Title of Invention	FLUORESCENCE READER
Abstract	Fluorescence leader (10) for an optical assay ar- rangement comprising a polymeric sample substrate (1) having a reaction site-surface and a substrate surface (3), the fluorescence reader comprising a light source (5) arranged to illuminate the reaction site-surface through the substrate surface, and a detec- tor device (6) arranged to detect fluorescent light emitted from said reaction site-surface and transmitted through the substrate surface, the substrate surface provided with total-intemal-reflec- tion suppressing means (15).

Title of Invention

FLUORESCENCE READER

Abstract

Fluorescence leader (10) for an optical assay ar- rangement comprising a polymeric sample substrate (1) having a reaction site-surface and a substrate surface (3), the fluorescence reader comprising a light source (5) arranged to illuminate the reaction site-surface through the substrate surface, and a detec- tor device (6) arranged to detect fluorescent light emitted from said reaction site-surface and transmitted through the substrate surface, the substrate surface provided with total-intemal-reflec- tion suppressing means (15).

Full Text	Fluorescence Reader TECHNICAL FIELD OF THE INVENTION The present invention relates to an improved fluorescence/phosphorescence reader for a sample substrate in an optical assay arrangement. The invention also relates to the use of said fluorescence/phosphorescense reader, and to a sample substrate adapted for said fluorescence reader. BACKGROUND OF THE INVENTION Analytical and diagnostic determinations can be performed on liquid samples by means of optical assays based on the detection of analytes in a sample, such as e.g. nucleic acids, peptides, proteins, antibodies, hormones, or drugs. One important' application of optical assays is the field of immunology, in which the analyte is detected with the aid of a specific antibody, which is capable of binding to the analyte to form optically detectable complexes, e.g. by labeling the analyte with a fluorophore, or by providing a fluorophore-labeled antibody before the optical detection. The detection of the fluorophores may be performed by mean's of a fluorescence reader, which is capable of illuminating the assay support substrate with an exerting light source and of detecting the fluorescent light emitted from the fluorophores. An optical assay involving detection of fluorescent light emitted from fluorophores is performed by an optical assay arrangement comprising a sample supporting substrate and a fluorescence reader. The fluorescence reader comprises a source and detector for electromagnetic radiation within the optical wavelength region (i.e. between approximately 4 0 nm and 1 mm), and suitable optical filters and wave-guiding means. The sample support comprises a substrate of e.g. a polymeric material having a high optical transmittance in the wavelength ranges of me exciting light and of the emitted lighr, and may also have a high absorbance of other wavelengths. The substrate is provided with one or more reaction site areas, comprising spots and/or lines of crobe molecules, e.g. of an antibody, providing binding sites for molecules of the analyte, i.e. the target molecules, that may be present in a sample. The substrate may further be provided with a pattern of protruding microstructures forming e.g. micro pillars or micro posts, which may be arranged to form a capillary flow path for the sample. When the sample is brought in contact with the capture molecules on the support surface, and the fluorescent or phosphorescent antibody detection conjugate, optically detectable spots or lines will be formed. Fluorescent or phosphorescent light will. be emitted when the substrate is illuminated with the exciting light source of the fluorescence reader, thereby indicating that a reaction has occurred between the target molecules of the sample and the probe molecules of "the reaction sites. Fluorescence and phosphorescence may be defined as the emission of electromagnetic radiation resulting from absorbed exciting electromagnetic radiation, the fluorescent light lasting less than 1 x 10-8 s after the excitation, and phosphorescent light lasting longer, i.e. is decaying more slowly after the exposure to the exciting light. In fluorescence (and phosphorescence), the exciting radiation normally has a shorter wavelength (i.e. higher energy) than the emitted radiation, although the reverse may be true for multi- photon fluorescence. The fluorescent behaviour may be studied in a sready state or time-resolved, and fluorescence spectroscopv involves e.g. single- and multi-photon fluorescence, FRET (fluorescence resonance energy transfer), and fluorescence up- conversion. In fluorescence assays, the wavelength of the exciting and the emitted radiation depends on the type of fluorophore, which may be of an organic or inorganic origin, e.g. cyanine dyes, fluorescir. dyes or nanocrystals. As an example, the cormon fluorophore Cy-5TW- (CE Healthcare) is typically excited with 649 nm, and the emitted light is measured at 670 nm. The difference in wavelength between the excitation maxima and the emittance maxima is commonly referred to as the Stokes shift, In optical assays, the concentration of an analyte in the sample may be determined by measuring the intensity of emitted fluorescent or phosphorescent light, by means of the detector device of a fluorescence reader, thereby enabling quantitative measurements. Consequently, the efficiency of the illumination of a reaction site area with exciting light, as well as the efficiency of the collection of the emitted fluorescent light, will have an effect on the performance of the optical assay, Further, the reaction sites on a substrate surface may be provided with an array of spots or lines of different probe molecules, binding different target molecules. Therefore, a fluorescence reader may be designed to be.capable of determining the presence of several analytes in a sample, by means of different fluorophores, or by using space separation of the probe molecules. A fluorescence reader can be arranged to perform the detection of fluorescent emitted light by scanning the reaction site area or to detect an image of the entire reaction-site area as a two- dimensional array of pixels. A scanning fluorescence reader scans the surface of the sample substrate by a relative movement between the optical means and ths sample substrate, and the optical means preferably comprises a narrowband exciting light source, such as a laser, a LED or a white light source provided with spectral filters, from which the light is focused on each individual detection site. The emitted fluorescent light from each detection site is focused on an optical detector, such as e.g. a photodiode or a PMT (photomultiplisr tube). An imaging fluorescence reader is capable of detecting a two-dimensional array of pixels and the optical means comprises an exciting light source for illuminating a large part of the surface area (or the entire surface area) of the sample substrate, and a detector capable of detecting emitted light from the entire. detection site-area simultaneously, e.g. a CCD (Charged-Coupled Device)-imager, which utilizes MOS (Metal-0n-3emiconductor) - technology. A prior art optical reader is described in WO 01/5755 0.1, which discloses optical imaging of 'an analyte containing sample on a transparent substrate. The optical reader comprises an exciting energy source to stimulate emission of detectable light from the sample, and.the substrate is provided with a reflective surface located below the sample to reflect the emitted light into the detection means. A light detecting optical device is disclosed in WO 99/4 6596, comprising a light conducting body coupled to a slide, thereby improving the light collecting efficacy. WO 03/103835 describes prior art sample substrates provided with protruding micro posts arranged to form a capillary flow path for the sample. It is an object of this invention to provide an improved fluorescence/phosphorescence reader capable of an efficient illumination of the reaction site area of the substrate and an efficient collection and detection of the emitted light, thereby achieving a high performance optical assay arrangement. DESCRIPTION OF THE INVENTION These and other objects are achieved by the fluorescence reader and sample substrate according to the attached claims. The claims relate to a fluorescence reader for a sample substrate having a reaction site-surface and an opposing substrate surface. The fluorescence reader comprises an exciting light source positioned to inject exciting light rays into the siabstrate surface, and a detector device positioned to detect the fluorescent light emitted from the reaction site surface and transmitted through t:he substrate surface. The substrate surface is provided with total-internal-reflection-sappressing means located in the optical path of the emitted fluorescent light to increase the transmission through the substrate -surface for detection by the detection device, and the total-internal- reflection suppressing meaans may be arranged to release emittad fluorescent light trapped within the substrate for detection by the. detection device. Further, the light source may be arranged to inject exciting light rays into the substrate surface with an incidence angle relative the normal to the reaction site surface substantially coinciding with the maximum emission angle of the emitted fluorescent light Thus, the substrate surface may be provided with incidence-angle-controlling means located in the optical path of the exciting light rays. The incidence-angle-controlling means may comprise a surface relief structured entry-section designed to admit exciting light into the substrate with an enlarged incidence angle relative the normal to the reaction sits surface, and the relief structure may comprise a diffractive or a refractive structure. The total-internal-reflection suppressing means may comprise a surface relief-structured exit-section designed to diffract or refract the emitted fluorescent light rays out of the substrate,, and it may further be designed to focus the emitted light rays, tha surface relief structured exit section comprising a diffractive or a refractive structure. The dssign of ths surfacs relief structure may be arranged to vary over the surface of the exit-section in correspondence with the varying emission angle of the imoinging fluorescent light, and the position and extension of the exit-section may determine the position and extension of the analysed reaction site area. additionally, a light collecting lens device may be positioned to receive the emitted fluorescent light transmitted. through the substrate surface. Alternatively, a light-collecting body may by located in close proximity to a surface relief structured exit section provided on said substrate surface. The total-internal-reflection suppressing means may comprise a light-collecting body positioned in optically wetting contact with said substrate surface. The light-collecting body may be designed to collect and transmit light by means of total-internal-reflection, and may be substantially ellipsoid-shaped. It may also be provided with an input port for the exciting light, and/or at least one output port, and the refractive index may substantially correspond to, or be larger than, the refractive index of the substrate. The total-internal-reflection suppressing means may, alternatively, comprise an optically wetting layer having a suitable refractive index, and at least a portion of said optically wetting layer may be attached to a lens device arranged to focus the emitted fluorescent light on the detector device. The refractive index may be higher than the refractive index of the substrate, and lower than the refractive index of the lens device. Further, at least a portion of said optically wetting layer ntay be attached to the detector device. The incidence angle controlling means may also comprise an optically wetting layer having a suitable refractive index, and the optically wetting layer may comprise a soft polymeric material. Further, the detector device may be provided with spectral filtering means arranged to prevent detection of the wavelengths of the exciting light, and/or with polarization filtering means. The light source may also he provided with spectral filtering means arranged to present transmission of the wavelengths coinciding with the fluorescence emission, and the excitation and the measurement of the emission may be performed in different geometrical planes. The claims also relate to the use of the fluorescence reader in an optical assay arrangement, Further, the claims relate to a sample substrate with a reaction site surface and an opposing substrate surface, the sample substrate adapted for a fluorescence reader according to this invention, The sample substrate may be made of a polymeric material, and the reaction site surface may be arranged to form lines and/or spots of fluorophores. Further, the reaction site surface may be provided with a pattern of protruding microstructures, e.g. micro posts enabling a capillary flow. The substrate surface may be provided with a surface relief structured exit section configured to suppress the total internal reflection of emitted fluorescence light rays, and/or with a surface relief structured entry section configured to enlarge the incidence angle of the exciting light rays, the surface reliefs comprising a diffractive or a refractive structure. Other features and further advantages of the invention will be apparent from the non-limiting embodiments of the invention disclosed in the following description and figures, as well as from, the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will now be described in more detail and with reference the drawings, of. which: figure 1 is a perspective side view of a sample substrate, indicating the reaction site surface and the opposing substrate surface, figure 2 illustrates the emission asymmetry of a fluorophore located on a reaction site surface of a polymeric sample substrate, figure ,3 is a graph schematically illustrating the fluorescence emission lobe of a fluorophore, figure 4 is a graph schematically.Illustrating tha fluorescence excitation efficiency at two different incidence angles of the exciting light, figure 5 schematically illustrates a cross section of a sample substrate and an exciting light source and a detector for the emitted fluorescent light, indicating the angle of incidence and the angle of emission,, figure 6 is a view of a surface relief structure comprising both a total-internal-refiection-suppressing exit section and a incidence angle controlling entry section, provided on a substrate surface of a sample substrate,. figure 7 schematically illustrates an enlarged example, of the surface relief structure, figure 8a schematically illustrates a cross section of a sabstrate provided with a surface relief structured entry section and exit section, figure 8b illustrates tha substrate of figure 8a, further provided with a light collecting lens device for the emitted light, figure 9 illustrates a fluorescence reader comprising a total-intenal-reflection suppressing light-collecting body provided with an input port functioning as a light directing device, and figure 10 shows an arrangement comprising a detector and lens, the lens provided with a total-internal-reflection suppressing wetting layer in physical contact with the substrate surface. DESCRIPTION OF PREFERRED EMBODIMENTS The term "fluorescence reader" is defined as a reader capable of exciting and detecting both fluorescent and phosphorescent light, and the term "fluorescent light" is defined to hereinafter refer to both fluorescent and phosphorescent light. Other terms and expressions used in the description and in the claims are meant to have the meaning normally used by a person skilled in the art. An improved performance is achieved in an optical assay arrangement comprising a polymeric sample substrate having a reaction site surface provided with a fluorophoric layer, the fluorophores forming e.g. lines or spots on the surface, and an opposing substrate surface, by means of a fluorescence reader according to the invention. The fluorescence reader comprises an exciting light source, e.g. a LED positioned to illuminate the substrate surface, and a detector, e.g. a photodiode, arranged to detect the fluorescent light emitted from the fluorophoric layer. The detector is positioned to collect and detect the emitted fluorescent light transmitted through the substrate surface, and the light source is arranged to inject exciting light into the substrate surface. A light ray impinging on a surface will be specularly reflected from said surface when the angle of incidence of the light ray relative the normal to the surface exceeds the critical anale for total internal reflection, which depends on the relationship between the refractive index of the material on both sides of the surface. The refraction of the light ray passing from a first medium, having the refractive index n1, into a second medium, with refractive index n2, is defined by the well-known relationship of Snell's Law: n1 * sin αin. =n2 * sin αrer (1) αin is the angle to the surface normal of the light ray in the first medium impinging on the surface between the first and the second medium, and αref is the angle to the surface normal of the refracted light ray propagating in the second medium. When aref = 90 degrees, total internal reflection occurs on the surface, and sin αraf = 1. From the relationship (1) above, it follows that: sin αin = n2/nl * sin αref => sin αin(TIR) = n2/nl => αin(TIR) = sin-1(n2/nl) , where αin(TIR) indicates the critical angle for total internal reflection. Thus, total-internal-reflection occurs when nl is larger than n.2 and αin exceeds the critical angle for total internal reflection, i.. e. αin > αin(TRA) In a conventional fluorescence reader suitable for a dielectric substrate provided with a fluorophoric layer, most of the light emitted from the fluorophoric layer will be directed into the substrate, and a large portion of this emitted fluorescent light will impinge on the substrate surface with an