WO2014165879A1

WO2014165879A1 - Micro fluorescence-detecting device and method therefor

Info

Publication number: WO2014165879A1
Application number: PCT/AT2014/000066
Authority: WO
Inventors: Eduard Gilli; Frank Reil; Stefan Köstler; Volker Schmidt
Original assignee: Joanneum Research Forschungsgesellschaft Mbh
Priority date: 2013-04-12
Filing date: 2014-03-31
Publication date: 2014-10-16
Also published as: AT513859A4; AT513859B1

Abstract

The invention relates to a micro fluorescence-detecting device (1), comprising a light source (2) for generating linearly polarised excitation light (4), at least one prism (6), to a base surface (7) of which a carrier for a material to be excited or analysed is connected, and a detection unit (13) arranged parallel to the carrier for the material to be excited or analysed, the micro fluorescence-detecting device being characterised in that the at least one prism (6) and the carrier for the material to be excited or analysed are designed as a one-piece microchip, wherein a side of the microchip (15) that forms the base surface (7) of the prism (6) that forms the carrier for the material to be excited or analysed, on which carrier the material to be excited or analysed is arranged in the region of the total internal reflection of the excitation light (4), and in that both lateral surfaces (5, 8) of the prism (6) are arranged at Brewster's angle to the excitation light (4).

Description

MICRO FLUORESCENT DETECTION DEVICE AND METHOD

THEREFOR

The present invention relates to a micro-fluorescence detection device comprising a light source for generating linearly polarized excitation light, at least one prism with the base surface of which a carrier for a material to be excited or analyzed is connected, and one parallel to the carrier for the excitation or to be analyzed And a method for detecting fluorescent light in a micro fluorescence detection device, a material disposed, to be excited or analyzed on a support connected to a base surface of a prism, wherein linearly polarized light from an excitation light source is directed to a side surface of the prism wherein at the base surface of the prism, the material to be excited or to be analyzed for the emission of fluorescent light with simultaneous reflection of the excitation light is excited and that of the to be excited or to ana luminescent material detected fluorescent light is evaluated in a detection unit.

In recent years, a variety of different fluorescence detection devices have become known. Both microlens and / or microgitter systems and structures using waveguides and TIRF excitation have been described. Among them, for example, US 2010/0225915 describes a prism for inducing a Brewster angular transmission and a fluorescence detection device for increasing a signal-to-noise ratio thereof in which structure evanescent waves are generated when light is applied to fluorescent material, wel When applied to a sample surface, it is incident at an angle greater than a critical angle. The evanescent waves are used here as fluorescence excitation light to induce total internal reflection of light such that light passes through the prism at a Brewster angle. A disadvantage of such a device is that the light entry is not at a Brewster angle but normal to the prism surface resulting unwanted reflections of the excitation light on the light entry side result, and that at least between the prism and a support for the sample to be analyzed, an interface exists at which unwanted light scattering can take place. Furthermore, in order to connect the support for the sample to be analyzed with the integrated prism used an immersion medium to achieve the necessary total reflection at the surface of the support.

Other systems in which different materials for the waveguide, i. For example, the prism and the sample carrier, on which the material to be excited or analyzed is applied, are widely known.

A. Brandenburg et al., Sensor. ACTUAT. B-Chem. (2009), 139, 245-249 have for the first time used polymer films as waveguides which have been formed with a hot start embossing and decoupling prism for the excitation light. The light source in this system comprises a laser diode and the system further comprises beam splitters, apertures and a stepper motor driven control mirror in which after filtering the fluorescent light, the punctiform field has been detected by an uncooled CCD camera.

Due to the ever increasing demand for miniaturized devices employing direct detection of, in particular, biomolecules or pathogens in applications such as diagnostics, food testing or environmental monitoring, the so-called "lab-on-a-chip" or "micro total analysis systems (μTAS ) have been developed to perform the entire process from sample preparation, measurement, reaction, incubation, etc., to detection on the same chip, however, early such miniaturized fluorescence devices have been built on glass substrates or silicon substrates and production technologies have been required in the field the electronics and semiconductor industries are applied, whereby such micro-fluorescence detection devices are not only expensive but also complicated and expensive to produce and, moreover, the systems are not yet sufficiently miniaturized.

