EP2227684A2

EP2227684A2 - Receptacle, and method for the detection of fluorescence

Info

Publication number: EP2227684A2
Application number: EP08761398A
Authority: EP
Inventors: Thomas Ruckstuhl; Stefan Seeger
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-04-30
Filing date: 2008-06-27
Publication date: 2010-09-15
Also published as: AU2008244225A1; CN101910823A; DE102007020610A1; WO2008132247A3; JP2011525612A; US20100136709A1; WO2008132247A8; WO2008132247A2

Abstract

The invention relates to a liquid receptacle comprising a bottom and sidewalls for holding a liquid. The bottom encompasses a flat sensor surface that is in contact with the liquid when the receptacle is filled, a light-incident area that is located below the sensor surface and is suitable for focusing light onto the sensor surface, a light emergence area, and a cover area that is suitable for reflecting light from the sensor surface such that the light can emerge through the light emergence area. The invention further relates to a method for qualitatively or quantitatively determining an analyte in such a liquid receptacle. In said method, excitation light is focused onto the sensor surface via the light-incident area such that a luminescent marker which characterizes the analyte is excited, and the generated luminescence is then reflected onto the cover surface and is detected after emerging through the light emergence area. The invention also relates to an analysis device comprising a holder for a liquid receptacle, a light source that is disposed such that the light thereof can be focused onto the sensor surface of the liquid receptacle via the light-incident area, and a detector which is arranged in such a way as to be able to detect the light emerging from the light emergence area of the liquid receptacle.

Description

CONTAINER AND METHOD FOR DETECTING FLUORESCENCE

The present invention describes reaction vessels with integrated light harvesting optics that can be used for detection of surface bound fluorescent molecules. In bioanalysis, solid-phase-based assays in which the substances to be detected are concentrated from the solution via immobilized receptors on the surface play a central role. By means of the affinity-based reaction on the surface, biological substances can be detected very selectively, even from a mixture. Typical receptors are antibodies and DNA molecules. An example of particularly high relevance is antigen detection via high-affinity antibodies. For fluorescence-based detection, the so-called sandwich test is frequently used, with a capture antibody, the receptor, binding the antigen to be detected to the surface. A second fluorescently labeled antibody also binds to the antigen and allows sensitive detection of the complexes. The antigen concentration can be quantified by an intensity measurement of the bound fluorescence markers. Due to its high sensitivity, fluorescence detection is one of the most important detection techniques in biotechnology.

The quantification of low levels of biomaterials places high demands on the detection technique in terms of sensitivity, safety and cost. For the detection of biomarkers, e.g. arise in connection with cancer or cardiovascular diseases, ever lower detection limits are sought. For medical applications are at the same time robustness and

Reproducibility of the analysis of great importance. In addition, the ever-increasing number of such tests requires measurements with the least possible expenditure of material and time. For the performance of a sensitive measurement method, the signal-to-noise ratio is of central importance. This is especially true for fluorescence measurement, where a technological improvement in the ratio of fluorescence intensity and noise leads to low detection limits and / or material and time savings. In the detection of binding assays by fluorescence methods, the signal-to-noise ratio is optimized by maximizing the fluorescence collection of surface-bound molecules while minimizing the light collection of all sources of oxygen.

The binding reaction is usually carried out at an interface between an aqueous solution and a transparent measuring substrate of glass or plastic material, which is coated with receptor molecules. An important source of interference is the fluorescence signal of freely diffusing molecules in the sample solution, which can superimpose the fluorescence signal of the molecules bound to the surface, thus making it difficult to determine the concentration. This can be prevented by washing steps, but this is time consuming and expensive. For real time measurement of the

To enable binding reactions and to circumvent complex rinsing steps, the surface-selective fluorescence detection is of decisive advantage. The aim here is to limit the detection volume as far as possible to the surface and thus to exclude fluorescence from unbound molecules from the detection.