angle exceeding said critical angle for total internal reflection. When a conventional polymeric substrate is used, with a typical refractive index between 1.5 and 1.6, total internal reflection will occur, and a large fraction of the emitted light will be trapped within the substrate, i.e. undergo multiple total internal reflections, thereby not reaching the detector. According to this invention, total-internai-reflection suppressing means is provided on the substrate surface to release the trapped fluorescent light from the substrate, thereby allowing a larger fraction of the emitted fluorescent light to reach the detector. The total-internal-reflection suppressing means is configured to diffract or refract the light ravs, or to remove the difference in redactive index. Thereby, emitted fluorescent light rays having an angle relative the normal to the substrate surface that exceeds the critical angle for total internal reflection, will be allowed to escape through the surface, and reach the detector. The exciting light rays from a light source propagating in air and impinging on the surface of the polymeric substrate will be refracted into the substrate with a direction given by the relationship (1), from which it-follows that: sin αref = n1/n2 x sin αin , where αref is the angle between the refracted light ray within the 'substrate and the surface normal, and αin, is the angle between the impinging light ray in air and said surface normal. Consequently, the angle of the refracted light is limited by the fact that nl/n2 will be approximately 1/1.55 = 0.65, and that αin will be less than 90 degrees, resulting in that αref Figure 1 illustrates schematically a sample substrate 1 for an optical assay arrangement, the sample substrate having a reaction site surface 2 and a substrate.surface 3, located- opposite each other. The reaction site surface of the substrate may further be provided with a pattern of protruding microstructures, e.g. micro posts (not illustrated), which may be arranged to form a capillary flow path for the sample. The size of the substrate is preferably adapted to form a suitable carrier for one or more reaction site areas for an ootical assay, and a prior art microarray slide is rectangular, with a size of 25 mm x 75 mm. The thickness of the substrate may be e.g. approximately 1 mm and the width and length e.g. between approximately 1 mm and 100 mm, forming an approximately rectangular or quadratic surface ares. The material of the substrate may be a thermoplastic polymer, e.g. a cycloolefinpolymer or a cycloolefincopolynter, having suitable optical properties, e.g. regarding the transparency of the optical radiation, with an index of refraction typically between 1.5 and 1.6. Apolymeric substrate may be manufactured by a polymer replication of a master structure, e.g. by injection molding. However, other substrate material may be used, e.g.. glass or silicon, having suitable optical properties. Figure 2 illustrates schematically the excited electromagnetic rays emitted from a fluorophore -4 located on the reaction site surface 2 of a dielectric sample substrate 1. The emission is asymmetric due to the different optical properties of air and 3 the substrate material, and the intensity of the emitted light is larger within the substrate than in the air. Since the substrate has a high transmittance of the wavelengths of the emitted rays, it is advantageous to detect the excited fluorescence light from the substrate side. Figure 3 is a graph schematically illustrating the intensity of the different emission angles from a thin layer of the fluorophore Cy-5™, the emission angle defined as the angle to the surface normal. (However, for a given emission angle, light will be emitted over 360°.) The graph indicates that the fluorescence emission is highly anisotropic, with a peak at an angle of approximately 50 degrees. This is an angle that is larger than the critical angle for total internal reflection for the substrate/air interfacing surface, since the critical angle of total internal reflection for a light ray propagating in a substrate material with a refractive index of 1.55 and impinging on the substrate/air interfacing surface is 40 degrees, according to the relationship (1) above. Therefore, most of the fluorescence light emitted within the substrate will impinge on the inner side of the subsrrate surface with an angle exceeding the critical angle for total internal reflection, and will be trapped inside the substrate. This light will, consequently, not reach a detector located outside the substrate surface in a controlled way. Some of the light trapped inside the substrate will eventually be refracted out of the substrate at the substrate edges, but this light will normally not reach the detector. If the reaction site surface is provided with protruding microstructures these may also affect the optical conditions for total internal reflection. However, in a fluorescence reader according to this invention, the substrate surface is provided with total-internal-reflection suppressing means in order to release the captured totai- internal-reflected rays by diffracting or refracting the rays, or by changing the refractive index-difference between the substrate material and air. Thereby, the detector device will be able to collect and detect a larger portion of the emitted fluorescence light, achieving a more reliable detection and a higher performance. Figure 4 is a graph schematically illustrating the excitation efficiency dependency on the excitation incidence angle, showing the intensity of the emitted fluorescent light at two different angles of incidence of the illumination, i.e. 50 degrees and 180 degrees from the surface normal, respectively. The graph indicates' the intensity at different emission angles in the interval between 30 and 60 degrees, with the light approaching the fluorophores from the substrate surface with an incidence angle of 50 degrees relative the surface normal, and the intensity at different emission angles with an incidence angle of 180 degrees, i.e. light approaching the fluorophores in a direction in parallel with said surface normal. Apparently, the excitation efficiency is larger at an incidence angle of 50 degrees, i.e. an angle of incidence that substantially coincides with the emission peak, which is illustrated in figure 3. According to one embodiment of this invention, en increased emission efficiency is achieved by selecting the angle of incidence of the exciting radiation to coincide with the maximum fluorescence emission angle. This is accomplished by arranging a light source to direct exciting light rays into a suystrate 1 to illuminate the fluorophoric layer 4 with an angle of incidence coinciding with the maximum emission angle. However, light rays impinging on the substrate surface 3 can not refract into the surface with an angle that is higher than the one given by the relationship (1) : sin αref = n1/n2 * sin αin. Since αin is less than 90 degrees, sin αin is less than 1. The air/substrate surface will typically have a value of nl/n2 being 1/1.55 = 0.65. Therefore, sin αref degrees. Since the incidence angle relative the fluorophoric ) layer corresponds to the angle of refraction, the incidence angle will also be less than 4 0 degrees, which is smaller than the desired incidence angle of 50 degrees. In order to enlarge the angle of incidence of the exciting light rays, the fluorescence reader, according to this embodiment of the invention, will illuminate the substrate surface 3 through incidence-angle-controlling means provided on the substrate surface in the optical path of the impinging exciting light rays, thereby further increasing the performance by achieving an enlarged incidence angle relative the surface normal. The incidence angle controlling means will enlarge the angle of refraction/ and, accordingly, also the angle of incidence, by diffracting or refracting the injected light rays, or by changing the refractive index difference between the substrate material and air. Figure 5 schematically illustrates an optical assay arrangement, comprising a sample substrate provided with a fiuorophoric layer 4 of e.g. Cy-5™ or TransFluoSpheres™ (Invitrogen Corporation). The fluorescence reader comprises a light source 5 illuminating the substrate surface 3 and a defector device 6 detecting light transmitted throucrh said surface 3, the surface normal indicated by the dashed lir.e, The intensity cf the fluorescent light emitted from the fluorophoric layer 4 has a peak at the maximum emission angle 6, which is approximately 50 degrees, relative the indicated surface normal. Since tnis angl- is larger tnan the critical angle of total internal reflection, which is approximately 40 degrees, at the inner side of the substrate surface 3, a large portion of the emitted fluorescent light will be trapped inside the substrate, therefore not reaching the detector 6. Thus, the. detected intensity can be increased by applying total-internal-reflection suppressing means on the substrate surface, in the path of the emitted light rays, thereby releasing the trapped light rays from the substrate. in ordsr to achieve an enhanced emission, the light source 5 should inject exciting light rays into the substrate with an incidence angle 7, relative the indicated surface" normal, substantially corresponding to said maximum emission angle 8 of 50 degrees. However, this angle cannot be obtained due to the refractive index relationship between air and the substrate material, In order to enlarge the incidence angle and increase the emission/ incidence-angle-controlling means can be provided on the substrate surface, in the optical path of the injected light rays. In a fluorescence reader according to this invention, total- internsl-reflection suppressing means are provided on the substrate surface in the optical path of the emitted fluorescent light, in order to reduce the total-internal-reflection and release the trapped emitted light, preferably in the path of the maximum emission lobe of the emitted light. Additionally, in order to further increase the fluorescence emission, incidence- light -controlling means may be provided on the substrate surface in the path of the exciting light rays, thereby enlarging the incidente angle of the exciting light inside substrate to achieve an incidence anqle substantially coinciding with the maximum emission angle of the fluorescence light. Figures 5-Ba schematically illustrates embodiments according to 5 first aspect c-f the invention, in which the total internal reflection suppressing means and the incidence angle controlling means are configured to diffract or refract the light rays in a desired direction. The total internal reflection suppressing means and the incidence angle controlling means comprise a relief structure 9 applied on the substrate surface 3 of the sample substrate, in the optical path of the emitted fluorescent light rays and of the exciting light rays. The surface relief structure is designed to be diftractive or refractive, and may comprise e.g. a grating or a facet-structure, providing a surface on which the light rays will diffract or refract into the desired directions. Figure 6 illustrates a first exemplary embodiment, in which the total-internal-reflection suppressing means and the incidence- angle-controlling means comprises a surface relief structure 9, divided in three sections, of which the first section 11 constitutes the incidence-angle-controlling means comprising an entry-section forming an inner channel designed to admit exciting light rays into the substrate with a suitable incidence angle, and the outer sections 12 constitutes total-internal- reflection suppressing means forming exit-sections allowing fluorescence light rays with an angle exceeding the critical angle for total internal reflection to be transmitted through the substrate surface. The exit-sections 12 are designed to release emitted fluorescent light rays having ar. emission angle both below and above the critical angle for total internal reflection from the substrate, and the entry-section 11 is designed to enlarge the incidence angle of the exciting rays inside the substrate, to exceed the refractive angle given by the relationship between the refractive indices of air and of the substrate. The position of the entry-section and the exit-sections, respectively, is preferably adapted to the location of the fluorophores on the reaction sits surface, e.g. by locating the entry section 11 directly under the fiucroohores. since the fluorescent light is emitted with a low intersity at small angles of emission 8, and by locating the exit sections 12 to surround the entry section, especially in the path of the maximum emission lobe of the fluorescent light. A further advantage with this invention is that a specific area of the reaction site area can be analysed by only detecting emitted fluorescent light from a specific, limited area of the surface relief structure, or by applying the total-internal reflection suppressing means to only cover a specific, limited area of the substrate surface. The surface reliefs may be produced e.g. by e-beam lithography,, grating ruling engines, holographic interference methods, diamond point turning, silicon micromachining etc, and the relief structure may e.g. be sinusoidal, triangular, or trapezoidal. The surface relief may be transferred onto a polymer substrate by replication methods from a master structure, like injection moulding. The surface reliefs can e.g. be formed as one- or two-dimensional pattern of facets, which may be designed to transmit the light rays undeviatsd or to focus the emitted light onto the detector device. Figure 7 illustrates a facet structure, having the facet angle a, depth D, and pitch, P, of which the depth and pitch are in the sub µm-range, up to several hundreds of µm. If a coherent light source, e.g. a laser, is used, the surface relief will be of diffractive nature. If a non-coherent light source, e.g. a LSD, is used, the surface relief will be of refractive nature. The light rays emitted from the fluorophores will impinge on the substrate surface with an incidence angle that varies over the suistrate surface depending on the emission angel and or. the position relative the fluorophores, e.g. of a line of fluorophores located directly above an inner channel 11, illustrated in figure 6. Therefore, according to a further embodiment of the relief structure, the design of the diffractive or refractive structure is arranged to vary over the surface of the exit section, in correspondence with the incidence angle of the impinging emitted light rays, on order to further improve the total-internal-reflection suppressing function. According to a further embodiment of a fluorescence reader, in which the position of the light source is fixed relative the substrate, the design of the diffractive or refractive structure is arranged to vary over the surface of the entry section in correspondence with the intensity distribution of the exciting light beam, or in order to achieve focusing of the exciting light. Figure 8a is a cross-sectional view of a sample substrate, with a fluorophoric layer 4 and a substrate surface 3 provided with a surface relief structure 9, of which one section is functioning as total internal reflection suppressing means, and another section is functioning as incidence angle controlling means, through which exciting light rays and fluorescent emitted light rays are transmitted, respectively. Figure 8b illustrates a further embodiment of a fluorescence reader according to the first aspect, showing the sample substrate according to figure 8a, further provided with a light collecting lens 13 positioned to receive the fluorescent light transmitted through the surface relief 9, in order to increase the intensity of the emitted light reaching the detector device (not illustrated) and thereby further improving the performance. Figure 9 schematically illustrates a fluorescence reader 10, suitable for an optical assay arrangement comprising a sample substrate 1 provided with a fiuorophoric layer 4, according to a second espect of the irvention, in which the total-internal- reflection suppressing means comprises a light collecting body 15 of a suitable material, e.g. a polymeric material or glass. According to a first embodiment, the light-collecting body 15 has a refractive index that corresponds to, or is larger than, the refractive index of the substrate material. The light- collecting body is positioned in physical contact .with the surface, preferably in optically wetting contact, in order to ""open up" the substrate surface and to allow the fluorescent light rsys having an emission angle that exceeds the critical angle for. total internal reflection to escape the substrate and enter into the body 12. Additionally, the shape of the light- collecting body will collect and focus the fluorescent light rays, e.g. by means of total-internal-reflection, on the inner surface of the body, to reach the detector device 6, mounted on the light-collecting body. Thus, the illustrated light- collecting body acts both as a total-internal-reflection suppressor and as a light collector. Thereby, the intensity of emitted fluorescent light reaching the detector device 6 will increase, and the performance of the optical assay arrangement will be improved. According to a second embodiment, the light collecting body is provided with an input port 14 functioning as a light-directing device, which is arranged to control the direction of the light rays from the light source to guide the exciting light into the substrate 1, e.g. to achieve an incidence angle substantially coinciding with the maximum emission angle of the fluorescent light. The optical properties of the light-collecting body 15 allows exciting light rays to enter the substrate with an appropriate incidence angle relative the fiuorophoric layer provided on the reaction site surface. Tnereby, the fiuorophoric layer is more favorable excited, and more fluorescent light will be emitted, thereby further improving the performance of the optical as say arrangement. According to further embodiments, the light collecting body 15 is provided with one or more output ports (not illustrated in the figure), arranged to e.g. function as a beam dump for specularly reflected exciting light rays. Figure 10 schematically illustrates a fluorescence reader suitable for an optical assay.arrangement comprising a sample substrate 1 provided with a fluorophoric layer 4, according to a third aspect of this invention, in which the total-internal- reflection suppressing means 16 is configured to change the refractive index-difference at the surface, by comprising an optically wetting layer of a suitable material. Since sin αin(TIR) = n2/n1, as follows from the relationship (1) above, more fluorescence light can be transmitted through the surface if the refractive index difference is reduced, such that n2/nl is increased. According to a first embodiment, the fluorescence reader comprises a light source 5, a detector 6 and a lens device 17, which is positioned to focus emitted, fluorescent light onto the detector 6. The lens device 17 is provided with total-internal- reflection suppressing means 16 comprising a layer of a suitable material, having a suitable refractive, positioned in physical contact with the substrate surface 3. The material of the layer is preferably a soft polymer, e.g. silicon, epoxy, polyeruthane or acrylate, which is applied as an optically wetting layer on the surface of the lens device 17, with no air between the layer and the surface. Thereby, the light rays propagating to the lens will not pass any air layer, which would cause reflection losses to occur at high angles. An additional advantage with selecting a soft polymeric material is that it is capable of forming an optically wetting layer on a surface that is not completely even or clane. Further, the material of the layer is selected to have suitable light transmitting properties, e.g. low fluorescence and low scattering of the transmitted emitted or exciting light rays, and the thickness of the layer 16 can be between a few µm and a few mm. A suitable value of the refractive index of the layer is between the values of the refractive index of the polymeric substrate material and of the lens device, respectively, the refractive index of the lens device being the largest. The total-internal-reflecting suppressing optically wetting layer 16 docked in physical contact with the substrate surface 3 releases the fluorescence rays from being trapped into the substrate, and enables a larger portion of the emitted rays to be collected by the detector 6. The light source 5 is arranged to illuminate the substrate surface 3 of the sample substrate 1, and the detector device 6 is arranged to detect fluorescent light emitted from the fluorophoric layer 4 on the reaction site surface of the sample substrate and transmitted through the substrate surface 3. The light source 5 injects exciting light rays into substrate, and the maximum emission angle of the fluorescent light normally exceeds the angle for total internal reflection. However, the optically wetting layer 16 is applied as a total-internal- reflection suppressing means on the lens device 17 in order to increase the intensity of the light escaping through the surface by increasing the angle for total internal reflection. According to an alternative embodiment, not illustrated in the figure, the optically wetting layer 16 is applied directly on a detector device 6, in case the detector device is adapted to collect the emitted light without any focusing means. Preferably, the detector device is positioned such that the Optically wetting layer is docked in physical contact with the substrate surface 3. Further, the optically wetting layer 15 mey cover substantially the entire lens 17 or detector device 6, or, alternatively, a suitable part of -he surface of the detector 5 or lens 17, depending on the configuration of the optical assay arrangement. According to an alternative embodiment, not illustrated in the figure, incidence angle controlling means is applied on the substrate surface 3, in order to enlarge the incidence angle of the exciting light rays relative the surface normal by changing the diffractive-index difference at the surface. Since the incidence angle corresponds to the angle of refraction, which is given by the relationship sin αref = n1/n.2 * sin αin, a change in the refractive index difference will affect the incidence angle. The incidence angle controlling means, according to this embodiment, comprises an optically wetting layer of a material, having a suitable refractive index, applied, on the substrate surface in the optical path of the injected-exciting light rays. If the refractive index of the optically "wetting layer is larger than the refractive index of air, n1/n2 will increase, which will result in an enlarged αref and an enlarged incidence angle. The resulting incidence angle may preferably coincide with the maximum emission angle of the fluorescent, such that more fluorescent light will be emitted. The optically wetting layer may, alternatively, be applied on the surface of a light- directing device (not illustrated in the figure) arranged to control the direction of the exciting light rays. According to a fourth aspect of the invention, the total- internal-reflection suppressing means comprises both a surface relief structure 9, according to the first aspect of the invention, as described with reference to figures 6-8, and a light collecting body 15 according to the second aspect, as described with reference to figure 9. The light collecting body is preferably located in close proximity to the substrate surface, e.g. at a distance less than approximately 1 mm from the surface. 3y combining a surface relief structure 9 and a light collecting body 25, an improved suppression of the total- internai-reflection is achieved, while an improved collection of the emitted light is provided by means of the light collecting body. In order to further increase the performance of the optical assay .arrangement, a fluorescence reader implemented according to any of the aspects of the invention, as described above, is provided with suitable filter, e.g. spectral filter and polarization filters, in order to prevent any exciting light from reaching the detector. Since a part of the exciting light rays will be reflected, transmitted through the substrate and released from the substrate surface to be detected by the detector device, the detector device is preferably provided by spectral filters removing the wavelengths of the exciting light rays. However, the spectrum of the exciting light rays, which may be light from e.g. a laser diode or a light emitting diode (LED) , normally has a tail that extends into the wavelength region of the emitted fluorescent light, which depends on the type of fluorophores in the fluorophoric layer. Since the wavelengths of the fluorescent light cannot be removed by detector 'filters, the exciting light source is preferably provided with spectral filters removing the tail. Thus, according to further exemplary embodiments of the fluorescence reader,.the detector is provided with spectral.filters removing the wavelengths of the exciting light rays and of any other unwanted light sources, and the light source is provided with spectral filters removing the wavelengths coinciding with the fluorescent light. Therefore, the cut-off frequency of the detector filtering means has to be adapted to the cut-off frequency of the light source filtering means, avoiding any overlap between the wavelength ranges of the exciting light and the detected light, thereby further improving the efficiency of the fluorescence reader. IN ordr to Drevent any exciting light from reaching the detector, another exemolary embodiment of the fluorescence geometrical planes for rhe excising light and for the measurement of the emitted, fluorescent light, e.g. a geometric yz-plans for the excitation and a geometric xz-plane for the measurement of the emission. According to a further embodiment of the fluorescence reader, the exciting light rays can be prevented from reaching the detector by providing the light source, if needed, and the detector with polarizing filters. If the sample substrate has a low birefringence, one exemplary setup would be to align the two polarizers perpendicularly to each other.. Thereby, only fluorescent light polarized in parallel with the polarizing filter of the detector will be detected. Alternatively, if the birefringence is varying and not negligible, one of the polarizers has to be rotatable to a suitable angle to remove the exciting light, thereby achieving a further improved performance of the fluorescence reader. Thus, by providing a scanning or an imaging fluorescence reader with suitable total-internal-reflection suppressing means, incidence-light controlling means, lenses and light-directing devices, as well of spectral and/or polarizing filtering, as described above, a high performance optical assay arrangement can be achieved. However, the invention is not restricted to the described embodiments in the figures, but may be varied freely within the scope of the appended claims. CLAIMS 1. A fluorescence reader (10) for a sample substrate (1) having a reaction site-surface (2) and an opposing substrate surface (3) , the fluorescence reader comprising an exciting light source (5) positioned to inject exciting light rays into the substrate surface (3), and a detector device (6) positioned to detect the fluorescent light emitted from the reaction site surface and transmitted through the substrate surface, characterized in that said substrate surface (3) is provided with total-internal- reflection-suppressing means (12, 15, 16) located in the optical path of the emitted fluorescent lighr to increase the transmission through the substrate surface fox detection by the detection device (6) . 2. A fluorescence reader according to claim 1, characterised in that said total-internal-reflection suppressing means is arranged to release emitted fluorescent light trapped within the substrate. (2) for detection by the detection device (6) . 3. A fluorescence reader according to claim 1 or 2, characterized in that said light source (5) is arranged to inject exciting light rays into the substrate surface (3) with an incidence angle (7) relative the normal to the reaction site surface substantially coinciding with the maximum emission angle (8) of the emitted fluorescent light. 4 . A fluorescence reader according to any of the preceding claims, characterized in that said substrate surface (3) is provided with incidence-angle-contrailing means (11) located in the optical path of -he exciting light rays. 5. A fluorescence reader according to claim 4, characterized in that said incidence-angle-controling means (11) comprises a surface relief structured entry-section (9) designed to admit exciting light into the substrate with an enlarged incidence angle .(7) relative the normal to the- reaction site surface. 6. A fluorescence reader according to claim 4 or 5, characterised in that said surface relief structured entry- section (11) comprises a diffractive structure. 7. A fluorescence reader according to claim 4 or 5, characterized in that said surface relief structured entry- section (11) comprises a refractive structure. 8. A fluorescence reader according to any of the preceding claims, characterized in that said total-internal- reflection suppressing means comprises a surface relief- structured exit-section (12) designed to diffract or refract the emitted fluorescent light rays out of the substrate. 9. A fluorescence reader according to claim 8, characterized in that said exit section (12) is designed no focus the emitted light rays. 10. A fluorescence reader according to claim 8 or 9, characterized in that said surface relief structured exit section (12) comprises a diffractive structure. 11. A fluorescence reader according to claim 3 or 9, characterized in that said surface relief structured exit section (12) comprises a refractive structure. 12. A fluorescence reader according any of the claims 8 - 11, characterized in that the assign of the surface relief structure is arranged to vary over the surface of the exit- section (12) in correspondence with the varying emission angle (8) of the impinging fluorescent light. 13. A fluorescence reader according to any of the claims 8 - 12, characterized in that the position and extension of the exit-section (12) determines the position and extension of the analysed reaction site area. 14. A fluorescence reader according to any of the preceding claims, characterized in that a light collecting lens device (13) is positioned to receive the emitted fluorescent light transmitted through the substrate surface. 15. A fluorescence reader according to any of the claims 8- 13, characterized in that a light-collecting body (15) is located in close proximity to a surface relief structured exit section (9) provided on said substrate surface. 16. A fluorescence reader according to any of the claims 1- 7, characterized in that said total-internal-reflection suppressing means comprises a light-collecting body (15) positioned in optically wetting contact with said substrate surface. 17. A fluorescence reader according to claim 15 or 16, characterised in that said light-collecting (15) body is designed to collect and transmit light by means of rotal- intern.al-ref lection. 18. A fluorescence reader according to any of the claims 15 - 17, characterised in that said light-collecting body (15) is substantially ellipsoid-shaped. 19. A fluorescence reader according to any of the claims 15 - 18, characterized in that said light collecting body (15) is provided with an input port (14) for the exciting light. 20 A fluorescence reader according to any of the claims 15 - 19, characterized in that said light collecting body (15) is provided with at least one output port. 21. A fluorescence reader according to claim 16 - 20, characterized in that said light-collecting body (15)' has a refractive index that substantially corresponds to, or is larger than, the refractive index of the substrate (1) . 22. A fluorescence reader according to any of the claims 1- 7, characterized in that said total-internal-reflection suppressing means comprises an optically wetting layer (16) having a suitable refractive index. 23. A fluorescence reader according to claim 22, characterized in that at least a portion of said optically wetting' layer is attached to a lens device (17-) arranged to focus the emitted fluorescent light on the detectox device (6). 24. A fluorescence reader according claim 23, characterised in that said optically wetting layer (16) has a refractive index that is higher than the refractive index of the substrate (1), and lower than the refractive index of the lens device (17 25. A fluorescence reader according ~o claim 22, characterized in that at least a portion of said optically wetting layer is attached to the detector device (6). 26. A fluorescence reader according to any of the claims 1- 4, characterized in that said incidence angle controlling means comprises an optically wetting layer having a suitable refractive index. 27. A fluorescence reader according to any of the claims 22-26, characterized in that said optically wetting layer. (16) comprises a soft polymeric material. 28. A fluorescence reader according to any of the preceding claims, characterized in that said detector device (6) is provided with spectral filtering means arranged to prevent detection of the wavelengths of the exciting light. 29. A fluorescence reader according to any of the preceding claims, characterized in that said detector device (6) is provided with polarization filtering means. 30. A fluorescence reader according to any of the preceding claims, characterized in that said light source (5) is provided with spectral filtering means arranged to prevent transmission of the wavelengths coinciding with the fluorescence emission. 31. A fluorescence reader according to any of the preceding claims,, characterized in that the excitation and the measurement of the emission is performed in different geometrical planes. 32. Use of a fluorescence reader according to any of the preceding claims in an optical assay arrangement. 33. A sample substrate (1) with a reaction site surface (2) and an opposing substrate surface (3) , characterised in that said sample substrate (1) is adapted for a fluorescence reader according to any of the claims 1-31. 34. A sample substrate according to claim 33, characterised in that it is made of a polymeric material. 35. A sample substrate according to claim 33 or 34, characterized in that the reaction site surface (2) is arranged to form lines and/or spots of fluorophores. 36. A sample substrate according to any of the claims 33 - 35, characterized in that the reaction site surface (2) is provided with a pattern of protruding microstructures. 37. A sample substrate according to claim-3 6, characterized in that said protruding microstructures comprises micro posts enabling a capillary flow. 38. A sample substrate according to any of the claims 33 - 37, characterized in that the substrate surface (3) is provided with a surface relief structured exit section (12) configured to suppress the total internal reflection of emitted fluorescence light rays. 39. A sample substrate according to any of the claims 33 - 38, characterized in that the substrate surface (3) is provided with a surface relief structured entry section (11) configured to enlarge the incidence angle (7) of the exciting light rays. 40. A sample substrate according to any of the claims 38 or 39, characterized in that surface relief is a diffractive structure. 41. A sample substrate according ro any of the claims 38 or 39,. characterized in that surface relief is a refractive structure. Fluorescence leader (10) for an optical assay ar- rangement comprising a polymeric sample substrate (1) having a reaction site-surface and a substrate surface (3), the fluorescence reader comprising a light source (5) arranged to illuminate the reaction site-surface through the substrate surface, and a detec- tor device (6) arranged to detect fluorescent light emitted from said reaction site-surface and transmitted through the substrate surface, the substrate surface provided with total-intemal-reflec- tion suppressing means (15).

