angle of incidence, which is essentially perpendicular, impinges on a mirror-coated inclined-side-surface (62) having an angle (70) to the bottom surface (63) essentially equal to 45°. The mirror-coated inclined-side-surface (62) further directs the light beams to a sensing portion (65), which is adapted to transmit the light beams, which are further directed to a RDOE (68) disposed on a second side-surface (76) having an angle (80) less than 45° to the bottom surface (63). The RDOE diffracts and directs the light beams towards an output coupling surface (69) of the solid transparent member (61). The light beams are further refracted from the output coupling surface (69) and focussed onto an external detector surface (71) with spatial separation of different wavelengths. The output coupling surface (69) joins the bottom surface (63) at a position (69')-

For LSPR sensing, the LSPR spectral position is highly dependent on the composition, size or shape of the nano-particles, as well as the refractive index of the dielectric medium in the sensing portion. Using gold nano-particles with sizes from 1 to 50 nm, the LSPR wavelength range is typically in the range from 400 to 700 nm.

Figs. 1 and 2 illustrate schematically by ray-tracing in a cross-sectional view the diffraction of a light beam at two different wavelengths, a short wavelength within the plurality of wavelengths illustrated with solid rays (4a; 24a; 44a; 64a) and a long wavelength within the plurality of wavelengths illustrated with dashed rays (4b; 24b; 44b; 64b). A set of three rays illustrates schematically the light beam width in the plane of incidence. The output coupling surface (9; 29; 49; 69) has a shape, which in combination with the spatial variation of the grating spacing of the RDOE (8; 28; 48; 68) enables the light beams (4; 24; 44; 64) to be focussed onto the external detector surface (11; 31 ; 51 ; 71) for the plurality of wavelengths. In the ray-tracing, each set of three rays [the three solid rays (4a; 24a; 44a; 64a) have a short wavelength (1 and the three dashed rays (4b; 24b; 44b; 64b) have a long wavelength (1₂ > l within the plurality of wavelengths] being diffracted from the RDOE (8; 28; 48; 68) are incident on essentially the same position on the detector surface (11 ; 31; 51 ; 71). The spectral resolution (Δ1) within the wavelength range (λ₂ ~ ) of the spectral sensor is determined by the distance between the two positions on the detector surface of the two sets of rays (s) (17; 37; 57; 77) and the width of the light beam at the particular wavelength (As) on the detector surface , i.e.

Δλ = ^ (λ₂ - λ₁) (1) where As(l) and s(l) are wavelength dependent. Optimization of the spectral resolution involves minimizing Δ1 according to equation (1). For a given input angle to the RDOE, which in the case of the SPR sensor needs to match the surface plasmon resonance and a desirable wavelength range (λ₂ the grating spacing of the RDOE and the shape of the output coupling surface are adjusted in order to obtain a minimum in As/s within the wavelength range. A minimization procedure may be based on computing the least squared summation (or integration) of As(l)/s(l) .

The thickness of the spectroscopic sensor and the number of reflections between the bottom surface and top surface can be varied in order to match the separation between the light source and the detector surface. In the embodiments illustrated in Figs. 1-2, only a few reflections have been illustrated. The thickness of the solid transparent member (18; 38; 58; 78) is preferably in the range from 0.5 to 20 mm, more preferably from 1 to 10 mm and even more preferably from 1.5 to 5 mm. The length of optical area of the solid transparent member measured from the centre of the inclined- side-surface (2; 22; 42; 62) to the centre of the RDOE (8; 28; 48; 68) is preferably in the range from 5 mm to 120 mm, more preferably in the range from 10 to 70 mm, and even more preferably from 15 to 40 mm.

The angle of incidence (Θ) of the light beam to the top surface (14; 34; 54) and the bottom surface (3; 23; 43) should preferably be larger than the critical angle (9_C) of the solid transparent member (1; 21 ; 41), i.e. Θ > 9_C = arcsin(^) (2) where arcsin is the arcsine function, n₀ is the refractive index surrounding the solid transparent member, which is usually air and equal to 1, and n_g is the refractive index of the solid transparent member, which for a typical polymer material is approximately equal to 1.5. The minimum angle for the propagating light beam according to (2) is 9_C « 40°, which is less than a typical 9_res for an SPR sensor of the embodiments in Fig. l(a-b) being in the range from 60° to 75° for a sensing portion comprising an aqueous solution with a refractive index close to 1.33.