The emission of fluorescence requires the optical excitation with light of suitable wavelength. The excitation light induces scattering and autofluorescence in the sample and substrate. Particularly problematic is that portion of the light which spectrally overlaps with the emission of the fluorescent dye to be detected and can not be blocked by wavelength filters. Since a significant portion of this light is generated in the measuring substrate, this contribution is suppressed by reducing the Detektionsvolumens in the substrate material. This can be achieved by spatial filtering of the fluorescence signal. This exploits the fact that fluorescence and scattered light arise spatially separated and thus can be separated from the optical system. Due to the different optical paths of fluorescence and scattering, the latter can be strongly suppressed by geometric means, eg with a pinhole. In summary, the detection volume of the fluorescence measurement results in simple design goals. First, the fluorescence collection of the optical system at the interface should be as high as possible. Second, light collection should be as low as possible both within the aqueous sample and within the substrate.

Close to the interface of two dielectric materials, such as between water (refractive index nl »1.33} and glass (n2« 1.52) has a significant influence on the fluorescence emission properties, unlike the fluorescence emission within a homogeneous medium, the emission from the surface is not isotropic but has a strong maximum in the direction of the critical angle of total reflection α _c , where

α _c = aresine (nl / n 2).

For the water / glass interface, a _{c is} «61 °. Fluorescent molecules bound at the interface emit about 74% of the light into the glass. In this case, 34% of the total emission is above the critical angle α _c . The fluorescence emission above the critical angle (supercritical angle fluorescence) is of particular importance for binding assays. It takes place exclusively by molecules which are located directly in front of the interface, ie at a distance from the substrate much smaller than the emission wavelength. Consequently, a fluorescence collection that exclusively allows is limited to the region above the critical angle, a surface-selective detection. The contribution of unbound fluorescent dyes can thus be almost completely suppressed, which allows, for example, real-time measurements of binding reactions.

The conventional method of surface-selective fluorescence measurement is carried out via a so-called evanescent excitation of the interface. The excitation light strikes the interface above the critical angle and is totally internally reflected in the measuring substrate. On the sample side of the interface, a thin excitation layer is thus generated, with which surface-bound molecules can be selectively excited to fluoresce. However, this method is technically complicated and makes miniaturization more difficult.

Ruckstuhl and Seeger describe a method of efficiently collecting fluorescence emission above the critical angle (PCT / EP099 / 1548). An optical waveguide, made of glass or plastic, has a lateral surface, which is the

Light collimated by an internal reflection. The use of a lateral surface of parabolic shape collimates the fluorescence into a parallel beam and makes further processing of the signal particularly easy. The collimated fluorescence can be focused through a pinhole which serves as a spatial filter. The diaphragm reduces the detection volume within the substrate and filters out the scattered light / autofluorescence induced there by the excitation light. Thus, the collimator achieves a very high signal-to-noise ratio and even allows the detection of individual molecules. Even with exclusive fluorescence collection above the critical angle, the collection efficiency of the collimator is more than 30%, well above the levels of conventional detection systems. Fluorescence sensors based on fiber optics achieve collection efficiencies of 1%. Refraction-based single lenses are up to a numerical aperture (N. A,) of about 0.6 and produce after all efficiencies by 5%. However, conventional lenses or lens systems mainly collect fluorescence below the critical angle and thus do not allow surface-selective fluorescence collection.

In bioanalytics, small standardized reaction vessels are often used, such as test tubes or cuvettes for single measurements and microtiter plates for higher throughput measurements. On microtiter plates, a large number of reaction vessels, so-called wells, are arranged in lattice-like fashion over an area of ~ 7 ^× 11 cm ² , which allows standard 96, 384 or 1526 independent measurements. The wells are typically read out sequentially, which requires a fast shift of the plate between measurements. By incorporating the supercritical fluorescence collimator into microtiter plates, the signal yield can be significantly improved compared to conventional plates. In addition, real-time measurements of high throughput binding reactions are made possible. However, the collimator must be miniaturized to a few millimeters in diameter.

The excellent light collection property of the collimator is limited to a limited area around the optical axis. As the distance of the fluorescence emission from the optical axis decreases, the quality of the light condensing and the collection efficiency deteriorate. This is analogous to fluorescence microscopy, where high numerical aperture optics achieve high collection efficiency and sensitivity, but only within a relatively small area in the ocular space. To fully exploit the performance of the collimator, the excitation light must be focused on the surface about the optical axis of the collimator. The size of the usable area and the accuracy with which the excitation light has to be centered on the optical axis depend on the size of the Collimator off. Miniaturization of the collimator reduces the usable area and thus increases the precision requirements. This increases the requirement and cost of the motorized shifting devices, adds more time and can also have a negative impact on the robustness and reproducibility of the measurements.