Full Text

Fluorescence Reader
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an improved
fluorescence/phosphorescence reader for a sample substrate in an
optical assay arrangement. The invention also relates to the use
of said fluorescence/phosphorescense reader, and to a sample
substrate adapted for said fluorescence reader.
BACKGROUND OF THE INVENTION
Analytical and diagnostic determinations can be performed on
liquid samples by means of optical assays based on the detection
of analytes in a sample, such as e.g. nucleic acids, peptides,
proteins, antibodies, hormones, or drugs. One important'
application of optical assays is the field of immunology, in
which the analyte is detected with the aid of a specific
antibody, which is capable of binding to the analyte to form
optically detectable complexes, e.g. by labeling the analyte
with a fluorophore, or by providing a fluorophore-labeled
antibody before the optical detection. The detection of the
fluorophores may be performed by mean's of a fluorescence reader,
which is capable of illuminating the assay support substrate
with an exerting light source and of detecting the fluorescent
light emitted from the fluorophores.
An optical assay involving detection of fluorescent light
emitted from fluorophores is performed by an optical assay
arrangement comprising a sample supporting substrate and a
fluorescence reader. The fluorescence reader comprises a source
and detector for electromagnetic radiation within the optical
wavelength region (i.e. between approximately 4 0 nm and 1 mm),
and suitable optical filters and wave-guiding means. The sample
support comprises a substrate of e.g. a polymeric material
having a high optical transmittance in the wavelength ranges of
me exciting light and of the emitted lighr, and may also have a
high absorbance of other wavelengths. The substrate is provided

