The present invention describes reaction vessels having integrated light collection optics, which may be used for the detection of surface-bound fluorescent molecules. In bioanalytics, a central role is played by solid phase-based assays in which the substances to be detected are increasingly concentrated from the solution by immobilized receptors on the surface. By means of the affinity-based reaction at the surface, the biological substances may be detected in a highly selective way, even in a mixture. Typical receptors are antibodies and DNA-molecules. An example of particularly high relevance is the detection of antigens via highly affine antibodies. Often, the so-called sandwich test is used for a fluorescence-based detection, wherein an intercepting antibody, the receptor, binds the antigen to be detected to the surface. A second fluorescently marked antibody also binds to the antigen and allows the sensitive detection of the complex. The antigen concentration may be quantified by an intensity measurement of the bound fluorescent marker. Due to the high sensitivity that can be achieved, the fluorescence detection is one of the most important detection techniques in biotechnology.
The quantification of small amounts of biological materials places high requirements on the detection technique as regards sensitivity, safety and costs. For the detection of bio markers, which are, for example, generated in conjunction with cancer or cardiovascular diseases, ever lower detection limits are aimed for. For medical applications both robustness and reproducibility of the analysis are of great importance. Moreover, the continuously increasing number of such tests requires measurements involving as little material and time as possible. The signal-to-noise ratio is of central importance for the effectiveness of a sensitive measurement method.
This is particularly true for a fluorescence measurement in which a technological improvement of the ratio of fluorescence intensity and noise results in low detection limits and/or material and time savings. In the detection of binding assays by means of fluorescence methods, the optimisation of the signal-to-noise ratio is done by maximizing the fluorescence collection from the surface-bound molecules while at the same time minimizing the light collection of all sources of interference (noise).
The binding reaction is usually performed at an interface between an aqueous solution and a transparent measurement substrate made of glass or plastic material, which is coated with receptor molecules. An important source of noise is the fluorescence signal of freely diffusing molecules in the assay solution, which may overlap the fluorescence signal of the surface-bound molecules and, thus, renders the determination of the concentration more difficult. This may be avoided by steps of washing which involve time and costs, though. In order to enable a real-time measurement of the binding reaction and to avoid elaborate rinsing (washing) steps, the surface-selective fluorescence detection offers a decisive advantage. Here, it is essential to restrict as far as possible the detection volume to the surface so as to exclude from detection the fluorescence arising from unbound molecules.
The emission of fluorescence requires optical excitation with light of a suitable wavelength. The excitation light induces scattering and also fluorescence in the sample and the substrate. In particular, problems arise from that portion of the light which spectrally overlaps the emission of the fluorescent dye to be detected and which cannot be blocked by means of a wavelength filter. As a significant part of this light is generated in the measurement substrate, suppression of the contribution is performed by reducing the detection volume in the substrate material. This may be effected by a spatial filtering of the fluorescence signal. Here, the circumstance is exploited that fluorescence and scattered light are generated in a spatially separate manner and, thus, may be separated by the optical system. By means of the different optical paths of fluorescence and scattering, the latter may be strongly suppressed in a geometrical way, for example by means of an aperture. In summary, simple design goals result for the detection volume of the fluorescence measurements: firstly, the fluorescence detection of the optical system at the interface should be as high as possible. Secondly, the light collection both within the aqueous sample and within the substrate should be as low as possible.
The closeness of the interface of two dielectric materials, such as between water (refractive index n1≈1.33) and glass (n2≈1.52) has a significant influence upon the properties of the fluorescence emission. In contrast to the fluorescence irradiation within a homogenous medium, the emission of the surface is not isotropic but features a strong maximum in the direction of the critical angle of total reflection αc, wherein
αc=arcsin(n1/n2).
For the water/glass interface αc≈61°. Fluorescent molecules which are bound to the surface irradiate about 74% of the light into the glass. 34% of the total emission is effected above the critical angle αc. The fluorescence emission above the critical angle (supercritical angle fluorescence) is of paramount importance for binding assays. It occurs exclusively by molecules which are located directly in front of the interface, that is in a distance to the substrate that is significantly smaller than the emission wavelength. Consequently, a fluorescence collection restricted exclusively to a region above the critical angle allows a surface selective detection. The contribution of unbound fluorescent dies may, thus, be suppressed almost completely, which allows real-time measurement of binding reactions, for example.