The present invention relates to methods of fluorescence collection above the critical angle in inexpensive liquid containers, such as test tubes and microtiter plates made of plastic. This is achieved by a novel collimator and the integration of the element into the bottom of the vessel. An improvement is in particular the integration of the focusing optics for the excitation light into the vessel bottom. With a convex surface arranged below the analyte-vessel bottom interface, excitation light near the optical axis of the collimator can be focused onto the interface. This leads to a significant increase in the allowed tolerances in the centering of the excitation light on the optical axis of the collimator. This results, inter alia, in the advantage that with sequential readout of several reactions, the displacement of the vessels above the detection system can be done less with less precision, without a negative effect on the sensitivity and reproducibility of the measurements. The manual or automated change of the container between two measurements can thus be faster and / or realized with less expensive components.

Figure 1 shows an embodiment of the invention. The collimator 1 shown is part of a liquid container. For the focusing of the excitation light, a convex-shaped surface 2 is integrated on the underside of the collimator 1. The convex surface 2 is preferably arranged rotationally symmetrical about the optical axis of the collimator and focuses the light preferably close to the axis on the opposite Sensor surface 3, which is in contact with the liquid analyte. For binding assays, the sensor surface 3 can be coated with receptor molecules. The fluorescence emitted at large angles into the waveguide material is reflected by the lateral surface of the collimator 4 and leaves the collimator through the lower-side light exit surface 5. The collimating of the lateral surface can be based on the total internal reflection, if it originates from a medium having a refractive index of less than 1.1 is surrounded, eg air with a refractive index of 1.0. The lateral surface can also be metallically mirrored. The lateral surface is preferably convex in such a way that the fluorescence after exiting the collimator is focused around the optical axis 9. The use of a parabolic lateral surface is particularly advantageous because it allows the fluorescence to be collimated into an approximately parallel beam. The lateral surface 4 can be directly adjacent to the light exit surface 5, but also to a further surface 6, which can be used for example for mounting purposes of the element. With the opaque aperture 7 can be prevented that excitation light outside the light entrance surface 2 penetrates into the collimator 1. In addition, the angular range of the fluorescence collection can be limited upwards by selecting the outer diameter of the diaphragm 7. With an opaque aperture 8, the angular range of the fluorescence light collection can be limited downwards, preferably above the critical angle α _c . The angular range of the light collection can, however, also be limited downwards without diaphragm 8, namely by suitable choice of the outer diameter of the lateral surface 4. The area of the sensor surface 3 is at least in the region which is excited by the light source. Preferably, the planar region has a diameter of more than 100 microns. The size of the collimator is preferably adapted to the respective size of the analyte container. Typically, a diameter between 2 and 15 millimeters. If a parabolic lateral surface 4 is selected, then it lies Focal length preferably in the range of 0.4 to 3 millimeters. The focal point of the parabolic lateral surface is then preferably on the sensor surface, so that the fluorescence striking the lateral surface is collimated to form an approximately parallel beam.

The distance of the convex light entry surface to the sensor surface is preferably 2 millimeters to 20 millimeters. The diameter of the light entry surface 2 is generally smaller than the inner diameter of the lateral surface 4 and is preferably in the range of 0.5 to 6 millimeters. For the cost-effective mass production of the collimator are particularly injection-moldable optical plastics, such as PMMA, PC, PS, Zeonor or Zeonex.