The present invention of a spectroscopic sensor also includes embodiments for gas sensing utilizing SPR and/or LSPR. In this case, the refractive index of the sensing portion comprising gas or air is close to 1.0, and 9_res may be as low as «43°, which is very close to the critical angle for embodiments of polymer materials with a refractive index of «1.5. For these embodiments, at positions where the light beams are internally reflected, it may therefore be advantageous to coat the surfaces by a highly reflective layer like a metal film, a dielectric coating with high effective refractive index or the like. The width of the solid transparent member of the present invention is perpendicular to the plane of the schematic drawings illustrated in Figs. 1-2. The width of the solid sensor body should preferably be similar to or larger than the width of the detector surface. The external detector surface may be the top surface of a detector array (11a; 31a; 51a; 71a) such as two-dimensional image sensors including charge-coupled-device (CCD) image sensors and CMOS image sensors. The detector array (11a; 31a; 51a; 71a) may further comprise optics to image the focussed light ray onto the pixels of the detector.

The light source (12; 32; 52; 72) may comprise one or more white light emitting diodes (LEDs) comprising pixels of red, green and blue LEDs with wavelengths being emitted between 420 nm and 780 nm (see e.g. US pat. appl. 2010/0067004, Fig. 10). The light source may further comprise collimating optics ensuring that the light beams reflected from the mirror-coated inclined-side-surface (2; 22; 42; 62) are collimated. The present invention also comprises embodiments where the light source is a photodiode array, such as an LED display with a wavelength emitting region approximately between 420 and 780 nm, but the scope of the present invention is not limited to this wavelength range. The advantage of using an LED display is that each pixel in the diode array can be turned on or off electronically, which can be controlled by software or firmware. Part of the incident light beam can be turned on and off, enabling sequentially illuminating a part of the sensing portion, which increases the number of sensing elements to be measured. The sensing portion may comprise an array of biosensor and/or chemical sensor elements aligned in the plane of incidence of the light beams. For instance, in Figs. 1(a) and (b), a sensor element may be disposed on top of each of the three rays of the light beam illustrated schematically (4; 24). Turning on and off each light ray, one by one photodiode of a photodiode array (not shown in Fig. 1), each sensor element can be measured individually. The array of biosensor and chemical sensor elements may also be aligned perpendicular to the plane of incidence of the light beams with a width matching the width of the detector surface.

Fig. 3 illustrates an example of a design of an output coupling surface (9; 29; 49; 69) being employed in the embodiments illustrated in Figs. 1-2. The output coupling surface illustrated is a curved surface having a circular cylindrical shape with a radius (R) and a line centre of the circle (0). The present invention also covers output coupling surfaces, which are plane surfaces. The present invention further covers shapes of curved surfaces including hyperbolic cylindrical shapes and spherical shapes. The curved surface may be concave or convex, but concave curved surfaces as illustrated in Fig. 3 are generally needed in order to minimize Eq. (1).

The solid transparent member of the sensor unit is made in a transparent dielectric material such as glass or polymer, but preferably in a polymer because of low production costs. The RDOE (8; 28; 48; 68) in the solid transparent member can be produced according to a method published in int. patent appl. no. WO 02/08800, which describes the formation of a surface relief pattern adapted to be replicated onto a substantially plane surface of a transparent member to form a reflective diffractive optical element.

In the present invention, the reflective diffractive optical elements comprises grooves with a variation in the spacing (a_grat), which combined with the shape of the output coupling surface (9; 29; 49; 69) enables minimization of Eq. (1), and for each wavelength within the plurality of wavelengths the collimated beams incident to the RDOE are focussed onto a line or curve on the external detector surface. The spatial separation of the lines or curves for the wavelengths determines the spectral resolution of the spectroscopic sensor of the present invention according to Eq. (1). The grating of the RDOE is preferably blazed for a wavelength close to the angle of incidence (ø).

Fig. 4 illustrates an example of a design of a spectroscopic sensor according to the present invention. It should be noted that it is only an example and does not limit the scope of the present invention. Fig. 4(a) illustrates ray tracing from an incoming collimated light beam with a width of 1 mm onto the bottom surface of a solid transparent member of an embodiment of the present invention. A rectangular coordinate system with the units of mm is depicted for the rays and the solid

transparent member. The light beam is reflected from an inclined-side-surface, and multiply reflected between a bottom surface and a top surface and illuminates a reflective diffractive optical element (with a schematic illustration), where it is diffracted and directed (the first order diffracted beam) towards an output coupling surface and focussed onto a detector plane. The parameters used in the calculations are, a thickness of the solid transparent member, d=2 mm, an angle between the inclined-side-surface and the bottom surface, a=34°, yielding an angle of incidence, θ = 2 = 68°. The distance between the bottom surface and the detector plane is, L=23 mm, the output coupling surface has a circular shape with a radius of R=60 mm and a centre of the circle being positioned at (x, y) = (26 mm, -35 mm). Rays are illustrated for a

wavelength of 700 nm (solid rays after the RDOE) and 600 nm (dashed rays after the RDOE) .

