Radiant energy – Luminophor irradiation
Reexamination Certificate
1999-05-03
2001-08-14
Hannaher, Constantine (Department: 2878)
Radiant energy
Luminophor irradiation
C250S459100
Reexamination Certificate
active
06274872
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a device for carrying out, in particular, quantitative fluorescence-marked affinity tests by means of evanescent field excitation. This may involve a wide variety of known biochemical assays of general receptor/ligand systems, such as antibody/antigen, lectin/carbohydrate, DNA or RNA/complementary nucleic acid, DNA or RNA/protein, hormone receptor, enzyme/enzyme cofactors, [sic] protein or protein A/immunoglobin [sic] or avidin/biotin. Preferably however, antibody/antigen systems are evaluated.
2. Description of the Prior Art
Fluorescence immunotests, or fluorescence immunosensors, use an antibody/antigen system and have long been used widely. They are used primarily to quantify an unknown amount of a particular chemical or biochemical substance in a liquid sample matrix. In this context, antibodies are bound selectively to the substance to be determined. The substance to be determined is referred to by the person skilled in the art as an antigen. In fluorescence immunotests, the analyte-specific antibodies are marked with a marking substance which is optically excited at a particular substance-specific wavelength &lgr;
ex
and the fluorescent light with a different wavelength, which is generally longer, is used with a suitable detector with evaluation of the fluorescent light intensity. The use of evanescent field excitation when implementing such fluorescence immunotests, or respectively the fluorescence immunosensors, already belongs to the prior art. For example, a variety of solutions have already been described in WO 94/27137, by R. A. Badlay, R. A. L. Drake, I. A. Shanks, F. R. S., A. M. Smith and P. R. Stephenson in “Optical biosensors for immunoassays: fluorescence capillary-fill device”, Phil. Trans. R. soc. Lund. B 316, 143 to 160 (1987) and D. Christensen, S. Dyer, D. Flowers and J. Herron, “Analysis of Exitation [sic] and Collection Geometries for Planar Waveguide Immunosensors”, Proc. SPIE-Int. Soc. Opt. Eng. Vol. 1986, Fiber Optic Sensors in Medical Diagnostics, 2 to 8 (1993). However, the known solutions generally have the disadvantage that they require relatively great outlay in order for the light needed to generate the fluorescence to be coupled into an optical fiber or for the fluorescent light to be extracted, which form an essential part of hitherto customarily used devices.
Further, U.S. Pat. No. 3,939,350 describes a solution in which fluorescence immunoassays are carried out by means of evanescent field excitation.
In this case, light from a light source is directed at an angle through a prism onto an interface, so that total reflection takes place and the fluorescence caused in a sample can be measured with a detector. The entire sample volume is in this case accommodated in a sealed closed space, so that on account of the relatively large sample volume only diffusion-controlled end-point detection can be carried out and this is susceptible to error.
WO 90/05295 describes an optical biosensor system in which [lacuna] an elaborate optical system excitation light can be directed onto sensitive regions of a likewise elaborate channel system, through which the sample volume is fed by means of controlled valves and pumps, and the fluorescent light emerging through windows from the sensitive regions can be re-directed onto a detector with a view to intensity measurement. Besides the aforementioned disadvantageous complex and elaborate structure, this system requires, before and after a test is carried out, cleaning both of the pumps and of the entire channel system in order to preclude the possibility of subsequent measurement errors.
WO 90/06503 describes a sensor in which excitation light is directed at a suitable angle through an optically transparent substrate onto an interface to form an optically transparent buffer layer, over which an extra waveguide layer, to which the analytes to be determined are in turn bound, is applied.
The refractive index of the buffer layer is in this case lower than that of the substrate and of the waveguide. If a suitable choice is made for the angle of the excitation light, total reflection takes place at the substrate/buffer boundary layer [sic] and, by means of the resulting evanescent field, the excitation light is coupled into the waveguide lying over the buffer layer. The light coupled into the waveguide is guided by means of total reflection in the waveguide, and the resulting evanescent field is correspondingly employed for fluorescence excitation.
The sample may be accommodated in one or more cavities, the only restriction on the corresponding dimensioning of such a cavity being that its size permits the sample to be transported into the cavities by means of capillary force. After the sample has been taken in by the cavities, no further flow or movement of the sample takes place.
WO 89/09408 A1 discloses a similar solution, which once more uses the light source for the excitation light and the detector for the fluorescent light on the same side. The sample to be detected is accommodated in a cavity between a waveguide and a cover plate. Here [sic] again no further flow or movement of the sample takes place after the sample has been taken up.
SUMMARY OF THE INVENTION
The object of the invention is therefore to provide the possibility of carrying out quantitative fluorescence-marked affinity tests, with a variety of known biochemical assays, with a very simply constructed device.
According to the invention, this object is achieved by the features in the characterizing part of claim
1
for the device, and the features of claim
25
for the method. Advantageous refinements and developments of the invention result from use of the features contained in the dependent claims.
With the device designed in accordance with the invention, it is possible to carry out fluorescence immunotests in a variety of procedures (assays) and further quantitative fluorescence-marked affinity tests. This provides the opportunity, on the one hand, for carrying out competitive assays, and sandwich assays as well as other known assay forms may further be used.
The procedure adopted when working with the device according to the invention is similar to that already known in the prior art. In this context, a fluorophore is used as marking substance and analyte-specific antibodies are marked using it. The bound fluorophore [lacuna] excited with evanescent field excitation and the fluorescent intensity which this has caused makes it possible to quantify the marked antibodies, and it thus becomes possible to quantify the analyte as well.
In the device according to the invention, light from a light source is directed at an angle &agr; onto the interface between two media with different refractive indices. In this context, a light source is selected which emits almost monochromatic light having a wavelength which is suitable for exciting the marking substance, in this case the fluorophore. Suitable light sources for this include, in particular, laser diodes since they have a suitable beam profile and sufficient optical power, together with a small overall size and low energy consumption.
Other light sources which emit monochromatic light may, however, also be employed.
In this case, care should then be taken that all the optically transparent objects are each transparent to the wavelengths which are used.
The angle &agr; at which the emitted light is delivered to the interface determines, besides the refractive index of the material arranged in the optical path in front of the interface and the material which follows, together with the wavelength of the light, the penetration depth d for the evanescent field. In this case, the refractive index n
1
of the material which is arranged in the optical path in front of the interface must permit total reflection at the interface, and should therefore be greater than the refractive index n
2
of the other material arranged thereafter, at the wavelengths of the
Gabor Otilia
Hannaher Constantine
ICB Institut fuer Chemo- und Biosensorik Menster E.V.
Marshall & Melhorn LLC
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