with one or more reaction site areas, comprising spots and/or
lines of crobe molecules, e.g. of an antibody, providing binding
sites for molecules of the analyte, i.e. the target molecules,
that may be present in a sample. The substrate may further be
provided with a pattern of protruding microstructures forming
e.g. micro pillars or micro posts, which may be arranged to form
a capillary flow path for the sample.
When the sample is brought in contact with the capture molecules
on the support surface, and the fluorescent or phosphorescent
antibody detection conjugate, optically detectable spots or
lines will be formed. Fluorescent or phosphorescent light will.
be emitted when the substrate is illuminated with the exciting
light source of the fluorescence reader, thereby indicating that
a reaction has occurred between the target molecules of the
sample and the probe molecules of "the reaction sites.
Fluorescence and phosphorescence may be defined as the emission
of electromagnetic radiation resulting from absorbed exciting
electromagnetic radiation, the fluorescent light lasting less
than 1 x 10-8 s after the excitation, and phosphorescent light
lasting longer, i.e. is decaying more slowly after the exposure
to the exciting light.
In fluorescence (and phosphorescence), the exciting radiation
normally has a shorter wavelength (i.e. higher energy) than the
emitted radiation, although the reverse may be true for multi-
photon fluorescence. The fluorescent behaviour may be studied in
a sready state or time-resolved, and fluorescence spectroscopv
involves e.g. single- and multi-photon fluorescence, FRET
(fluorescence resonance energy transfer), and fluorescence up-
conversion. In fluorescence assays, the wavelength of the
exciting and the emitted radiation depends on the type of
fluorophore, which may be of an organic or inorganic origin,
e.g. cyanine dyes, fluorescir. dyes or nanocrystals. As an
example, the cormon fluorophore Cy-5TW- (CE Healthcare) is

typically excited with 649 nm, and the emitted light is measured
at 670 nm. The difference in wavelength between the excitation
maxima and the emittance maxima is commonly referred to as the
Stokes shift,
In optical assays, the concentration of an analyte in the sample
may be determined by measuring the intensity of emitted
fluorescent or phosphorescent light, by means of the detector
device of a fluorescence reader, thereby enabling quantitative
measurements. Consequently, the efficiency of the illumination
of a reaction site area with exciting light, as well as the
efficiency of the collection of the emitted fluorescent light,
will have an effect on the performance of the optical assay,
Further, the reaction sites on a substrate surface may be
provided with an array of spots or lines of different probe
molecules, binding different target molecules. Therefore, a
fluorescence reader may be designed to be.capable of determining
the presence of several analytes in a sample, by means of
different fluorophores, or by using space separation of the
probe molecules.
A fluorescence reader can be arranged to perform the detection
of fluorescent emitted light by scanning the reaction site area
or to detect an image of the entire reaction-site area as a two-
dimensional array of pixels. A scanning fluorescence reader
scans the surface of the sample substrate by a relative movement
between the optical means and ths sample substrate, and the
optical means preferably comprises a narrowband exciting light
source, such as a laser, a LED or a white light source provided
with spectral filters, from which the light is focused on each
individual detection site. The emitted fluorescent light from
each detection site is focused on an optical detector, such as
e.g. a photodiode or a PMT (photomultiplisr tube). An imaging
fluorescence reader is capable of detecting a two-dimensional
array of pixels and the optical means comprises an exciting

light source for illuminating a large part of the surface area
(or the entire surface area) of the sample substrate, and a
detector capable of detecting emitted light from the entire.
detection site-area simultaneously, e.g. a CCD (Charged-Coupled
Device)-imager, which utilizes MOS (Metal-0n-3emiconductor) -
technology.
A prior art optical reader is described in WO 01/5755 0.1, which
discloses optical imaging of 'an analyte containing sample on a
transparent substrate. The optical reader comprises an exciting
energy source to stimulate emission of detectable light from the
sample, and.the substrate is provided with a reflective surface
located below the sample to reflect the emitted light into the
detection means.
A light detecting optical device is disclosed in WO 99/4 6596,
comprising a light conducting body coupled to a slide, thereby
improving the light collecting efficacy.
WO 03/103835 describes prior art sample substrates provided with
protruding micro posts arranged to form a capillary flow path
for the sample.
It is an object of this invention to provide an improved
fluorescence/phosphorescence reader capable of an efficient
illumination of the reaction site area of the substrate and an
efficient collection and detection of the emitted light, thereby
achieving a high performance optical assay arrangement.
DESCRIPTION OF THE INVENTION
These and other objects are achieved by the fluorescence reader
and sample substrate according to the attached claims.
The claims relate to a fluorescence reader for a sample
substrate having a reaction site-surface and an opposing
substrate surface. The fluorescence reader comprises an exciting

light source positioned to inject exciting light rays into the
siabstrate surface, and a detector device positioned to detect
the fluorescent light emitted from the reaction site surface and
transmitted through t:he substrate surface. The substrate surface
is provided with total-internal-reflection-sappressing means
located in the optical path of the emitted fluorescent light to
increase the transmission through the substrate -surface for
detection by the detection device, and the total-internal-
reflection suppressing meaans may be arranged to release emittad
fluorescent light trapped within the substrate for detection by
the. detection device.
Further, the light source may be arranged to inject exciting
light rays into the substrate surface with an incidence angle
relative the normal to the reaction site surface substantially
coinciding with the maximum emission angle of the emitted
fluorescent light Thus, the substrate surface may be provided
with incidence-angle-controlling means located in the optical
path of the exciting light rays.
The incidence-angle-controlling means may comprise a surface
relief structured entry-section designed to admit exciting light
into the substrate with an enlarged incidence angle relative the
normal to the reaction sits surface, and the relief structure
may comprise a diffractive or a refractive structure.
The total-internal-reflection suppressing means may comprise a
surface relief-structured exit-section designed to diffract or
refract the emitted fluorescent light rays out of the substrate,,
and it may further be designed to focus the emitted light rays,
tha surface relief structured exit section comprising a
diffractive or a refractive structure.
The dssign of ths surfacs relief structure may be arranged to
vary over the surface of the exit-section in correspondence with
the varying emission angle of the imoinging fluorescent light,

and the position and extension of the exit-section may determine
the position and extension of the analysed reaction site area.
additionally, a light collecting lens device may be positioned
to receive the emitted fluorescent light transmitted. through the
substrate surface. Alternatively, a light-collecting body may by
located in close proximity to a surface relief structured exit
section provided on said substrate surface.
The total-internal-reflection suppressing means may comprise a
light-collecting body positioned in optically wetting contact
with said substrate surface.
The light-collecting body may be designed to collect and
transmit light by means of total-internal-reflection, and may be
substantially ellipsoid-shaped. It may also be provided with an
input port for the exciting light, and/or at least one output
port, and the refractive index may substantially correspond to,
or be larger than, the refractive index of the substrate.
The total-internal-reflection suppressing means may,
alternatively, comprise an optically wetting layer having a
suitable refractive index, and at least a portion of said
optically wetting layer may be attached to a lens device
arranged to focus the emitted fluorescent light on the detector
device. The refractive index may be higher than the refractive
index of the substrate, and lower than the refractive index of
the lens device. Further, at least a portion of said optically
wetting layer ntay be attached to the detector device. The
incidence angle controlling means may also comprise an optically
wetting layer having a suitable refractive index, and the
optically wetting layer may comprise a soft polymeric material.
Further, the detector device may be provided with spectral
filtering means arranged to prevent detection of the wavelengths
of the exciting light, and/or with polarization filtering means.

The light source may also he provided with spectral filtering
means arranged to present transmission of the wavelengths
coinciding with the fluorescence emission, and the excitation
and the measurement of the emission may be performed in
different geometrical planes.
The claims also relate to the use of the fluorescence reader in
an optical assay arrangement,
Further, the claims relate to a sample substrate with a reaction
site surface and an opposing substrate surface, the sample
substrate adapted for a fluorescence reader according to this
invention,
The sample substrate may be made of a polymeric material, and
the reaction site surface may be arranged to form lines and/or
spots of fluorophores. Further, the reaction site surface may be
provided with a pattern of protruding microstructures, e.g.
micro posts enabling a capillary flow.
The substrate surface may be provided with a surface relief
structured exit section configured to suppress the total
internal reflection of emitted fluorescence light rays, and/or
with a surface relief structured entry section configured to
enlarge the incidence angle of the exciting light rays, the
surface reliefs comprising a diffractive or a refractive
structure.
Other features and further advantages of the invention will be
apparent from the non-limiting embodiments of the invention
disclosed in the following description and figures, as well as
from, the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will now be described in more detail and
with reference the drawings, of. which:
figure 1 is a perspective side view of a sample substrate,
indicating the reaction site surface and the opposing substrate
surface,
figure 2 illustrates the emission asymmetry of a fluorophore
located on a reaction site surface of a polymeric sample
substrate,
figure ,3 is a graph schematically illustrating the fluorescence
emission lobe of a fluorophore,
figure 4 is a graph schematically.Illustrating tha fluorescence
excitation efficiency at two different incidence angles of the
exciting light,
figure 5 schematically illustrates a cross section of a sample
substrate and an exciting light source and a detector for the
emitted fluorescent light, indicating the angle of incidence and
the angle of emission,,
figure 6 is a view of a surface relief structure comprising both
a total-internal-refiection-suppressing exit section and a
incidence angle controlling entry section, provided on a
substrate surface of a sample substrate,.
figure 7 schematically illustrates an enlarged example, of the
surface relief structure,
figure 8a schematically illustrates a cross section of a
sabstrate provided with a surface relief structured entry
section and exit section,
figure 8b illustrates tha substrate of figure 8a, further
provided with a light collecting lens device for the emitted
light,
figure 9 illustrates a fluorescence reader comprising a
total-intenal-reflection suppressing light-collecting body
provided with an input port functioning as a light directing
device, and