Recently, attempts have been made to integrate such microanalysis devices in plastic chips, which on the one hand have high transparency and, on the other hand, low auto fluorescence. A common problem with such devices, both on glass or silicon substrates, as well as on plastic substrates, is that a separation of the fluorescent light from the excitation light must be done by means of a spectrometer or by means of optical filters, which proves to be unsatisfactory , The most favorable is an effective, geometric separation between fluorescent light and excitation light, since the intensity of the excitation light usually by a Many times higher than the intensity of the fluorescence light generated, which is a big problem in terms of measurement accuracy, especially in miniaturized systems, since usually not inconsiderable amounts of scattered light disturb the measurement or falsify.

The present invention now aims to provide a miniaturized fluorescence detection device and a method for detecting fluorescent light, in which as much excitation light is coupled into a fluorescent dye of a material to be excited or analyzed and at the same time as little excitation light reaches the detector, to achieve an effective, geometric separation of fluorescent light and excitation light and thus an increased measurement accuracy. Furthermore, the invention aims to minimize the unwanted reflection of the excitation light when entering and exiting the prism and a subsequent possible scattering of excitation light and thus after an effective excitation of the fluorescent material in a material to be analyzed or to be detected Material to measure the fluorescent light sufficiently sensitive and accurate with a spatially resolving detector.

To achieve this object, the device according to the invention is characterized in that the at least one prism and the carrier for the material to be excited or analyzed are formed as a monolithic microchip, wherein a base surface of the prism forming side of the microchip the carrier for the material to be excited or analyzed on which the material to be excited or analyzed in the total reflection of the excitation light is arranged and that both side surfaces of the prism are arranged in the Brewster angle to the excitation light. By forming the at least one prism and the support for the material to be excited or analyzed as a one-piece microchip, there is no interface in the device between the support for the material to be excited or analyzed and the optical waveguide, namely the prism, whereby an undesired recharge. Flexions or stray light is avoided. Further, by providing the material to be excited or analyzed on which a base surface of the prism-forming side of the microchip is mounted as a carrier, attenuated total reflection (ATR) excitation is achieved. In this case, the material to be excited or analyzed on the side of the prism is illuminated at an angle which is greater than the critical angle of total reflection, for which reason no excitation light intensity emanates from the prism in the region of the material to be excited or analyzed. penetrates, but is completely reflected inside the prism. The substance to be stimulated or to be analyzed, which is simultaneously an absorbing substance for the excitation light and is applied to the same base surface of the prism, is thus excited by an evanescent wave emerging in the near field and causes the emission of fluorescent light, which fluorescence light, subsequently in the Detection unit is evaluated. Furthermore, by locating both side faces of the prism at Brewster's angle to the excitation light, the excitation light is coupled into or out of the prism, substantially without producing any reflections. For an improvement or for a further reduction of the reflections of the excitation light in this case is linearly polarized light, which may for example come from a laser diode as a light source whose polarization plane is set parallel to the plane of incidence, ie p-polarized light in the Brewster angle on the side surfaces of the prism. A further increase in the efficiency of the coupling-in and coupling-out of the excitation light without undesired reflection of the excitation light when light enters and exits is hereby achieved according to the invention in that the microchip of the fluorescence detection device is formed from a non-absorbing material which is suitable for p polarized light is not reflective at the Brewster angle. Such a configuration thus maximizes the coupling and decoupling of the excitation light and at the same time minimizes or completely eliminates the scattered light, which is why, in particular, fluorescent light of the material to be excited or analyzed can be exactly detected without any scattered light.

By, as corresponds to a development of the invention, the fluorescence detection device is designed such that the base of the microchip representing the carrier for the material to be excited or analyzed is structured and that the structuring comprises one or more components or structures Includes microfluidic structures and channels, micro-optical structures, electrodes, valves or membranes, it is possible to provide a so-called lab-on-a-chip system in a simple manner, in which the number of interfaces between the material to be excited or to be analyzed on a minimum is reduced, whereby as much excitation light is coupled into the luminescent dye contained in the material to be excited or analyzed. Moreover, with such a device when used for fluid materials, the use of immersion liquids can be avoided. By, as corresponds to a development of the invention, the material to be excited or analyzed as a grid of fluorescent dots on the carrier forming side or the base surface of the microchip is applied, it is possible with simultaneous effective excitation with a conventional spatially resolving detector, such as a CCD camera, sufficiently sensitive to measure the fluorescence of the material to be excited or analyzed. An assignment of the individually to be measured points to a specific analyte or to be measured material or parameter succeeds with a conventional software, which is not part of the invention. By, as corresponds to a development of the invention, a diameter of each point of the grid of fluorescent dots is 0.05 to 1 mm, in particular 0, 1 and 0.4 mm, with simultaneous homogeneous irradiation and a quantification of the average intensity of individual sensor points can be achieved. The larger the grid of fluorescent dots and the larger the dot diameter, the more inhomogeneous the results, so that particularly homogeneous and meaningful measured values can be achieved.