The conventional method of a surface selective fluorescence measurement is performed by means of a so-called evanescent excitation of the interface. Here, the excitation light is incident onto the interface above the critical angle and is totally internally reflected within the measurement substrate. Thus, on the sample side of the interface a thin excitation layer is generated, by which the surface-bound molecules may selectively be excited to fluoresce. This method is, however, technically elaborate and hinders miniaturization.
Ruckstuhl and Seeger describe a method to collect fluorescence emission above the critical angle in a very efficient manner (PCT/EP099/1548). An optical waveguide made of glass or plastic comprises a shell surface (boundary surface) which collimates light by internal reflection. The use of a shell surface having a parabolic shape collimates the fluorescence into a parallel beam (bundle) of rays and facilitates further processing of the signal. The collimated fluorescence may be focussed by an aperture serving as a spatial filter. The aperture reduces the detection volume within the substrate and filters the scattered light/autofluorescence induced by the exciting light therein. Thus, the collimator achieves a very high signal-to-noise ratio and even allows the detection of individual molecules. Even the fluorescence is collected exclusively above the critical angle, the collection efficiency of the collimator is more than 30%, which is significantly higher than the values of common detection systems. Fluorescence sensors based upon fiber optics achieve collection efficiencies around 1%. Refraction-based single lenses may be produced up to a numerical aperture (N.A.) of about 0.6 and at least achieve efficiencies around 5%. Conventional lenses or lens systems, however, predominantly collect fluorescence below the critical angle and, thus, do not allow for a surface selective fluorescence collection.
In the field of bioanalytics, small standardized reaction vessels are also used such as test tubes or cuvettes for single measurements and microtiter plates for measurements with higher throughput. In the microtiter plates a plurality of reaction vessels, so-called wells, are arranged in a grid on a surface of 7×11 cm2, which by default allows for 9, 384 or 1526 independent measurements. The wells are usually read out sequentially, requiring a fast displacement of the plate between the measurements. By integrating the collimator for supercritical fluorescence in the micro titer plates, the signal yield may be significantly improved with respect to conventional plates. Moreover, real-time measurements of binding reactions with high throughput are rendered possible. However, to that end the collimator has to be miniaturized down to a few millimetres in diameter.
The excellent light collection capability of the collimator is restricted to a limited region around the optical axis. With increasing distance of the fluorescence emission from the optical axis, quality of light bundling and collection efficiency are deteriorated. This is analogous to fluorescence microscopy in which optics (lenses) of a high numeric aperture achieve a high collection efficiency and sensitivity, but only within a relatively small region in the object space. In order to fully exploit the performance of the collimator, the exciting light has to be bundled on the surface around the optical axis of the collimator. The size of the useful area and the precision with which the exciting light has to be centered onto the optical axis depend upon the size of the collimator. A miniaturization of the collimator leads to a reduction of the useful surface and, thus, increases precision requirements. This increases the requirements and costs of the motorized displacement means, leads to an increased time requirement and may have a negative influence on the robustness and reproducibility of the measurements.
The present invention concerns methods of fluorescence collection above the critical angle in cost-effective liquid containers, such as test tubes and microtiter plates made of plastic. This is achieved by means of a novel collimator and by integrating the element into the receptacle bottom (floor). An improvement is constituted in particular be the integration of the focussing optics for the excitation light into the base of the receptacle. By means of a convex surface arranged below the interface between analyte and receptacle bottom the exciting light may be focussed onto the interface in vicinity of the optical axis of the collimator. This results in a significant increase in the allowed tolerances when centering the exciting light onto the optical axis of the collimator. It results, inter alia, in the advantage that in a sequential readout of several reactions the displacement of the receptacles above the detection system may be smaller and may be effected with a lower precision without negative effects on the sensitivity or the reproducibility of the measurements. The manual or automatized change of the receptacles in between two measurements may, thereby occur faster and/or be realized with cheaper components.
In an embodiment of the invention, the convex face 2 has an aspherical shape. The curvature of the surface may be chosen so that the excitation light is diffraction-limitedly focussed within the waveguide material. As shown in
In an embodiment of the invention, the diameter of the convex face 2 is chosen smaller than the cross section of the collimated excitation beam 13 (
In an embodiment of the invention, the diameter of the convex face 2 is larger than the cross section of the collimated excitation beam (
In an embodiment of the invention, a transparent, preferably flat (planar) substrate made of plastic or glass, for instance a microscopy cover glass, is integrated into the receptacle bottom. This is shown in
A further embodiment of the invention is shown in
In an embodiment of the invention, the flat substrate is the bottom of a microliter plate through which the fluorescence is detected (
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP08/58302 | 6/27/2008 | WO | 00 | 1/25/2010 |