figure 10 shows an arrangement comprising a detector and lens,
the lens provided with a total-internal-reflection suppressing
wetting layer in physical contact with the substrate surface.
DESCRIPTION OF PREFERRED EMBODIMENTS
The term "fluorescence reader" is defined as a reader capable of
exciting and detecting both fluorescent and phosphorescent
light, and the term "fluorescent light" is defined to
hereinafter refer to both fluorescent and phosphorescent light.
Other terms and expressions used in the description and in the
claims are meant to have the meaning normally used by a person
skilled in the art.
An improved performance is achieved in an optical assay
arrangement comprising a polymeric sample substrate having a
reaction site surface provided with a fluorophoric layer, the
fluorophores forming e.g. lines or spots on the surface, and an
opposing substrate surface, by means of a fluorescence reader
according to the invention. The fluorescence reader comprises an
exciting light source, e.g. a LED positioned to illuminate the
substrate surface, and a detector, e.g. a photodiode, arranged
to detect the fluorescent light emitted from the fluorophoric
layer. The detector is positioned to collect and detect the
emitted fluorescent light transmitted through the substrate
surface, and the light source is arranged to inject exciting
light into the substrate surface.
A light ray impinging on a surface will be specularly reflected
from said surface when the angle of incidence of the light ray
relative the normal to the surface exceeds the critical anale
for total internal reflection, which depends on the relationship
between the refractive index of the material on both sides of
the surface. The refraction of the light ray passing from a
first medium, having the refractive index n1, into a second
medium, with refractive index n2, is defined by the well-known
relationship of Snell's Law:

n1 * sin αin. =n2 * sin αrer (1)
αin is the angle to the surface normal of the light ray in the
first medium impinging on the surface between the first and the
second medium, and αref is the angle to the surface normal of the
refracted light ray propagating in the second medium. When
aref = 90 degrees, total internal reflection occurs on the
surface, and sin αraf = 1. From the relationship (1) above, it
follows that:
sin αin = n2/nl * sin αref => sin αin(TIR) = n2/nl =>
αin(TIR) = sin-1(n2/nl) , where αin(TIR) indicates the critical angle
for total internal reflection.
Thus, total-internal-reflection occurs when nl is larger than n.2
and αin exceeds the critical angle for total internal reflection,
i.. e. αin > αin(TRA)
In a conventional fluorescence reader suitable for a dielectric
substrate provided with a fluorophoric layer, most of the light
emitted from the fluorophoric layer will be directed into the
substrate, and a large portion of this emitted fluorescent light
will impinge on the substrate surface with an angle exceeding
said critical angle for total internal reflection. When a
conventional polymeric substrate is used, with a typical
refractive index between 1.5 and 1.6, total internal reflection
will occur, and a large fraction of the emitted light will be
trapped within the substrate, i.e. undergo multiple total
internal reflections, thereby not reaching the detector.
According to this invention, total-internai-reflection
suppressing means is provided on the substrate surface to
release the trapped fluorescent light from the substrate,
thereby allowing a larger fraction of the emitted fluorescent
light to reach the detector. The total-internal-reflection
suppressing means is configured to diffract or refract the light
ravs, or to remove the difference in redactive index. Thereby,

emitted fluorescent light rays having an angle relative the
normal to the substrate surface that exceeds the critical angle
for total internal reflection, will be allowed to escape through
the surface, and reach the detector.
The exciting light rays from a light source propagating in air
and impinging on the surface of the polymeric substrate will be
refracted into the substrate with a direction given by the
relationship (1), from which it-follows that:
sin αref = n1/n2 x sin αin , where αref is the angle between the
refracted light ray within the 'substrate and the surface normal,
and αin, is the angle between the impinging light ray in air and
said surface normal. Consequently, the angle of the refracted
light is limited by the fact that nl/n2 will be approximately
1/1.55 = 0.65, and that αin will be less than 90 degrees,
resulting in that αref Figure 1 illustrates schematically a sample substrate 1 for an
optical assay arrangement, the sample substrate having a
reaction site surface 2 and a substrate.surface 3, located-
opposite each other. The reaction site surface of the substrate
may further be provided with a pattern of protruding
microstructures, e.g. micro posts (not illustrated), which may
be arranged to form a capillary flow path for the sample. The
size of the substrate is preferably adapted to form a suitable
carrier for one or more reaction site areas for an ootical
assay, and a prior art microarray slide is rectangular, with a
size of 25 mm x 75 mm. The thickness of the substrate may be
e.g. approximately 1 mm and the width and length e.g. between
approximately 1 mm and 100 mm, forming an approximately
rectangular or quadratic surface ares. The material of the
substrate may be a thermoplastic polymer, e.g. a
cycloolefinpolymer or a cycloolefincopolynter, having suitable
optical properties, e.g. regarding the transparency of the
optical radiation, with an index of refraction typically between

1.5 and 1.6. Apolymeric substrate may be manufactured by a
polymer replication of a master structure, e.g. by injection
molding. However, other substrate material may be used, e.g..
glass or silicon, having suitable optical properties.
Figure 2 illustrates schematically the excited electromagnetic
rays emitted from a fluorophore -4 located on the reaction site
surface 2 of a dielectric sample substrate 1. The emission is
asymmetric due to the different optical properties of air and
3 the substrate material, and the intensity of the emitted light
is larger within the substrate than in the air. Since the
substrate has a high transmittance of the wavelengths of the
emitted rays, it is advantageous to detect the excited
fluorescence light from the substrate side.
Figure 3 is a graph schematically illustrating the intensity of
the different emission angles from a thin layer of the
fluorophore Cy-5™, the emission angle defined as the angle to
the surface normal. (However, for a given emission angle, light
will be emitted over 360°.) The graph indicates that the
fluorescence emission is highly anisotropic, with a peak at an
angle of approximately 50 degrees. This is an angle that is
larger than the critical angle for total internal reflection for
the substrate/air interfacing surface, since the critical angle
of total internal reflection for a light ray propagating in a
substrate material with a refractive index of 1.55 and impinging
on the substrate/air interfacing surface is 40 degrees,
according to the relationship (1) above. Therefore, most of the
fluorescence light emitted within the substrate will impinge on
the inner side of the subsrrate surface with an angle exceeding
the critical angle for total internal reflection, and will be
trapped inside the substrate. This light will, consequently, not
reach a detector located outside the substrate surface in a
controlled way. Some of the light trapped inside the substrate
will eventually be refracted out of the substrate at the

substrate edges, but this light will normally not reach the
detector. If the reaction site surface is provided with
protruding microstructures these may also affect the optical
conditions for total internal reflection.
However, in a fluorescence reader according to this invention,
the substrate surface is provided with total-internal-reflection
suppressing means in order to release the captured totai-
internal-reflected rays by diffracting or refracting the rays,
or by changing the refractive index-difference between the
substrate material and air. Thereby, the detector device will be
able to collect and detect a larger portion of the emitted
fluorescence light, achieving a more reliable detection and a
higher performance.
Figure 4 is a graph schematically illustrating the excitation
efficiency dependency on the excitation incidence angle, showing
the intensity of the emitted fluorescent light at two different
angles of incidence of the illumination, i.e. 50 degrees and 180
degrees from the surface normal, respectively. The graph
indicates' the intensity at different emission angles in the
interval between 30 and 60 degrees, with the light approaching
the fluorophores from the substrate surface with an incidence
angle of 50 degrees relative the surface normal, and the
intensity at different emission angles with an incidence angle
of 180 degrees, i.e. light approaching the fluorophores in a
direction in parallel with said surface normal. Apparently, the
excitation efficiency is larger at an incidence angle of 50
degrees, i.e. an angle of incidence that substantially coincides
with the emission peak, which is illustrated in figure 3.
According to one embodiment of this invention, en increased
emission efficiency is achieved by selecting the angle of
incidence of the exciting radiation to coincide with the maximum
fluorescence emission angle. This is accomplished by arranging a
light source to direct exciting light rays into a suystrate 1 to

illuminate the fluorophoric layer 4 with an angle of incidence
coinciding with the maximum emission angle. However, light rays
impinging on the substrate surface 3 can not refract into the
surface with an angle that is higher than the one given by the
relationship (1) : sin αref = n1/n2 * sin αin. Since αin is less
than 90 degrees, sin αin is less than 1. The air/substrate
surface will typically have a value of nl/n2 being 1/1.55 =
0.65. Therefore, sin αref degrees. Since the incidence angle relative the fluorophoric
) layer corresponds to the angle of refraction, the incidence
angle will also be less than 4 0 degrees, which is smaller than
the desired incidence angle of 50 degrees. In order to enlarge
the angle of incidence of the exciting light rays, the
fluorescence reader, according to this embodiment of the
invention, will illuminate the substrate surface 3 through
incidence-angle-controlling means provided on the substrate
surface in the optical path of the impinging exciting light
rays, thereby further increasing the performance by achieving an
enlarged incidence angle relative the surface normal. The
incidence angle controlling means will enlarge the angle of
refraction/ and, accordingly, also the angle of incidence, by
diffracting or refracting the injected light rays, or by
changing the refractive index difference between the substrate
material and air.
Figure 5 schematically illustrates an optical assay arrangement,
comprising a sample substrate provided with a fiuorophoric layer
4 of e.g. Cy-5™ or TransFluoSpheres™ (Invitrogen Corporation).
The fluorescence reader comprises a light source 5 illuminating
the substrate surface 3 and a defector device 6 detecting light
transmitted throucrh said surface 3, the surface normal indicated
by the dashed lir.e, The intensity cf the fluorescent light
emitted from the fluorophoric layer 4 has a peak at the maximum
emission angle 6, which is approximately 50 degrees, relative
the indicated surface normal. Since tnis angl- is larger tnan

the critical angle of total internal reflection, which is
approximately 40 degrees, at the inner side of the substrate
surface 3, a large portion of the emitted fluorescent light will
be trapped inside the substrate, therefore not reaching the
detector 6. Thus, the. detected intensity can be increased by
applying total-internal-reflection suppressing means on the
substrate surface, in the path of the emitted light rays,
thereby releasing the trapped light rays from the substrate.
in ordsr to achieve an enhanced emission, the light source 5
should inject exciting light rays into the substrate with an
incidence angle 7, relative the indicated surface" normal,
substantially corresponding to said maximum emission angle 8 of
50 degrees. However, this angle cannot be obtained due to the
refractive index relationship between air and the substrate
material, In order to enlarge the incidence angle and increase
the emission/ incidence-angle-controlling means can be provided
on the substrate surface, in the optical path of the injected
light rays.
In a fluorescence reader according to this invention, total-
internsl-reflection suppressing means are provided on the
substrate surface in the optical path of the emitted fluorescent
light, in order to reduce the total-internal-reflection and
release the trapped emitted light, preferably in the path of the
maximum emission lobe of the emitted light. Additionally, in
order to further increase the fluorescence emission, incidence-
light -controlling means may be provided on the substrate surface
in the path of the exciting light rays, thereby enlarging the
incidente angle of the exciting light inside substrate to
achieve an incidence anqle substantially coinciding with the
maximum emission angle of the fluorescence light.
Figures 5-Ba schematically illustrates embodiments according to
5 first aspect c-f the invention, in which the total internal
reflection suppressing means and the incidence angle controlling