In order to allow, in particular, a simultaneous excitation of a plurality of materials or materials to be excited, according to a development the device is designed such that a plurality of prisms is arranged on a common microchip and that the prisms are integrated into a surface of the microchip. The plurality of prisms arranged on a common microchip makes it possible to provide a plurality of carriers for material to be analyzed or stimulated, and in particular, for example, a plurality of microfluidic channels which are cut into the prism base area. With such a design, it is possible with a single light source, to provide a plurality of illuminated detection zones available and thus to provide a small-sized device for the simultaneous measurement of several mutually identical or different materials or detection zones available. According to a further development, the fluorescence detection device can be designed in this case such that the plurality of

Prisms is arranged parallel to each other with directed to the excitation light side surfaces. Another variant is achieved in that the plurality of prisms is arranged one behind the other and a light incidence of the excitation light on the prisms takes place one after the other. By arranging a plurality of prisms parallel to one another with side faces directed towards the excitation light, it is possible to obtain a plurality of mutually different samples or a plurality of mutually identical samples be displayed simultaneously with excitation light to emissions of fluorescent light. Such a device is preferred, for example, for an application for the examination of biological fluids or the like, since only very small amounts of substance are contained in the sample and with such a configuration a simultaneous, yet very sensitive measurement of several parameters can be achieved a plurality of prisms is arranged one behind the other.

As is the case with a further development of the invention, the micro-fluorescence detection device can be developed such that the detection unit is arranged opposite to the prism of the prism. In particular, by arranging the detection unit opposite the vertex of the prism, it is possible to provide an extremely small-sized device in comparison with a variant in which the detection unit is arranged opposite the base surface of the prism. Furthermore, with a micro-fluorescence detection device in which the detection unit is arranged opposite the apex of the prism, it is possible to provide a device in which an extremely simple replacement of the biochip is made possible due to the ready accessibility thereof and thus the times and the outlay to the change, which are generally determined for single use, analysis chips compared to conventional designs are significantly reduced.

By, as corresponds to a development of the invention, the entire microchip of the micro-fluorescence detection device is made of one and the same material, in particular made of plastic, it is possible on the one hand to provide a non-absorbing medium for the p-polarized light and on the other hand can Such device simple, for example, in a single manufacturing step, such as injection molding,

Hot embossing and the like. Are manufactured, whereby a total of manufacturing costs for the production of disposable, analysis chips is significantly reduced. Furthermore, by making the entire microchip of one and the same material, it is possible to provide an inert carrier for the material to be analyzed while at the same time providing a prism which is suitable for the

Beam geometry, which is required for the apparatus of the present invention to provide required refractive indices.

According to a preferred development of the invention, the microchip, in particular the biochip, is made of a polymer material based on cycloolefin polymer (COP), cycloolefin copolymer (COC), polycarbonate (PC), polymethyl methacrylate (PMMA), poly (poly (meth) acrylate). styrene (PS), polyethylene terephthalate (PET), polyacrylate, polyurethane acrylate (PUA), polyuretane methacrylate (PUMA) or polypropylene (PP), in particular of cycloolefin polymer or copolymer (COP / COC), polycarbonate (PC), polymethyl methacrylate (PMMA) or polystyrene (PS) made. Such materials are not only chemically inert, but have the index of refraction and transparency required for the present invention and, because of their ease of processing, are suitable for mass production of microchips for a micro-fluorescence detection device according to the present invention. In order to protect in particular the material to be analyzed or the material to be stimulated against excessive photobleaching, the device is designed according to a development that it is arranged in a housing, in particular an aluminum housing or plastic housing and that inside the housing, a partition for a separation in a compartment containing the light source and a compartment containing the detection device is arranged. By the department of the housing of the micro-fluorescence detection device in a compartment containing the light source and a compartment containing the detection device, the light source can be operated continuously and at the same time the material to be analyzed or the material to be excited are protected from photobleaching. In particular, when the light source, such as a laser diode, is operated continuously, its lifetime is significantly increased over a light source that is frequently turned on and off.

