1. Field of the Invention
The invention relates to a substrate having a coating, in particular a dielectric coating, which substantially amplifies the fluorescent excitation of a material lying on this coated substrate. The invention also relates in particular to a sample carrier which is provided with a coating and amplifies the fluorescent excitation of a sample and therefore the fluorescent light signal to be detected, and which substantially improves the signal-to-noise ratio during the fluorescence measurement.
2. Description of Related Art
Fluorescence microscopy is a conventional technology, which is employed particularly in the field of molecular biology. It is used to analyze biological materials, for example nucleic acids (DNA, RNA) and proteins (enzymes etc.). The substances to be studied are labeled with fluorescent dyes and thus made visible in a fluorescence scanner or fluorescence microscope.
Particularly in the field of biochip technology, fluorescence spectroscopy is very important for the analysis of so-called microarrays. The DNA array technique is based, for example, on the hybridization of nucleic acids (Watson Crick base pairing). In this case, two complementary nucleic acid single strands combine via a hydrogen bridging bonds between their hydrophobic purine and pyrimidine bases, in order to minimize the water contact. It was Ed Southern who first applied the principle of using labeled nucleic acids to analyze other nucleic acids (Southern Blot).
Biochips typically consist of an organic film applied to a glass substrate, on which single-stranded DNA material has been applied in the form of points (about 100 to 150 μm diameter). Successful binding of the single-stranded DNA to be analyzed, which is provided with a fluorescent dye such as cyanine dyes, including Cy3 dyes and Cy5 dyes, to the single-stranded DNA applied in the form of points on the organic film can be detected by a fluorescent DNA dot. Instead of complete DNA, it is also possible to analyze short-chained molecules (oligos).
A high detection sensitivity is in any event a necessary prerequisite for the successful use of biochips as a responsive sensor system. This detection sensitivity is essentially determined by the strength of the signal to be detected and by the signal-to-noise ratio. Sources of perturbing background signals are, for example, autofluorescence of the substrate or impurities, or of the optical components lying in the excitation light, as well as intrinsic noise in the detector. The autofluorescence of the substrate or optical components can be substantially restricted by using non-fluorescent or low-fluorescence materials. Other possibilities for the elimination of perturbing noise signals during the measurement are offered by pulsed or modulated excitation.
There are furthermore solution approaches which relate to improving the substrates or sample carriers. For example, U.S. Pat. No. 5,552,272 proposes to provide a substrate with an antireflection layer which suppresses reflection of the excitation light with a particular wavelength, and therefore reduces the noise signal. According to U.S. Pat. No. 5,552,272, this antireflection layer has no effect on the generation of the fluorescent signal, and it is possible to achieve a stronger contrast between the signal and the noise. In this case, the maximum achievable signal-to-noise ratio SNRmax is limited to SNRmax=S0.5, where S is the strength of the fluorescent signal.
With a view to further improving the fluorescence excitation and the fluorescent emission detection, and therefore a higher measurement sensitivity, WO 98/53304 discloses a sample carrier consisting of a substrate with a reflective metal surface and a transparent dielectric layer on top. The transparent dielectric layer has a thickness such that the optical path length from the layer surface to the reflective surface of the substrate corresponds to an odd multiple of λ/4, λ being the wavelength of the excitation light. The thickness of the transparent layer is therefore determined by the wavelength of the excitation light, the refractive index n of the layer material and the angle of incidence of the beam. With such dimensioning of the dielectric layer and arrangement on a reflective metal surface, a maximum in the electric field amplitude due to the constructive superposition of the incident and reflected excitation light, and the resultant standing wave of the excitation light and the reflected excitation light of wavelength λ, is formed at the layer surface of the dielectric layer.
The reflective metal surface is obtained by the substrate itself being a metal, or being coated with a metal, and it is primarily used for reflecting the excitation light. Such reflective metals are preferably aluminum, silver, gold or rhodium, which are suitable for a wide spectrum of the excitation light. The transparent dielectric layer, on which the sample directly lies, acts as a kind of spacer layer from the reflective metal surface and makes it possible to position the sample in the region of a maximum of the electric field of the excitation beam. The sample can therefore be arranged in the maximum intensity region of the excitation and the fluorescence can be increased. The use of this sample carrier, however, is on the one hand restricted to a particular excitation wavelength (defined by the thickness of the spacer layer) and on the other hand limited by the susceptibility of the metal layers to oxidation.
U.S. Pat. No. 6,552,794 B2 discloses an optical detection method with improved measurement sensitivity having a substrate, provided with a reflective layer, as the sample carrier which reflects the fluorescent light emitted in the direction of the substrate by the sample, and having an optimized optical detection arrangement which can record both the fluorescent light emitted in its direction and the emitted fluorescent light reflected by the layer. With ideal reflection of the emitted fluorescent radiation, the measurement accuracy can then be improved by the maximum factor of 2. A substantial disadvantage of this arrangement is that it does not prevent the substrate from being excited and fluorescing as well, and thereby detrimentally affecting the measurement. The theoretical increase in the fluorescence is furthermore limited to a factor of 2.
The requirements for fluorescence detection have continued to increase, especially in the field of biochips. In most cases, it is not enough merely to establish that there is fluorescence, but it is also necessary to determine the intensity very accurately for comparative analysis with other biochips. The more accurate the measurements are, the more accurate the analysis can be.