means are configured to diffract or refract the light rays in a
desired direction. The total internal reflection suppressing
means and the incidence angle controlling means comprise a
relief structure 9 applied on the substrate surface 3 of the
sample substrate, in the optical path of the emitted fluorescent
light rays and of the exciting light rays. The surface relief
structure is designed to be diftractive or refractive, and may
comprise e.g. a grating or a facet-structure, providing a
surface on which the light rays will diffract or refract into
the desired directions.
Figure 6 illustrates a first exemplary embodiment, in which the
total-internal-reflection suppressing means and the incidence-
angle-controlling means comprises a surface relief structure 9,
divided in three sections, of which the first section 11
constitutes the incidence-angle-controlling means comprising an
entry-section forming an inner channel designed to admit
exciting light rays into the substrate with a suitable incidence
angle, and the outer sections 12 constitutes total-internal-
reflection suppressing means forming exit-sections allowing
fluorescence light rays with an angle exceeding the critical
angle for total internal reflection to be transmitted through
the substrate surface. The exit-sections 12 are designed to
release emitted fluorescent light rays having ar. emission angle
both below and above the critical angle for total internal
reflection from the substrate, and the entry-section 11 is
designed to enlarge the incidence angle of the exciting rays
inside the substrate, to exceed the refractive angle given by
the relationship between the refractive indices of air and of
the substrate.
The position of the entry-section and the exit-sections,
respectively, is preferably adapted to the location of the
fluorophores on the reaction sits surface, e.g. by locating the
entry section 11 directly under the fiucroohores. since the
fluorescent light is emitted with a low intersity at small

angles of emission 8, and by locating the exit sections 12 to
surround the entry section, especially in the path of the
maximum emission lobe of the fluorescent light.
A further advantage with this invention is that a specific area
of the reaction site area can be analysed by only detecting
emitted fluorescent light from a specific, limited area of the
surface relief structure, or by applying the total-internal
reflection suppressing means to only cover a specific, limited
area of the substrate surface.
The surface reliefs may be produced e.g. by e-beam lithography,,
grating ruling engines, holographic interference methods,
diamond point turning, silicon micromachining etc, and the
relief structure may e.g. be sinusoidal, triangular, or
trapezoidal. The surface relief may be transferred onto a
polymer substrate by replication methods from a master
structure, like injection moulding. The surface reliefs can e.g.
be formed as one- or two-dimensional pattern of facets, which
may be designed to transmit the light rays undeviatsd or to
focus the emitted light onto the detector device.
Figure 7 illustrates a facet structure, having the facet angle
a, depth D, and pitch, P, of which the depth and pitch are in
the sub µm-range, up to several hundreds of µm. If a coherent
light source, e.g. a laser, is used, the surface relief will be
of diffractive nature. If a non-coherent light source, e.g. a
LSD, is used, the surface relief will be of refractive nature.
The light rays emitted from the fluorophores will impinge on the
substrate surface with an incidence angle that varies over the
suistrate surface depending on the emission angel and or. the
position relative the fluorophores, e.g. of a line of
fluorophores located directly above an inner channel 11,
illustrated in figure 6. Therefore, according to a further

embodiment of the relief structure, the design of the
diffractive or refractive structure is arranged to vary over the
surface of the exit section, in correspondence with the
incidence angle of the impinging emitted light rays, on order to
further improve the total-internal-reflection suppressing
function.
According to a further embodiment of a fluorescence reader, in
which the position of the light source is fixed relative the
substrate, the design of the diffractive or refractive structure
is arranged to vary over the surface of the entry section in
correspondence with the intensity distribution of the exciting
light beam, or in order to achieve focusing of the exciting
light.
Figure 8a is a cross-sectional view of a sample substrate, with
a fluorophoric layer 4 and a substrate surface 3 provided with a
surface relief structure 9, of which one section is functioning
as total internal reflection suppressing means, and another
section is functioning as incidence angle controlling means,
through which exciting light rays and fluorescent emitted light
rays are transmitted, respectively.
Figure 8b illustrates a further embodiment of a fluorescence
reader according to the first aspect, showing the sample
substrate according to figure 8a, further provided with a light
collecting lens 13 positioned to receive the fluorescent light
transmitted through the surface relief 9, in order to increase
the intensity of the emitted light reaching the detector device
(not illustrated) and thereby further improving the performance.
Figure 9 schematically illustrates a fluorescence reader 10,
suitable for an optical assay arrangement comprising a sample
substrate 1 provided with a fiuorophoric layer 4, according to a
second espect of the irvention, in which the total-internal-
reflection suppressing means comprises a light collecting body

15 of a suitable material, e.g. a polymeric material or glass.
According to a first embodiment, the light-collecting body 15
has a refractive index that corresponds to, or is larger than,
the refractive index of the substrate material. The light-
collecting body is positioned in physical contact .with the
surface, preferably in optically wetting contact, in order to
""open up" the substrate surface and to allow the fluorescent
light rsys having an emission angle that exceeds the critical
angle for. total internal reflection to escape the substrate and
enter into the body 12. Additionally, the shape of the light-
collecting body will collect and focus the fluorescent light
rays, e.g. by means of total-internal-reflection, on the inner
surface of the body, to reach the detector device 6, mounted on
the light-collecting body. Thus, the illustrated light-
collecting body acts both as a total-internal-reflection
suppressor and as a light collector. Thereby, the intensity of
emitted fluorescent light reaching the detector device 6 will
increase, and the performance of the optical assay arrangement
will be improved.
According to a second embodiment, the light collecting body is
provided with an input port 14 functioning as a light-directing
device, which is arranged to control the direction of the light
rays from the light source to guide the exciting light into the
substrate 1, e.g. to achieve an incidence angle substantially
coinciding with the maximum emission angle of the fluorescent
light. The optical properties of the light-collecting body 15
allows exciting light rays to enter the substrate with an
appropriate incidence angle relative the fiuorophoric layer
provided on the reaction site surface. Tnereby, the fiuorophoric
layer is more favorable excited, and more fluorescent light will
be emitted, thereby further improving the performance of the
optical as say arrangement.
According to further embodiments, the light collecting body 15
is provided with one or more output ports (not illustrated in

the figure), arranged to e.g. function as a beam dump for
specularly reflected exciting light rays.
Figure 10 schematically illustrates a fluorescence reader
suitable for an optical assay.arrangement comprising a sample
substrate 1 provided with a fluorophoric layer 4, according to a
third aspect of this invention, in which the total-internal-
reflection suppressing means 16 is configured to change the
refractive index-difference at the surface, by comprising an
optically wetting layer of a suitable material. Since sin αin(TIR)
= n2/n1, as follows from the relationship (1) above, more
fluorescence light can be transmitted through the surface if the
refractive index difference is reduced, such that n2/nl is
increased.
According to a first embodiment, the fluorescence reader
comprises a light source 5, a detector 6 and a lens device 17,
which is positioned to focus emitted, fluorescent light onto the
detector 6. The lens device 17 is provided with total-internal-
reflection suppressing means 16 comprising a layer of a suitable
material, having a suitable refractive, positioned in physical
contact with the substrate surface 3. The material of the layer
is preferably a soft polymer, e.g. silicon, epoxy, polyeruthane
or acrylate, which is applied as an optically wetting layer on
the surface of the lens device 17, with no air between the layer
and the surface. Thereby, the light rays propagating to the lens
will not pass any air layer, which would cause reflection losses
to occur at high angles. An additional advantage with selecting
a soft polymeric material is that it is capable of forming an
optically wetting layer on a surface that is not completely even
or clane. Further, the material of the layer is selected to have
suitable light transmitting properties, e.g. low fluorescence
and low scattering of the transmitted emitted or exciting light
rays, and the thickness of the layer 16 can be between a few µm
and a few mm. A suitable value of the refractive index of the

layer is between the values of the refractive index of the
polymeric substrate material and of the lens device,
respectively, the refractive index of the lens device being the
largest. The total-internal-reflecting suppressing optically
wetting layer 16 docked in physical contact with the substrate
surface 3 releases the fluorescence rays from being trapped into
the substrate, and enables a larger portion of the emitted rays
to be collected by the detector 6.
The light source 5 is arranged to illuminate the substrate
surface 3 of the sample substrate 1, and the detector device 6
is arranged to detect fluorescent light emitted from the
fluorophoric layer 4 on the reaction site surface of the sample
substrate and transmitted through the substrate surface 3. The
light source 5 injects exciting light rays into substrate, and
the maximum emission angle of the fluorescent light normally
exceeds the angle for total internal reflection. However, the
optically wetting layer 16 is applied as a total-internal-
reflection suppressing means on the lens device 17 in order to
increase the intensity of the light escaping through the surface
by increasing the angle for total internal reflection.
According to an alternative embodiment, not illustrated in the
figure, the optically wetting layer 16 is applied directly on a
detector device 6, in case the detector device is adapted to
collect the emitted light without any focusing means.
Preferably, the detector device is positioned such that the
Optically wetting layer is docked in physical contact with the
substrate surface 3.
Further, the optically wetting layer 15 mey cover substantially
the entire lens 17 or detector device 6, or, alternatively, a
suitable part of -he surface of the detector 5 or lens 17,
depending on the configuration of the optical assay arrangement.

According to an alternative embodiment, not illustrated in the
figure, incidence angle controlling means is applied on the
substrate surface 3, in order to enlarge the incidence angle of
the exciting light rays relative the surface normal by changing
the diffractive-index difference at the surface. Since the
incidence angle corresponds to the angle of refraction, which is
given by the relationship sin αref = n1/n.2 * sin αin, a change in
the refractive index difference will affect the incidence angle.
The incidence angle controlling means, according to this
embodiment, comprises an optically wetting layer of a material,
having a suitable refractive index, applied, on the substrate
surface in the optical path of the injected-exciting light rays.
If the refractive index of the optically "wetting layer is larger
than the refractive index of air, n1/n2 will increase, which
will result in an enlarged αref and an enlarged incidence angle.
The resulting incidence angle may preferably coincide with the
maximum emission angle of the fluorescent, such that more
fluorescent light will be emitted. The optically wetting layer
may, alternatively, be applied on the surface of a light-
directing device (not illustrated in the figure) arranged to
control the direction of the exciting light rays.
According to a fourth aspect of the invention, the total-
internal-reflection suppressing means comprises both a surface
relief structure 9, according to the first aspect of the
invention, as described with reference to figures 6-8, and a
light collecting body 15 according to the second aspect, as
described with reference to figure 9. The light collecting body
is preferably located in close proximity to the substrate
surface, e.g. at a distance less than approximately 1 mm from
the surface. 3y combining a surface relief structure 9 and a
light collecting body 25, an improved suppression of the total-
internai-reflection is achieved, while an improved collection of
the emitted light is provided by means of the light collecting
body.