Fig. 4(b) illustrates a calculation of the position of the beam on the detector plane (at y = 0) as solid curve and the light beam width (As) on the detector plane (dashed curve) as function of wavelength. The resolution of the spectroscopic sensor is defined in Eq. (1). As observed from Fig. 4(b), the largest value of As/s = 0.2 mm/(7-(-9)

mm) = 0.0125. The corresponding wavelength resolution according to eq . (1) is 3.1 nm. Other effects also contribute to the resolution including non-perfect collimation of the light beam. In order to resolve a surface plasmon response a narrower wavelength range from 600 to 750 nm is usually acceptable.

The wavelength dependence of the refractive index of the solid member has not been taken into account in the present ray tracing. The design of the solid transparent member based on ray tracing can therefore be further optimized.

For the example of a design of an RDOE in Fig. 4(a), the grating spacing of the RDOE is monotonically increasing with decreasing position x and it can be approximated by a second order polynomial according to a(x) = c₂ (x— x₀)² + c_x {x— x₀) + c₀ where x₀ = 4.43 mm is the centre of the grating, c₂ =0.40 nm/mm², q = 13.8 nm/mm and c₂ =473.5 nm. Fig. 5 illustrates (a) a further embodiment of the present invention, where the incoming light beams are timely separated from an external light source such as a one- dimensional or two-dimensional white light emitting diode array (112). Each individual pixel in the diode array can be turned on and off, and the corresponding spectral response from the sensing portion (105) on the solid transparent member (101) is directed to a detector surface (111) of an external photo-detector or photo-detector array (111a). For a pre-determined position of a light ray on the detector surface (117), a corresponding ray with a particular wavelength (104a', 104b', 104c') originates from a light ray with a corresponding input position (104a, 104b, 104c) on the bottom surface (103). The spectral response from the sensing portion (105) can therefore be monitored by timely separating the incoming light beams, for example by turning on and off each individual pixels or sub-arrays of pixels on the external light source (112). Fig. 5(b) illustrates a plot of a calculation of the spectral distribution of light beams at the input to the bottom surface (103) along this surface for a predetermined position of a light ray on the external detector surface as function of wavelength. The origin on the ordinate axis in Fig. 5(b) has been chosen to be the centre of the wavelength range

corresponding to a position of an incoming light beam at the point marked '0' in Fig. 5(a).

In Fig. 5(a), a plurality of timely separated light beams each beam comprising a plurality of wavelengths (104a, 104b, 104c) originating from a light source (112) and collimated with a lens, lens system or lens array (113) enter the solid transparent member (101) of a spectroscopic sensor from the bottom surface (103) at an angle of incidence, which is essentially perpendicular, impinges onto a mirror-coated inclined-side-surface (102) having an angle (110) to the bottom surface of less than 45°. The light beams are reflected from the mirror-coated inclined-side-surface (102), further reflected from the bottom surface (103) and illuminates the backside surface of an electrically conducting film (107) disposed on the top surface (114) of the solid transparent member (101) and with a bio-sensing area comprising sensor elements (106) on the top. The light beams are further reflected from the bottom surface ( 103), diffracted from a reflective diffractive optical element (RDOE) (108) and directed towards an output coupling surface (109). The light beams are further refracted from the output coupling surface (109) of the solid transparent member and directed onto an external detector surface (111).