FIG. 2 shows a possible embodiment of the fluorescence measurement analyzer with the collimator 1 integrated in a vessel bottom. A light source 10 emits light for fluorescence excitation of a suitable wavelength. The light is sufficiently collimated by optical components 11 and brought to a suitable beam diameter. These components may include optical lenses, optical fibers, mirrors and apertures. The light is spectrally cleaned by Weilenlängenfilter 12. The excitation light is irradiated in the direction of its optical axis in the collimator. The fluorescence emitted above the critical angle emerges annularly as collimated radiation from the collimator. However, the collimator also collects fluorescence with the convex surface 2. The fluorescence in this angular range around the optical axis can also be emitted by molecules that are not bound to the interface 3. For a surface-selective measurement, therefore, fluorescence collected by the convex surface 2 must be completely blocked. For this purpose, a reflector element 14 is arranged below the collimator which separates fluorescence emitted near the axis from the supercritical fluorescence. In the case shown, the reflector element 14 directs the excitation light 13 to the optical Axis of the collimator and reflects the fluorescence collected by the convex surface 2 at the same time completely from the detection beam path. In a further embodiment, the reflector element may be such that the supercritical emitted fluorescence is mirrored, but excitation light and fluorescence collected near the axis are transmitted. In the detection beam path, optical components 15 are arranged which focus the supercritical fluorescence through a pinhole 16 and project onto the light-sensitive surface of a detector 17. The pinhole diaphragm serves for spatial filtering and is arranged in such a way that stray light or autofluorescence generated in the collimator is largely blocked and the fluorescence passes from the surface. Wavelength filters 18 are preferably included in the detection beam path.

In one embodiment of the invention, the convex surface 2 has an aspherical shape. The curvature of the surface can be selected such that the excitation light is focused diffraction-limited in the waveguide material. As shown in Figure 3, the focal length of the asphere can be chosen so that the focus is below or above the sensor surface 3. In this way, an excitation disc with a defined diameter can be produced on the interface, preferably with a diameter smaller than 300 micrometers.

In one embodiment of the invention, the diameter of the convex surface 2 is chosen smaller than the cross section of the collimated excitation beam 13 (Figure 4). The part of the excitation beam which hits the collimator outside the convex surface is blocked by the opaque aperture 7 at the collimator. If the excitation beam has a homogeneous intensity profile, ie constant intensity over the entire cross section, then a certain lateral offset between the optical axis of the collimator and the excitation beam has no influence on the excitation profile of the collimator Sensor surface 3. As a result, the fluorescence bound to the surface of the collimator can be reproducibly read without a high-precision lateral adjustment of the collimator. This is particularly advantageous for the fast sequential reading of multiple collimators. The

Sensor surface 3 is preferably excited in a region around the optical axis, preferably this region has a diameter smaller than 300 micrometers. However, small collimators of a few millimeters in diameter require even more accurate centering of the excitation light on the optical axis. By integrating the convex surface 2 into the bottom of the vessel, the excitation light incident on the sensor surface can be precisely centered on the optical axis, even with a lateral displacement of the collimated excitation beam 13 to the optical axis of the collimator of several hundred microns.

In one embodiment of the invention, the diameter of the convex surface 2 is larger than the cross section of the collimated excitation beam (Figure 5). Even with a certain lateral offset of collimator and excitation beam, the light strikes the sensor surface 3 of the collimator very center-centered.

In one embodiment of the invention, a transparent preferably planar substrate made of plastic or glass, for example a microscopy cover glass, is integrated in the bottom of the vessel. This is shown in Figure 6. The substrate 19 may be connected by an optical adhesive 20 with the collimator. The substrate, the optical adhesive and the collimator 1 preferably have similar refractive indices. The arrangement is chosen so that the fluorescence is excited and collected at the top of the substrate, ie the sensor surface 3 lies on the substrate. The use of a piano substrate has the following advantages: First, the microscope cover glass allows fluorescence measurements with very low background. Second, such glasses mass produced and are very cost effective. Third, the substrate prevents contact of the aqueous sample with the lateral surface 4. A gaseous environment 21 of the lateral surface allows loss-free collimation by total internal reflection. Fourth, glass is for the

Immobilization of receptor molecules particularly well suited. In the example shown, the collimator is integrated in a test tube 22.