In order to further increase the performance of the optical
assay .arrangement, a fluorescence reader implemented according
to any of the aspects of the invention, as described above, is
provided with suitable filter, e.g. spectral filter and
polarization filters, in order to prevent any exciting light
from reaching the detector. Since a part of the exciting light
rays will be reflected, transmitted through the substrate and
released from the substrate surface to be detected by the
detector device, the detector device is preferably provided by
spectral filters removing the wavelengths of the exciting light
rays. However, the spectrum of the exciting light rays, which
may be light from e.g. a laser diode or a light emitting diode
(LED) , normally has a tail that extends into the wavelength
region of the emitted fluorescent light, which depends on the
type of fluorophores in the fluorophoric layer. Since the
wavelengths of the fluorescent light cannot be removed by
detector 'filters, the exciting light source is preferably
provided with spectral filters removing the tail. Thus,
according to further exemplary embodiments of the fluorescence
reader,.the detector is provided with spectral.filters removing
the wavelengths of the exciting light rays and of any other
unwanted light sources, and the light source is provided with
spectral filters removing the wavelengths coinciding with the
fluorescent light. Therefore, the cut-off frequency of the
detector filtering means has to be adapted to the cut-off
frequency of the light source filtering means, avoiding any
overlap between the wavelength ranges of the exciting light and
the detected light, thereby further improving the efficiency of
the fluorescence reader.
IN ordr to Drevent any exciting light from reaching the
detector, another exemolary embodiment of the fluorescence
geometrical planes for rhe excising light and for the
measurement of the emitted, fluorescent light, e.g. a geometric

yz-plans for the excitation and a geometric xz-plane for the
measurement of the emission.
According to a further embodiment of the fluorescence reader,
the exciting light rays can be prevented from reaching the
detector by providing the light source, if needed, and the
detector with polarizing filters. If the sample substrate has a
low birefringence, one exemplary setup would be to align the two
polarizers perpendicularly to each other.. Thereby, only
fluorescent light polarized in parallel with the polarizing
filter of the detector will be detected. Alternatively, if the
birefringence is varying and not negligible, one of the
polarizers has to be rotatable to a suitable angle to remove the
exciting light, thereby achieving a further improved performance
of the fluorescence reader.
Thus, by providing a scanning or an imaging fluorescence reader
with suitable total-internal-reflection suppressing means,
incidence-light controlling means, lenses and light-directing
devices, as well of spectral and/or polarizing filtering, as
described above, a high performance optical assay arrangement
can be achieved.
However, the invention is not restricted to the described
embodiments in the figures, but may be varied freely within the
scope of the appended claims.

CLAIMS
1. A fluorescence reader (10) for a sample substrate (1)
having a reaction site-surface (2) and an opposing
substrate surface (3) , the fluorescence reader comprising
an exciting light source (5) positioned to inject exciting
light rays into the substrate surface (3), and a detector
device (6) positioned to detect the fluorescent light
emitted from the reaction site surface and transmitted
through the substrate surface, characterized in that said
substrate surface (3) is provided with total-internal-
reflection-suppressing means (12, 15, 16) located in the
optical path of the emitted fluorescent lighr to increase
the transmission through the substrate surface fox
detection by the detection device (6) .
2. A fluorescence reader according to claim 1, characterised
in that said total-internal-reflection suppressing means is
arranged to release emitted fluorescent light trapped
within the substrate. (2) for detection by the detection
device (6) .
3. A fluorescence reader according to claim 1 or 2,
characterized in that said light source (5) is arranged to
inject exciting light rays into the substrate surface (3)
with an incidence angle (7) relative the normal to the
reaction site surface substantially coinciding with the
maximum emission angle (8) of the emitted fluorescent
light.
4 . A fluorescence reader according to any of the preceding
claims, characterized in that said substrate surface (3) is
provided with incidence-angle-contrailing means (11)
located in the optical path of -he exciting light rays.

5. A fluorescence reader according to claim 4, characterized
in that said incidence-angle-controling means (11)
comprises a surface relief structured entry-section (9)
designed to admit exciting light into the substrate with an
enlarged incidence angle .(7) relative the normal to the-
reaction site surface.
6. A fluorescence reader according to claim 4 or 5,
characterised in that said surface relief structured entry-
section (11) comprises a diffractive structure.
7. A fluorescence reader according to claim 4 or 5,
characterized in that said surface relief structured entry-
section (11) comprises a refractive structure.
8. A fluorescence reader according to any of the preceding
claims, characterized in that said total-internal-
reflection suppressing means comprises a surface relief-
structured exit-section (12) designed to diffract or
refract the emitted fluorescent light rays out of the
substrate.
9. A fluorescence reader according to claim 8, characterized
in that said exit section (12) is designed no focus the
emitted light rays.
10. A fluorescence reader according to claim 8 or 9,
characterized in that said surface relief structured exit
section (12) comprises a diffractive structure.
11. A fluorescence reader according to claim 3 or 9,
characterized in that said surface relief structured exit
section (12) comprises a refractive structure.
12. A fluorescence reader according any of the claims 8 -
11, characterized in that the assign of the surface relief

structure is arranged to vary over the surface of the exit-
section (12) in correspondence with the varying emission
angle (8) of the impinging fluorescent light.
13. A fluorescence reader according to any of the claims 8
- 12, characterized in that the position and extension of
the exit-section (12) determines the position and extension
of the analysed reaction site area.
14. A fluorescence reader according to any of the preceding
claims, characterized in that a light collecting lens
device (13) is positioned to receive the emitted
fluorescent light transmitted through the substrate
surface.
15. A fluorescence reader according to any of the claims 8-
13, characterized in that a light-collecting body (15) is
located in close proximity to a surface relief structured
exit section (9) provided on said substrate surface.
16. A fluorescence reader according to any of the claims 1-
7, characterized in that said total-internal-reflection
suppressing means comprises a light-collecting body (15)
positioned in optically wetting contact with said substrate
surface.
17. A fluorescence reader according to claim 15 or 16,
characterised in that said light-collecting (15) body is
designed to collect and transmit light by means of rotal-
intern.al-ref lection.
18. A fluorescence reader according to any of the claims 15
- 17, characterised in that said light-collecting body (15)
is substantially ellipsoid-shaped.

19. A fluorescence reader according to any of the claims 15
- 18, characterized in that said light collecting body (15)
is provided with an input port (14) for the exciting light.
20 A fluorescence reader according to any of the claims 15
- 19, characterized in that said light collecting body (15)
is provided with at least one output port.
21. A fluorescence reader according to claim 16 - 20,
characterized in that said light-collecting body (15)' has a
refractive index that substantially corresponds to, or is
larger than, the refractive index of the substrate (1) .
22. A fluorescence reader according to any of the claims 1-
7, characterized in that said total-internal-reflection
suppressing means comprises an optically wetting layer (16)
having a suitable refractive index.
23. A fluorescence reader according to claim 22,
characterized in that at least a portion of said optically
wetting' layer is attached to a lens device (17-) arranged to
focus the emitted fluorescent light on the detectox device
(6).
24. A fluorescence reader according claim 23, characterised
in that said optically wetting layer (16) has a refractive
index that is higher than the refractive index of the
substrate (1), and lower than the refractive index of the
lens device (17
25. A fluorescence reader according ~o claim 22,
characterized in that at least a portion of said optically
wetting layer is attached to the detector device (6).
26. A fluorescence reader according to any of the claims 1-
4, characterized in that said incidence angle controlling

means comprises an optically wetting layer having a
suitable refractive index.
27. A fluorescence reader according to any of the claims
22-26, characterized in that said optically wetting layer.
(16) comprises a soft polymeric material.
28. A fluorescence reader according to any of the preceding
claims, characterized in that said detector device (6) is
provided with spectral filtering means arranged to prevent
detection of the wavelengths of the exciting light.
29. A fluorescence reader according to any of the preceding
claims, characterized in that said detector device (6) is
provided with polarization filtering means.
30. A fluorescence reader according to any of the preceding
claims, characterized in that said light source (5) is
provided with spectral filtering means arranged to prevent
transmission of the wavelengths coinciding with the
fluorescence emission.
31. A fluorescence reader according to any of the preceding
claims,, characterized in that the excitation and the
measurement of the emission is performed in different
geometrical planes.
32. Use of a fluorescence reader according to any of the
preceding claims in an optical assay arrangement.
33. A sample substrate (1) with a reaction site surface (2)
and an opposing substrate surface (3) , characterised in
that said sample substrate (1) is adapted for a
fluorescence reader according to any of the claims 1-31.

34. A sample substrate according to claim 33, characterised
in that it is made of a polymeric material.
35. A sample substrate according to claim 33 or 34,
characterized in that the reaction site surface (2) is
arranged to form lines and/or spots of fluorophores.

36. A sample substrate according to any of the claims 33 -
35, characterized in that the reaction site surface (2) is
provided with a pattern of protruding microstructures.
37. A sample substrate according to claim-3 6,
characterized in that said protruding microstructures
comprises micro posts enabling a capillary flow.
38. A sample substrate according to any of the claims 33 -
37, characterized in that the substrate surface (3) is
provided with a surface relief structured exit section (12)
configured to suppress the total internal reflection of
emitted fluorescence light rays.
39. A sample substrate according to any of the claims 33 -
38, characterized in that the substrate surface (3) is
provided with a surface relief structured entry section
(11) configured to enlarge the incidence angle (7) of the
exciting light rays.
40. A sample substrate according to any of the claims 38 or
39, characterized in that surface relief is a diffractive
structure.
41. A sample substrate according ro any of the claims 38 or
39,. characterized in that surface relief is a refractive
structure.

Fluorescence leader (10) for an optical assay ar-
rangement comprising a polymeric sample substrate (1) having a
reaction site-surface and a substrate surface (3), the fluorescence
reader comprising a light source (5) arranged to illuminate the
reaction site-surface through the substrate surface, and a detec-
tor device (6) arranged to detect fluorescent light emitted from
said reaction site-surface and transmitted through the substrate
surface, the substrate surface provided with total-intemal-reflec-
tion suppressing means (15).

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=X8LI9pkJiDe/vhKpph7nCw==&loc=wDBSZCsAt7zoiVrqcFJsRw==

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Patent Number

278120

Indian Patent Application Number

3084/KOLNP/2008

PG Journal Number

52/2016

Publication Date

16-Dec-2016

Grant Date

14-Dec-2016

Date of Filing

29-Jul-2008

Name of Patentee

AMIC AB

Applicant Address

UPPSALA SCIENCE PARK SE-75183 UPPSALA

Inventors:

#	Inventor's Name	Inventor's Address
1	OHMAN, OVE	ASPLUNDA, UPPSALA-NAS, SE-755 91 UPPSALA
2	BACKLUND, JOHAN	BARKEN BEATRICES GATA 22, SE-417 60 GOTEBORG
3	VILHELMSSON, KENNET	LODJURSVAGEN 13, SE-433 50 OJERSJO
4	LINDSTROM, TOMAS	GARDESVAGEN 25 B, SE-756 51 UPPSALA
5	MENDEL-HARTVIG, IB	RABENIUSVAGEN 28, SE-756 55 UPPSALA

PCT International Classification Number

G01N 21/64

PCT International Application Number

PCT/SE2007/000275

PCT International Filing date

2007-03-21

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	0600642-3	2006-03-22	Sweden