A polarization filter for enabling p-polarized light may be disposed either as integrated in the solid transparent member (101), such as onto the backside surface (103) or in front of the incoming light beams (104a, 104b, 104c). A wavelength selective filter for selective attenuation of specific wavelengths may be disposed either as integrated in the solid transparent member (101), such as onto the backside surface (103) or in front of the incoming light beams (104a, 104b, 104c). As illustrated in Fig. 5(a), each of the timely separated light beams with a plurality of wavelengths (104a, 104b, 104c) are diffracted at the RDOE with a diffraction angle depending on the wavelength and each of the timely separated light beams with a plurality of wavelengths are directed onto the external detector surface (111) with spatial separation of the different wavelengths. For each timely separated light beam, solely a ray with a narrow range of wavelengths is directed to a predetermined position on the detector surface (111). Other rays are directed to other positions on the detector surface or outside the detector surface. Each of the three rays (104a', 104'b, 104c') representing three different wavelengths in Fig. 5(a) therefore corresponds to a particular input position of a light beam (104a, 104b, 104c) on the bottom surface (103). The plot of a calculation in Fig. 5(b) has been made for an embodiment of the present invention with a thickness of the solid transparent member (118) of 2.5 mm, , an angle to the bottom surface (110) of 34° , a distance between the bottom surface (103) and the detector surface (111) of 6.0 mm, a constant grating spacing of the RDOE of 424 nm, a distance between the centre of the incoming light beam (104a) and the position, where it hits the RDOE (108) of 21.7 mm, and a refractive index of the solid transparent member of 1.526. The output coupling surface ( 109) is a plane surface. As observed in Fig. 5(b), for a predetermined position on the detector surface (111), the relationship between the position of the incoming light beam and detection of light of a particular wavelength is almost linear. Fig. 6 illustrates an example of application of an embodiment of the present invention of a spectroscopic sensor with (a) an illustration of the components of the application and (b) an illustration of the components assembled and an illustration of the ray trace of a light ray. The application utilizes a smartphone (90) as reader unit, an embodiment of the spectroscopic sensor of the present invention (94) and a clamp component (95) enabling attachment of the spectroscopic sensor at a suitable position on the

smartphone. Incoming light beams to the spectroscopic sensor (94) are created by means of light emission from an LCD (liquid crystal display) or OLED (organic light emitting-diode) touchscreen (92) and detection of the output light beams from the spectroscopic sensor is obtained by means of a CMOS image sensor (front camera) (91) on the smartphone.

The CMOS image sensor may be a one-dimensional, but it is preferably a two- dimensional image sensor with monochrome pixels, red-green-blue (RGB) pixels or red- green-blue-white (RGBW) pixels. In the case of an image sensor comprising RGB or RGBW pixels, the spectroscopic signals from the image sensor may be based on the digitized values from each of the R, G, B or W values or the spectroscopic signals may be based on the sum or a weighted sum of the digitized values or other computational algorithms based on the digitized values from the pixels. Analyses of samples in the sensing portion (93) comprising a bio-sensing area are performed by application software (an app) on the smartphone. An LCD or OLED touchscreen exhibits light emission with a typical wavelength range from 500 to 750 nm, which is within the range of wavelength interrogation of surface plasmons or localized surface plasmons as utilized by the embodiment of the present invention. The CMOS image sensor covers this wavelength range. As an alternative, the present invention may employ the flash of the smartphone and an image sensor positioned on the rear side of the smartphone (rear camera).

For the embodiment of the present invention as illustrated in Fig. 6(b), the application on the smartphone turns off deselected pixels and turns on selected pixels or sub-arrays of pixels enabling light beams, which are directed by the mirror-coated inclined-side- surface (96) to the sensing portion (93), where the light beams are further directed to a reflective diffractive optical element (RDOE) (97) and directed towards an output coupling surface (98), where the light beams are further refracted and directed onto the image sensor of the smartphone (91). Prior to making analyses of samples in the sensing portion (93), calibration may be made by taking signals from the image sensor with the light source turned off (dark signal) and by taking signals from the image sensor with an empty cell, i.e. without the sensing portion or with the sensing portion (93) being replaced by a white reference (reference signal). The mirror-coated inclined-side-surface (96) may be formed to enable collimation of the incoming light beams or alternatively, a lens or lens-arrays may be integrated into the clamp component (95). In addition, a polarization filter for enabling p-polarized incident light beams may also be disposed between the touchscreen (92) and the image sensor (91) on either the spectroscopic sensor (94) or in the clamp component (95). The clamp component is preferably made of a polymer material like acrylics, polycarbonate, polyetherimide or polyolefin such as cyclic olefin copolymer, but the material is not limited to these materials. In order to enable analyses of liquids, a microfluid or nanofluid system preferably driven by capillary forces may be disposed on the top of the sensing portion (not shown in Fig. 6).