In order to ensure that even for convergent or divergent bundles of rays of the excitation light, each individual light beam strikes the side surface of the prism at a Brewster angle, the invention is developed such that the side surfaces of the prism are formed as curved free-form surfaces.

The present invention further relates to a method for detecting fluorescent light in a micro-fluorescence detection device, in which the amount of interfering stray light impinging on a detection unit is minimized, while at the same time reliably and reproducibly reproducible results are achieved.

To achieve this object, the method according to the invention is essentially characterized in that the excitation light is irradiated at the Brewster angle on the side surface of the prism that the material to be excited or to be analyzed is arranged directly on the base surface of the prism at an angle which is greater as a critical angle of total reflection is excited to emit fluorescent light.

By irradiating the excitation light at the Brewster angle on the side face of the prism, it is ensured that the excitation light can be coupled into and out of the prism without unwanted reflection of the excitation light being generated from the prism during light entry and exit , whereby the measurement accuracy and in particular the signal-to-noise ratio of a measurement can be significantly improved. Further, by arranging the material to be excited or analyzed directly on the base surface of the prism and illuminated at an angle greater than the critical angle of total reflection, it is further ensured that no excitation intensity of the excitation light from the prism at the surface of the prism Prism, on which the to be detected or to be excited sample or the material to be excited is applied, penetrates, which in turn the signal-to-noise ratio of a measurement can be significantly improved and in particular exact and sharp measurement points or lines of the generated Fluorescent light can be obtained in a detection unit.

By simultaneously detecting a plurality of materials to be excited or analyzed, the speed of the measuring method is increased on the one hand and on the other hand it is ensured that a plurality of different substances to be analyzed are analyzed quickly or simply a plurality of mutually identical substances can be analyzed, whereby, after a corresponding communication, the measurement errors can be significantly reduced compared to conventional measurement methods.

If, as is the case with a further development of an invention, the method is carried out such that the plurality of materials to be excited or analyzed simultaneously are applied to a plurality of prism faces arranged parallel to one another or to be simultaneously detected If a plurality of materials to be stimulated or analyzed are applied to a plurality of prisms arranged in a row, the plurality of prisms are applied and excited in succession with one and the same excitation light, whereby one and the same sample can be excited several times and thus faulty Measurements or read-out errors after detection in the detection device tion can be withheld by direct comparison of the measurement results with certainty.

By applying the material to be excited or analyzed as a grid of fluorescent dots on the base surface of the prism, as is the case with a further development of the invention, it is possible with sufficient sensitivity to simultaneously excite with a conventional spatially resolving detector, such as a CCD camera Fluorescence of the material to be excited or analyzed. By virtue of the fact that, as is the case with a further development of the invention, the fluorescent dots are formed by binding fluorescently labeled molecules from a solution to capture or probe molecules fixed to the prism base surface, the apparatus permits the detection and quantification of samples present in the sample solution analytes. For this purpose, the fluorescent points are determined by the attachment / binding of fluorescence-dye-labeled molecules or particles, such as oligonucleotides, proteins, peptides, biomarkers, antibodies, antigens, etc., from the binding partner applied to the analyzing solution at the base surface of the prism, ie In turn corresponding complementary oligonucleotides, proteins, antigens, etc. are formed, the detection and quantification of existing in the sample solution analytes is possible.

By carrying out the method in such a way that at the same time a plurality of different parameters are analyzed by one or more materials to be stimulated or analyzed, various components, for example, can be used in biological samples Nucleic acid sequences, proteins, peptides, biomarkers, pathogens, viruses and the like can be detected and quantified by corresponding probe molecules are attached to the base surface of the prism for binding these sample components simultaneously or sequentially detected, whereby a variety of essential measurement signals are obtained quickly and easily ,

The invention will be explained in more detail with reference to embodiments shown in the drawing. In this show

1 is a schematic representation of a micro-fluorescence detection device according to the present invention. 2 shows a schematic representation of light incident at the Brewster angle on the side surfaces of a prism integrated in a carrier substrate.

