• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A micro-fluorescence detection device (1) comprising a light source (2) for generating linearly polarized excitation light (4), at least one prism (6) with the base surface (7) is connected to a carrier for a material to be excited or analyzed, and a parallel to the support for the material to be excited or analyzed material arranged detection unit (13) is characterized in that the at least one prism (6) and the carrier for the material to be excited or analyzed as a one-piece microchip (15) are formed, wherein a base surface (7) of the prism (6) forming side of the microchip (15) forms the support 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 (4) is arranged and that both Side surfaces (5, 8) of the prism (6) in the Brewster angle (BBy to the excitation light (4) are arranged and micro-Fluore comprising a light source (2) for generating linearly polarized excitation light (4), at least one prism (6) with the base surface (7) a support for a material to be excited or is connected, as well as a parallel to the carrier for the material to be excited or arranged to be arranged detection unit (13), characterized in that the at least one prism (6) and the support for the material to be excited or analyzed are formed as a one-piece microchip (15), wherein a base surface (7) the prism (6) forming side of the microchip (15) forms the support 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 (4) is arranged and that both side surfaces (5 , 8) of the prism (6) are arranged at the Brewster angle (63) to the excitation light (4) and methods for detecting fluorine szenzlicht.
    公开号:AT513859A4
    申请号:T2992013
    申请日:2013-04-12
    公开日:2014-08-15
    发明作者:Eduard Dr Gilli；Frank Dr Reil；Stefan Dr Köstler；Volker Dr Schmidt
    申请人:Joanneum Res Forschungsgmbh；
    IPC主号:
    专利说明:

    • " J · · · · »*: ** * '* • *» · · · · ··· ·· «• ····································································
    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 A material arranged detection unit, and a method for detecting fluorescent light in a micro-fluorescence detection device, arranged on a connected to a base surface of a prism carrier to be excited or analyzed material in which 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 to be stimulated or analyzed material for the emission of fluorescent light with simultaneous reflection of the excitation light is excited and that of the to be excited or to analyze sizing material detected fluorescent light is evaluated in a detection unit.
    In recent years, a variety of different fluorescence detection devices have become known. Both systems with micro-lites and / or micro-gratings 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 onto fluorescent material is applied to a sample surface is applied, at an angle greater than a critical angle is incident. 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, to connect the support for the sample to be analyzed with the prism integrated into the detection unit, an immersion medium must be used in order 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 is 2/24
    In this system, a laser diode and the system further includes beam splitters, apertures, and a through a stepper motor. English: driven control or regulating mirror, in which after filtering the fluorescent light, the punctiform field was detected by a non-cooled CCD camera, on.
    Due to the ever-increasing demand for miniaturized devices employing direct detection of particular biomolecules or pathogens in applications such as diagnostics, food testing, or environmental monitoring, the so-called " lab-on-a-chip " or " total microanalysis systems (μ-TAS) designed to perform the entire process from sample preparation, measurement, reaction, incubation, etc., to detection on one and the same chip. However, early such miniaturized fluorescence devices have been built on glass substrates or silicon substrates, and production technologies in the field of electronics and semiconductor industry have had to be used, whereby such micro fluorescence detection devices are not only expensive but also complicated and expensive to manufacture 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 autofluorescence. 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 here is an effective, geometric separation between fluorescent light and excitation light, since the intensity of the excitation light is usually higher by a multiple than the intensity of the generated fluorescent light, which is a major problem in terms of measurement accuracy, especially in miniaturized systems, as usually not inconsiderable amounts of stray light interfere with or falsify the measurement.