There are several advantages of combining a smartphone with the embodiment of the present invention as illustrated in Fig. 6. The spectroscopic sensor and the clamp component can be manufactured at a low price by standard moulding techniques such as injection moulding, compression moulding, or hot embossing and may be combined with evaporation and/or sputtering of thin films. The spectroscopic sensor system does not need development and marketing of a separate reader unit, because the smartphone acts as a reader and a smartphone application software can be tailored to analyse the sample in the sensing portion. The size of the spectroscopic sensor system is small and portable. The smartphone is coupled to an IT infrastructure, which makes it easy to wirelessly transfer measurements from the spectroscopic sensor. In addition, the spectroscopic sensor system is easy to use, since it employs the well-known user interface of a smartphone. The embodiments of the present invention can be used in a number of applications; as a diagnostic tool in point-of-care applications such as detection of infectious diseases, e.g. tuberculosis, influenza or dengue virus infection, in combination with therapy by monitoring serological markers for particular diseases, detection of pathogens in water or food, in analyses of the quality of drinking water and in quality control of food and beverages. It may also be used in discrimination between genuine food products and adulterated food products. In addition, the present invention may be used for the purpose of teaching the principles of optical spectroscopy, surface plasmon resonance and bio-sensing to high-school students or university students.

Claims

1. A spectroscopic sensor comprising an optical sensor unit comprising a solid transparent member, an inclined-side-surface disposed on the solid transparent member, said inclined- side-surface being adapted to receive incoming light beams of a plurality of wavelengths and being adapted to direct first light beams as essentially collimated light beams towards a reflective diffractive optical element disposed on the solid transparent member, and a sensing portion adapted to comprise a sample to be analysed, said sensing portion being positioned in an optical path between the incoming light beams and an external detector surface, wherein the reflective diffractive optical element is adapted to diffract and further direct second light beams to an output coupling surface disposed on the solid transparent member, said output coupling surface being adapted to direct third light beams onto the external detector surface, and wherein the reflective diffractive optical element and the output coupling surface are arranged to direct, with spatial separation of different wavelengths, the third light beams of the plurality of wavelengths onto the external detector surface.

2. A spectroscopic sensor according to claim 1, wherein the reflective diffractive optical element comprises a variation in a grating spacing, and wherein said variation in the grating structure and a shape of the output coupling surface in combination focus, with spatial separation of different wavelengths, the third light beams onto the external detector surface.

3. A spectroscopic sensor according to claim 1 or 2, further comprising collimating means being integrated into the solid transparent member such that the incoming light beams are directed as essentially collimated light beams towards the inclined-side- surface.

4. A spectroscopic sensor according to claim 3, wherein the collimating means comprises one or more collimating lens' or one or more collimating lens arrays.

5. A spectroscopic sensor according to any of claims 1-4, wherein the sensing portion is disposed on a surface of the solid transparent member.

6. A spectroscopic sensor according to claim 5, wherein the sensing portion comprises an electrically conducting material, such as gold, silver or aluminium for supporting surface plasmons.

7. A spectroscopic sensor according to claim 3, wherein the sensing portion is disposed in front of the collimating means.

8. A spectroscopic sensor according to claim 7, wherein the sensing portion comprises nano-particles comprising a conducting material, such as gold, silver or aluminium for supporting localized surface plasmons.

9. A spectroscopic sensor according to any of claims 1-8, wherein the plurality of wavelengths covers a spectral range from 400 nm to 700 nm, or from 550 nm to 800 nm.

10. A spectroscopic sensor according to any of claims 1-9, wherein the solid transparent member comprises a polymer material, such as acrylics, polycarbonate, polyetherimide or polyolefin, such as cyclic olefin copolymer.

11. A spectroscopic sensor according to any of claims 1-10, wherein the inclined-side- surface comprises an externally arranged reflective material, such as gold, silver, aluminium, or a dielectric reflective coating.

12. A spectroscopic sensor assembly comprising a spectroscopic sensor according to any of claims 1-11, the spectroscopic sensor assembly further comprising a light source emitting essentially white light.

13. A spectroscopic sensor assembly according to claim 12, wherein the light source comprises one or more white light emitting diodes or one or more white light emitting diode arrays, such as one or two-dimensional light emitting diode displays.

14. A spectroscopic sensor assembly according to claim 12 or 13, further comprising an external sensor comprising the external sensor surface, said external sensor comprising a one or a two-dimensional CMOS image sensor or CCD image sensor.

15. A method of determining biochemical and/or chemical substances within a sample using a spectroscopic sensor according to any of claims 1-11, or using a sensor assembly according to any of claims 12-14.

16. Use of a spectroscopic sensor according to any of claims 1-11 for determining biochemical and/or chemical substances within a sample.

17. Use of a spectroscopic sensor assembly according to any of claims 12-14 for determining biochemical and/or chemical substances within a sample.