Fig. 3 is a partial perspective view of a prism applied to a carrier substrate and the schematic representation of the passage of light through this prism.

4 shows a representation similar to FIG. 3 with a plurality of prisms integrated on a support,

5 shows a plurality of successively arranged prisms, which are gradually penetrated by excitation light, according to another embodiment of the invention and

6 shows a diagram relating to the fluorescence imaging and the temporal evaluation of the fluorescence intensity for a streptavidin concentration of 7 nM.

1 shows a schematic representation of a micro-fluorescence detection device 1, in which a bundle of linearly polarized light is applied from a light source 2, for example a laser diode, to a beam sharpener 3 and from this the schematically illustrated beam 4, which is a Polarization in a plane parallel to the plane of incidence 5 of the prism 6, is incident. The beam 4, which includes the Brewster angle with the plane of incidence 5 not shown in FIG. 1, is refracted inside the prism 6, is completely reflected at a base surface 7 of the prism 6 and enters at a second side surface 8 of the prism 6 again from the inside of the prism 6 at the Brewster angle. On the base surface 7, a plurality of fluorescent dots 9, in particular a grid of fluorescent dots 9 are applied in a microfluidic system, in particular in the area where the total reflection of the beam 4 takes place.

The microfluidic system is shown schematically in FIG. 1 through the depression 10, which is formed on the base surface 7 of the prism 6, and is intended, for example, to symbolize a microchannel. The fluorescent dots 9 can have a diameter of 0.05 to 1 in particular 0, 1 to 0.4 mm in such a configuration. By locating the points 9 or the grid of points 9 of the material to be excited or analyzed, it is prism-side, ie on the side of the surface 7 of the prism 6 at an angle which is greater than the critical angle of total reflection, illuminated, why no excitation light from the prism 6 penetrates. By virtue of the fact that an absorbing substance, in particular at points 9, is located on surface 7 of prism 6, it can be excited by an evanescent wave emerging in the near field and the fluorescent light 1 1 emitted after excitation is subsequently focused by a camera lens 12, which is a converging lens, again and evaluated by a detector 13, which is for example a CCD camera and an evaluation, not shown in Fig. 1. In order to avoid the occurrence of any stray light, several essential features are observed, on the one hand only an interface between the prism 6 and the sample to be analyzed 9 is present and thus an otherwise necessary composite of the prism with a separate carrier on which to analyze Molecules can be applied avoided.

Furthermore, it is essential that the prism 6 is formed of a non-absorbing medium, since non-absorbing media for p-polarized light, ie light, the plane of polarization is set parallel to plane of incidence 5 of the prism 6, ie for light, which in the Brewster- Angle incident on the plane of incidence 5 of the prism 6, are not reflective. Finally, the light is irradiated at the Brewster angle on the surface of the prism. The Brewster angle (qβ) is given here by the formula tan (qe)

ni and n2 are the refractive indices of the participating media, ie in the present case air and the material of the prism 6. By maintaining such a configuration, it is possible to perform a measurement of the emitted fluorescent light, which is absolutely free of unwanted reflections of the excitation light, so that the signal-to-noise ratio of the measurement is optimized. Finally, in the embodiment illustrated in FIG. 1, the detection unit 13 is arranged on the side of the vertex 14, in the present case a vertex surface, of the prism 6. Such a configuration is favored over the configuration also contemplated by the present invention, in which the detection unit 13 is disposed on the side of the sample 9 to be analyzed, because of its reduced space requirement, by arranging both the light source 2 and of the

Detection unit 13 on the side of the prism 6, an improved accessibility of the samples surface 7 of the prism is achieved and thus, for example, a replacement of the entire prism 6 is much easier possible than in an arrangement in which the light source 2 and the detection unit 13 on different sides of the prism 6 are arranged. FIG. 2 shows a schematic representation of a carrier with integrated prism, ie a microchip 15 according to the invention. In the microchip 15, the prism 6 is formed as part of the base surface of the microchip 15, and an interface between the chip 15 and the prism 6 does not exist, so that the number of optical interfaces is minimized.

In Fig. 4, the bundle of incident light beams 4 is further shown, which incident on the surface 5 of the prism 6 at the Brewster angle qe. In the interior of the prism 6, the incident light beam 4 is refracted to the base surface 7 and reflected therefrom and exits at the on the side surface 8 again at the Brewster angle qß from the prism 6. The prism 6 shown in FIG. 2 is a conventional prism 6 having a vertex and a base surface 7.