    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 an effective, geometric separation of fluorescent light and excitation light and thus to achieve 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 material to be detected to measure the fluorescence light sensitively and accurately with a spatially resolving detector. 3.24
    ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦··············································································· •
    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 one-piece (monolithic) microchip, wherein a base surface of the prism forming side of the microchip the carrier for the to be excited or material to be analyzed is formed, on which the material to be excited or analyzed is arranged in the region of the total reflection of the excitation loop and that both side surfaces of the prism are arranged at Brewster angle to the excitation light. Since the at least one prism and the carrier for the material to be excited or analyzed are formed as a one-piece microchip, there is no interface in the device between the carrier for the material to be excited or analyzed and the optical waveguide, namely the prism, as a result of which unwanted reflections or reflections are produced 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. Here, 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, so that no excitation light intensity from the prism in the region of the material to be excited or penetrated, but inside the prism is completely reflected. The substance to be excited or to be analyzed applied on the same base surface of the prism, which is at the same time an absorbing substance for the excitation light, is thus excited by an evanescent wave emerging in the near field and causes the emission of fluorescent light, which fluorescence light, subsequently evaluated in the detection unit By further arranging 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 enhancement or further reduction of reflections of the excitation light in this case, linearly polarized light, which may originate, for example, from a laser diode as the light source whose polarization plane is set parallel to the plane of incidence, ie p-polarized light at the Brewster angle irradiated 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 during light entry and exit 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 Brewster's angle. Such a configuration thus maximizes the coupling and decoupling of the excitation light while at the same time minimizing or completely avoiding 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. 4.24
    ♦ · · · · · · · · · · · · · · · · · · · · · · · · · ·
    By, as corresponds to a development of the invention, the fluorescence detection device is designed so that the carrier for the material to be excited or performing material representing base surface of the microchip is structured and that the structuring of one or more components or structures selected from micro-fluidic structures and channels, micro-optical structures, electrodes, valves or membranes, it is possible in a simple manner to provide a so-called lab-on-a-chip system in which the number of interfaces between the material to be excited or analyzed to 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 further development of the invention, the material to be excited or analyzed as a grid of fluorescent dots on the carrier ausbildenden side or the base surface of the microchip is applied, it is possible with simultaneous effective excitation with a conventional spatially resolving detector, like a CCD camera, it is 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 1mm, in particular 0.1 and 0.4 mm, with simultaneous homogeneous irradiation and a quantification of the average intensity of the 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 measurements can be achieved.
    In order to enable, 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 cut into the prism base surface. 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. 5/24 • · • - 5 · · · · · · · · · · · · · · · · · · · · · · ··· ·· ·· ··
    According to a development, the fluorescence detection device can in this case be designed such that the plurality of prisms is arranged parallel to one another with side faces directed toward the excitation light. 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 surfaces directed towards the excitation light, it is possible to display a plurality of mutually different samples or a plurality of mutually identical samples simultaneously with excitation light for 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 minute amounts of substance are contained in the sample and with such a configuration a simultaneous and yet very sensitive measurement of several parameters can be achieved by a plurality of prisms arranged one behind the other.
    As is the case with a development of the invention, the micro-fluorescence detection device can be developed in such a way that the detection unit is arranged opposite the vertex 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 which allows an extremely simple exchange of the biochip on account of its easy accessibility 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 For example, such a device can be easily manufactured, for example, in a single manufacturing step, such as injection molding, hot stamping and the like, thereby significantly reducing the manufacturing effort required to produce the single-use analysis chips. Furthermore, by making the entire microchip of one and the same material, it is possible to provide an inert support for the material to be analyzed and at the same time to provide a prism which is suitable for the beam geometry used for the device of the present invention required to have required refractive indices available to sites. 6/24
    •. •. · T · · · · · · · · · · · · · ♦♦
    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), polystyrene (PS), polyethylene terephthalate (PET), polyacrylate , Polyurethane acrylate (PUA), Polyuretanmeth-acrylate (PUMA), polypropylene (PP) made, in particular made of cycloolefin polymer or copolymer (COP / COC), polycarbonate (PC), polymethyl methacrylate (PMMA) or polystyrene (PS). 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. Through 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 aims at a method for detecting fluorescent light in a micro-fluorescence detection device, in which the amount of spurious scattered 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 inventive method is essentially characterized in that the excitation light is irradiated at the Brewster angle to the side surface of the prism that arranged directly on the base surface of the prism to be excited or analyzed material at an angle greater than 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 undesired reflection of the excitation light upon light entry and exit from the prism becomes, 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, 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 fluorescence light in a detection unit can be obtained.
    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 or a plurality can be analyzed by mutually identical substances, which can be significantly reduced after a corresponding averaging the measurement error compared to conventional measurement methods.
    If, as this corresponds to a development of an invention, the method is performed so that the simultaneously to be detected plurality of materials to be excited or analyzed on a plurality of mutually parallel base surfaces of prisms is applied, or that simultaneously detected plurality of to be excited or analyzed materials on a plurality of successively arranged base surfaces of prisms, the plurality of prisms with one and the same excitation light is applied and excited in succession, whereby one and the same sample can be excited several times and in this way erroneous measurements or readout Errors can be safely avoided after detection in the detection device by direct comparison of the results.
    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.