Another embodiment of the invention is shown in FIG. Here, the liquid container consists only of two components, vessel wall and acting as a vessel bottom collimator. The vessel wall is shaped such that it adjoins the sensor surface 3. In this way, contact of analyte fluid and lateral surface can be prevented. The vessel wall may preferably surround the collimator laterally. As a result, the optical lateral surface 4 is protected against contamination, e.g. in front of fingerprints of the user. It can also be prevented by using an opaque vessel wall that ambient light passes through the lateral surface in the collimator. Advantages of this embodiment are in particular the reduced manufacturing costs and reduced number of adhesive surfaces, which can be causes of quality fluctuations.

In one embodiment of the invention, the planar substrate is the bottom of a microtiter plate through which the fluorescence is detected (Figure 8) The collimators may be individually connected to the bottom of the microtiter plate or on one The collimators can have the lattice spacing of the wells, but can also be arranged more densely, which allows multiple measurements at several points of a well. for example, by a displacement unit 24, which moves the microtiter plate with the collimators perpendicular to the optical axis. Alternatively, the excitation beam can be translated.

Claims

claims

1 . Liquid container comprising a bottom and side walls for holding a liquid,

the soil comprises:

a) a planar sensor surface, which is in contact with the liquid in contact;

b) a light entry surface beneath the sensor surface adapted to focus light on the sensor surface;

c) a light exit surface;

d) a lateral surface, which is suitable to reflect light from the sensor surface, so that it can escape through the light exit surface.

2. A method for qualitative or quantitative analyte determination in a liquid container according to claim 1, wherein excitation light is focused on the light incident surface on the sensor surface, thereby a luminescence marker characterizing the analyte is excited, and then reflected the resulting luminescence on the mantle surface and after exiting through the Light exit surface is detected.

3. Analysis device comprising

a) a holder for a liquid container according to claim 1;

b) a light source which is arranged such that its light on the light entry surface on the Sensor surface of Flussigkeitsbehalters can be focused; such as

c) a detector, which is arranged such that it from the light exit surface of

Flussigkeitsbehalters can detect emerging light.

4. Use of a Flussigkeitsbehalters according to claim 1 for binding assays.

5. Flussigkeitsbehalter according to claim 1, characterized in that the light entry surface is convex.

6. Flussigkeitsbehaiter according to claim 1 and 5, characterized in that the light entry surface is shaped as an aspheric.

7. Flussigkeitsbehalter according to claim 1 and 5, characterized in that the light entry surface is spherically shaped,

8. Flussigkeitsbehalter according to claim 1, characterized in that the light entry surface is cylindrically shaped.

9. Flussigkeitsbehalter according to claim 1, characterized in that the light entry surface is a Fresnel lens.

10. Flussigkeitsbehalter according to claim 1, characterized in that a diffractive structure on the light entry surface is adapted to focus light on the sensor surface.

11. A liquid container according to claim 1, 4-10, characterized in that an opaque aperture is integrated in the bottom, with which the light entry can be laterally limited to the light entry surface.

12. A liquid container according to claim 1, 4-11, which contains recesses on the outside, which are adapted to fix the liquid container in the analysis device.

13. A liquid container according to claim 1, 4-11, which contains on the outside protruding structures, which are adapted to fix the liquid container in the analysis device.

14. A liquid container according to claim 1, wherein the bottom is glued into the container.

15. A liquid container according to claim 1, wherein the bottom is inserted into the container without adhesive.

16. A liquid container according to claim 1, wherein a transparent pianares substrate is integrated in the soil.

17. A liquid container according to claim 1, which is used as a test tube.

18. A liquid container according to claim 1, which is used as a microfluidic chip,

19. A plurality of liquid container according to claim 1, 4-18, which are arranged in a plane.

20. Arrangement of liquid containers according to claim 1, 4- 16, 19, which are the wells of a microtiter plate.

21. A method for determining analyte according to claim 2 in a liquid container according to claims 1, 4-20, wherein the excitation light completely illuminates the light entry surface and the light entry is bounded laterally by the opaque aperture.

22. A method for determining analyte according to claim 2 in a liquid container according to claims 1, 4-20, wherein the excitation light is guided in its entirety on the light entry surface.

23. A method for determining analyte according to claim 2 in a liquid container according to claims 1, 4-20, wherein only above the critical angle emitted by the sensor surface light passes through the light exit surface.

24. Analysis device according to claim 3, wherein a raster device allows the displacement of the liquid container.