In Fig. 3 is a similar view as shown in Fig. 2 in perspective, wherein the prism 6 is formed in Fig. 3 as a blunt prism 6 with two horizontal sides. Also in Fig. 3, the path of the light is indicated schematically by the line 16. In Fig. 3 as well as in Fig. 4, the base surface 7 as well as in Fig. 1 of the prism 6 is shown extended and thus represents the base of the microchip 15. In this case, the prism 6 is formed directly on the microchip 15, as this is shown schematically by the dotted line 17 in order to minimize the number of interfaces.

In FIG. 4, a plurality of prisms 6, which are formed analogously to those of FIG. 3, are arranged parallel to each other on a microchip 15, wherein each of the prisms 6 is acted upon by light, wherein the light path is again schematically designated 16. In this case, the light source 2, which acts on the plurality of prisms 6, may be a common light source 2 or a plurality of light sources 2 arranged parallel to one another may be used.

Finally, in the representation of FIG. 5, a plurality of prisms 6 are shown, which are arranged one behind the other and which are penetrated successively by a light bundle 4. Due to the fact that the incident light 6 again impinges on the surface 5 of the prism 6 at the Brewster angle qB and emerges from the first prism 6 exactly at the Brewster angle qβ, it is possible to apply all the prisms 6 with exactly the same light geometry. so that errors due to stray light, which may be caused by non-observance of the light geometry, are avoided. In the illustration of FIG. 5, recesses 10 are again shown in the base surface 7 of the prism 6, which are intended to symbolize microchannels in which fluid material can be guided. Needless to say that the Prismengeometrie can be chosen arbitrarily and that, for example, free-form prisms can be used, which are used to compensate for possible radiation divergences.

The invention will be further explained below with reference to an embodiment.

Example 1 :

A biochip according to FIG. 3 with external dimensions of 25 × 75 mm is produced by hot pressing or injection molding of COP material, namely a thermoplastic polyolefin resin marketed under the trade name Zeonor® 1060R (a trademark of Zeon Corporation), the chip incorporating a includes integrated prism whose angle of the side surfaces to the base surface amount to 33.2 °.

Biotin-functionalized bovine serum albumin (BSA) is imprinted on the base surface of this prism in the form of a dot-shaped raster. Subsequently, this chip is incorporated with another chip, in which a microfluidic channel system consisting of at least one channel, which allows a sample solution to be passed over the grid applied on the base surface of the prism and in which a liquid inlet and outlet is further incorporated, connected. In this way, a microchip, in particular a biochip, was prepared for the detection of biotin or biotin-binding proteins, such as streptavidin or neutravidin.

A sample or solution containing a certain amount of biotin and streptavidin labeled with the fluorescent dye Cy3 is then introduced into the microfluidic channel and the fluorescence intensity of the grid printed on the base surface of the prism is subsequently monitored with a CCD camera. A corresponding increase in

Fluorescence intensity with incubation time indicates binding of the Cy3-labeled streptavidin to the printed halftone dots of biotinylated BSA. The rate of this increase or the achievable fluorescence intensities depend on the concentration of biotin and streptavidin in the sample solution. The corresponding fluorescence imaging and the temporal evaluation of the fluorescence intensity are shown for a Cy3 streptavidin concentration of 7 nM in FIG.

Claims

P atent claims:

1. Micro-fluorescence detection device (1) comprising a light source (2) for generating linearly polarized excitation light (4), at least one prism (6) to whose base surface (7) a carrier for a material to be excited or analyzed is connected, and a Detection unit (13) arranged parallel to the carrier for the material to be excited or analyzed, characterized in that the at least one prism (6) and the carrier for the material to be excited or analyzed are designed as a one-piece microchip (15), with a base surface (7) side of the microchip (15) forming the prism (6) forms the carrier for the material to be excited or analyzed, on which the material to be excited or analyzed is arranged in the area of total reflection of the excitation light (4) and that both side surfaces (5, 8) of the prism (6) are arranged at the Brewster angle (qe) to the excitation light (4).

2. Micro-fluorescence detection device (1) according to claim 1, characterized in that the base surface (7) of the microchip (15) which represents the carrier for the material to be excited or analyzed is designed to be structured and that the structuring (10) contains one or more components or structures selected from microfluidic structures and channels, micro-optical structures, electrodes, valves or membranes.