    The fact that, as corresponds to a further development of the invention, the fluorescent dots are formed by binding of fluorescently labeled molecules from a solution on captured at the base surface of the prism capture or probe molecules, the device allows the detection and quantification of vorhan in the sample solution - 8/24
    ♦ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · '• • · ·· • t those analytes. For this purpose, the fluorescent dots are formed by the attachment / binding of fluorescent dyes labeled molecules or particles, such as oligonucleotides, proteins, peptides, biomarkers, antibodies, antigens, etc. from the solution to be analyzed on the prism base on the base of the binding partner, i. 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 a plurality of different parameters are simultaneously analyzed by one or more materials to be stimulated or analyzed, various constituents, for example in biological samples, such as nucleic acid sequences, proteins, peptides, for example , Biomarkers, pathogens, viruses and the like. Detected and quantified by corresponding probe molecules are attached to the base surface of the prism for binding these Probenbestantteile are detected simultaneously or sequentially, 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
    Fig. 1 is a schematic representation of a micro-fluorescence detection apparatus 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,
    Fig. 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 to a beam sharpener 3 from a light source 2, for example a laser diode, and from this the schematically illustrated beam 4, which has a beam 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. ···································································································································································································································· occurs at a second side surface 8 of the prism 6 again at the Brewster angle from the interior of the prism 6. On the base surface 7, in particular in the region where the total reflection of the beam 4 takes place, a plurality of fluorescent dots 9, in particular a grid of fluorescent dots 9 are applied in a micro-fluidic system. 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 may have a diameter of 0.05 to 1 in particular 0.1 to 0.4 mm in such a configuration. By locating the dots 9 or the grid of dots 9 of the material to be excited or analyzed, he / she is prism-side, i. 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, so that no excitation light from the prism 6 penetrates. By having an absorbing substance, in particular at points 9, on the surface 7 of the prism 6, it can be excited by an evanescent wave emerging in the near field and the fluorescence light 11 emitted after excitation is subsequently transmitted through a camera lens 12 represents a converging lens, in turn focused and by a detector 13, which is for example a CCD camera and one not shown in Fig. 1
    Evaluation electronics evaluated.
    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 be formed of a non-absorbing medium since non-absorbing media for p-polarized light, i. Lieht, whose polarization plane is set parallel to plane of incidence 5 of the prism 6, i. for light which impinges on the plane of incidence 5 of the prism 6 at Brewster angle, are not reflective. Finally, the light is irradiated at the Brewster angle on the surface of the prism. Of the
    Brewster's angle (0B) is defined here by the formula tan (ΘΒ) = n2 / n1, where ni and n2 are the refractive indices of the media involved, i. in the present case, air and the material of the prism 6 are.
    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 opposite to that of the present invention 10/24
    • «• •« · ····································································································································· is arranged on the side of the sample to be analyzed or 9, due to their reduced space requirements favors, since by arranging both the light source 2 and the detection unit 13 on the side of the prism 6 improved accessibility of the sample surface 7 of the Prism is achieved and thus, for example, an exchange of the entire prism 6 is much easier possible than in an arrangement in which the light source 2 and the detection unit 13 are arranged on different sides of the prism 6.
    In Fig. 2 is a schematic representation of a carrier with integrated prism, i. a microchip 15 according to the invention is shown. In the microchip 15, the prism 6 is part of the
    Base surface of the microchip 15 is formed 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 rays 4 is further shown, which incident on the surface 5 of the prism 6 at the Brewster angle ΘΒ. 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 ΘΒ 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, Here, the prism 6 is formed directly on the microchip 15, as 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 analogous 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 indicated by 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 illustration 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 und and exits the first prism 6 exactly at the Brewster angle ΘΒ, all the prisms 6 succeed in applying exactly the same light geometry. so that errors due to stray light, which may be caused by the non-compliance of the light geometry, are avoided. 11/24
    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 into 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 printed 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 biochip for the detection of biotin or biotin-binding proteins, such as streptavidin or neutravidin was prepared.
    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.
    权利要求:
    Claims (19)
    [1]


    1. Micro-fluorescence detection device (1) comprising a light source (2) for generating linearly polarized excitation light (4), at least one prism (6) with its base surface (7) is connected to a carrier for a material to be excited or analyzed, 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 formed as a one-piece microchip (15), wherein a base surface (7) of the prism (6) forming side of the microchip (15) forms the support 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 (4) is arranged and that both side surfaces (5, 8) of the prism (6) are arranged at the Brewster angle (ΘΒ) to the excitation light (4).
    [2]
    2. micro-fluorescence detection device (1) according to claim 1, characterized in that the carrier for the material to be excited or representing material representing base surface (7) of the microchip (15) is structured and that the structuring (10) one or more components or structures selected from microfluidic structures and channels, micro-optical structures, electrodes, valves or membranes.
    [3]
    3. Micro-fluorescence detection device according to claim 1 or 2, characterized in that the material to be excited or analyzed as a grid of fluorescent dots (9) on the base surface (7) of the microchip (15), in particular in microfluidic structures (10) and channels of the micro-chip (15) is applied.