3. Micro-fluorescence detection device according to claim 1 or 2, characterized in that the material to be excited or analyzed is applied as a grid of fluorescent dots (9) to the base surface (7) of the microchip (15), in particular in microfluidic structures (0). and channels of the microchip (15) is applied.

4. Micro-fluorescence detection device according to claim 3, characterized in that a diameter of each point of the grid of fluorescent points is 0.05 to 1 mm, in particular 0, 1 or 0.4 mm

5. Micro-fluorescence detection device (1) according to one of claims 1 to 4, characterized in that a plurality of prisms (6) is arranged on a common microchip (15) and that the prisms (6) are embedded in a surface of the microchip (15 ) are integrated.

6. Micro-fluorescence detection device (1) according to claim 5, characterized in that the plurality of prisms (6) are arranged parallel to one another with side surfaces (5) directed towards the excitation light (4).

7, micro-fluorescence detection device (1) according to claim 5, characterized in that the plurality of prisms (6) are arranged one behind the other and the excitation light (4) is incident one after the other.

8. Micro-fluorescence detection device (1) according to one of claims 1 to 7, characterized in that the detection unit (13) is arranged opposite the vertex (14) of the prism (6).

9. Micro-fluorescence detection device (1) according to one of claims 1 to 9, characterized in that the entire microchip (15) is formed from one and the same material, in particular from a plastic.

10. Micro-fluorescence detection device (1) according to claim 9, characterized in that the microchip (15) made of a polymer material based on cycloolefin polymer (COP), cycloolefin copolymer (COC), polycarbonate (PC), polymethyl methacrylate (PMMA), polystyrene (PS) , polyethylene terephthalate (PET), polyacrylate, polyurethane acrylate (PUA), polyurethane methacrylate (PUMA) or polypropylene (PP), in particular made of cycloolefin polymer or copolymer (COP/COC), polycarbonate (PC), polymethyl methacrylate (PMMA) or polystyrene (PS ) is manufactured.

1 1. Micro-fluorescence detection device (1) according to one of claims 1 to 10, characterized in that the micro-fluorescence detection device (1) is arranged in a housing, in particular an aluminum housing or plastic housing, and that inside the housing there is a partition for separation a compartment containing the light source (2) and a compartment containing the detection unit (13) are arranged.

12. Micro-fluorescence detection device (1) according to one of claims 1 to 11, characterized in that the side surfaces (5, 8) of the prism (6) are designed as curved free-form surfaces.

13. Method for detecting fluorescent light in a micro-fluorescence detection device (1), one connected to a base surface (7) of a prism (6). Material arranged on a carrier to be excited or analyzed, in which linearly polarized light (4) is directed from an excitation light source (2) onto a side surface (5) of the prism (6), whereby on the base surface (7) of the prism (6). The material to be excited or analyzed is excited to emit fluorescent light with simultaneous reflection of the excitation light and the fluorescent light emitted by the material to be excited or analyzed is evaluated in a detection unit (13), characterized in that the excitation light is at the Brewster angle (q _B ) is irradiated onto the side surface (5) of the prism (6) and that the material to be excited or analyzed, which is arranged directly on the base surface (7) of the prism (6), is at an angle that is greater than a critical angle of total reflection, is stimulated to emit fluorescent light.

14. The method according to claim 13, characterized in that a plurality of materials to be excited or analyzed are detected at the same time.

15. The method according to claim 13 or 14, characterized in that the simultaneously detected plurality of materials to be excited or analyzed is applied to a plurality of base surfaces (7) of prisms (6) arranged parallel to one another.

16. The method according to claim 13, 14 or 15, characterized in that the simultaneously detected plurality of materials to be excited or analyzed is applied to a plurality of base surfaces (7) of prisms (6) arranged one behind the other.

17. The method according to any one of claims 13 to 16, characterized in that the material to be excited or analyzed is applied as a grid of fluorescent dots on the base surface (7) of the prism (6).

18. The method according to claim 17, characterized in that the fluorescent points are formed by binding fluorescently marked molecules from a solution to catcher or probe molecules fixed to the base surface (7) of the prism (6).

19. The method according to any one of claims 13 to 18, characterized in that a plurality of different parameters of one or more materials to be excited or analyzed are analyzed at the same time.