    [4]
    4. micro-fluorescence detection device according to claim 3, characterized in that a diameter of each point of the grid of fluorescent dots 0.05 to 1 mm, in particular 0.1 or 0.4 mm
    [5]
    5. micro-fluorescence detection device (1) according to one of claims 1 to 4, characterized in that a plurality of prisms (6) on a common microchip (15) is arranged and that the prisms (6) in a surface of the microchip (15 ) are integrated.
    [6]
    6. micro-fluorescence detection device (1) according to claim 6, characterized in that the plurality of prisms (6) parallel to each other with the excitation light (4) directed side surfaces (5) is arranged.
    [7]
    7. micro-fluorescence detection device (1) according to claim 6, characterized in that the plurality of prisms (6) is arranged one behind the other and a light incidence of the excitation light (4) takes place successively. 13/24
    [8]
    8. micro-fluorescence detection device (1) according to one of claims 1 to 8, characterized in that the detection unit (13) is arranged opposite the apex (14) of the prism (6).
    [9]
    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]
    The micro-fluorescence detection device (1) according to claim 10, characterized in that the microchip (15) is 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), polyuretane methacrylate (PUMA), polypropylene (PP), and the like. In particular of cycloolefin polymer or copolymer (CÖP / COC), polycarbonate (PC), polymethylmethacrylate (PMMA) or polystyrene (PS) is made.
    [11]
    11. micro-fluorescence detection device (1) according to one of claims 1 to 10, characterized in that the micro-fluorescence detection device (1) in a housing, in particular aluminum housing or plastic housing is arranged and that in the interior of the housing, a partition for separation into a Compartment containing the light source (2) and a compartment containing the detection unit (13) is arranged.
    [12]
    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 formed as curved freeform surfaces.
    [13]
    13. A method for the detection of fluorescent light in a micro-fluorescence detection device (1), arranged on a with a base surface (7) of a prism (6) connected carrier, to be excited or analyzed material in which linearly polarized light (4) of a Exciting light source (2) on a side surface (5) of the prism (6) is directed, wherein at the base surface (7) of the prism (6) the excited or to be analyzed material for the emission of fluorescent light with simultaneous reflection of the excitation light is excited and that of the fluorescence light emitted to be excited or analyzed material in a detection unit (13) is evaluated, characterized in that the excitation light at the Brewster angle (ΘΒ) on the side surface (5) of the prism (6) is irradiated and that directly on the base surface (7) of the prism (6) arranged to be excited or analyzed material at an angle greater as a critical angle of total reflection is excited to emit fluorescent light.
    [14]
    14. The method according to claim 13, characterized in that at the same time a plurality of materials to be excited or to be analyzed is detected.
    [15]
    15. The method according to claim 13, wherein the plurality of materials to be excited or to be analyzed are applied on a 14/24 plurality of base surfaces (7) of prisms (6) arranged parallel to one another.
    [16]
    16. The method of claim 13, 14 or 15, characterized in that the simultaneously to be detected plurality of materials to be excited or to be analyzed on a plurality of successively arranged base surfaces (7) of prisms (6) is applied.
    [17]
    17. The method according to any one of claims 13 to 16, characterized in that the material to be excited or to be analyzed as a grid of fluorescent dots on the base surface (7) of the prism (6) is applied.
    [18]
    18. The method according to claim 17, characterized in that the fluorescent points are formed by binding of fluorescently labeled molecules from a solution on the base surface (7) of the prism (6) fixed catcher or probe molecules.
    [19]
    19. The method according to any one of claims 13 to 18, characterized in that the method is performed so that simultaneously a plurality of different parameters of one or more materials to be analyzed or analyzed to be analyzed.

    by: Cunow Patentanwalts KG Vienna, April 12, 2013 15/24
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    同族专利:
    公开号 | 公开日
    WO2014165879A1|2014-10-16|
    AT513859B1|2014-08-15|
    引用文献:
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    KR101067348B1|2009-03-04|2011-09-23|한국과학기술원|The prism inducing Brewster's angle transmission and apparatus for fluorescence detection for enhancement of signal to noise ratio used to thereof|DE102017115661A1|2017-07-12|2019-01-17|Endress+Hauser Conducta Gmbh+Co. Kg|Optical sensor|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    AT2992013A|AT513859B1|2013-04-12|2013-04-12|Micro-fluorescence detection device and method for detection|AT2992013A| AT513859B1|2013-04-12|2013-04-12|Micro-fluorescence detection device and method for detection|
    PCT/AT2014/000066| WO2014165879A1|2013-04-12|2014-03-31|Micro fluorescence-detecting device and method therefor|
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