Radiant energy – Luminophor irradiation
Reexamination Certificate
2000-11-14
2002-08-20
Hannaher, Constantine (Department: 2878)
Radiant energy
Luminophor irradiation
C250S252100
Reexamination Certificate
active
06437345
ABSTRACT:
This invention relates to an optical sensing unit. More particularly, it relates to a luminescence-based chemical and biochemical optical sensing unit and to the uses thereof.
In human diagnostics, an increasing demand for the detection of extremely low concentrations of biochemically relevant molecules in small sample volumes has triggered research efforts towards more sensitive and selective sensors. Optical sensors are favored because of their chemical stability and ease of fabrication. Bioaffinity sensors based on luminescence excitation schemes combine (bio-)chemical selectivity due to the application of recognition elements specifically binding the analyte molecules, with spatial selectivity originating from evanescent field excitation techniques. Common to the various evanescent field sensing methods that have been developed, interaction with the analyte molecules is restricted to the penetration depth of the evanescent field, thus emphasizing the processes occurring at the sensing surface or within the sensing layer and discriminating processes in the bulk medium.
The combination of fiber-shaped evanescent field sensors with bioaffinity assays, using fluorescent tracer probes for signal generation, has proven capability and is widely used. A detection limit of 7.5×10
−14
M fluorescein-labeled complimentary DNA in a DNA hybridization assay using multimode fibers has been reported. On the other hand, in recent years evanescent field sensors with planar transducer geometries have been adapted for the detection of biomolecules using the principle of effective refractive index changes such as surface plasmon resonance, grating couplers, and interferometers. They are associated with the attractive feature of direct sensing, without the necessity of using any labels. However, the signals of these devices are directly associated with the adsorbed molecular mass which limits the sensitivity of these configurations. Typically, detected concentrations hardly range below 10
−10
M.
To match the goal of extremely low detection limits in demand by gene probe analysis as well as the diagnostics of diseases and infections, it has been proposed to use single-mode metal oxide waveguides as transducers for luminescence-based bioaffinity sensors. This transducer geometry offers advantages due to ease of production of planar chips, sensor handling, increased excitation efficiency of the luminescence labels, and fluid handling of minute sample volumes. The features of a planar evanescent field transducer for a luminescence detection scheme and the design of a sensor system based on such waveguides are described in a paper by D. Neuschäfer et al, entitled “Planar waveguides as efficient transducers for bioaffinity sensors”, Proc. SPIE, Vol. 2836 (1996). The sensor described uses a single-mode planar waveguide consisting of a tantalum pentoxide waveguiding film deposited on a glass substrate. For luminescence detection, in general, a “volume detection” configuration shown in
FIG. 1
is used. In this case, the bottom half-sphere part of the luminescence light, which is excited by the evanescent field and then isotropically emitted, is collected underneath the sensor chip, using a high numerical aperture lens or lens system. Two identical interference filters are used for discrimination of excitation light. Signal detection is performed using either photodiodes in combination with high-gain amplifiers, or a selected photomultiplier in combination with a photon-counting unit. As an alternative, the part of the luminescence signal which is coupled back into the waveguiding film may be collected using a second, outcoupling grating (not shown). This is known as “grating detection”. The angular separation of outcoupled light of different wavelengths offers the additional feature of simultaneous determination of the- transmitted excitation and the emitted luminescence light intensities, It is also possible to combine the two methods in one device to provide simultaneous “volume” and “grating” detection. A detailed comparison of the two methods is discussed by G. L. Duveneck et al, in a paper entitled “A novel generation of luminescence-based biosensors: single-mode planar waveguide sensors”, Proc. SPIE, Vol. 2928 (1996).
In the present application, the term “measurement field” refers to the smallest area of a sensor field capable of discrimination by a photoelectric detector used to detect luminescence. The present invention addresses the need for simultaneous, spatially selective excitation and highly sensitive detection of luminescence signals from an array of measurement fields. Conventional bioaffinity sensors typically rely upon macroscopic imaging of emitted luminescence from a single, large measurement field. A direct transfer of this technique to arrays of measurement fields suffers from inherent optical crosstalk and optical pick-up of background radiation to the extent that the detection limit is often not sufficiently low for many applications. Furthermore, when macroscope optical elements are used to provide a degree of lateral resolution, the distance between the measurement field and the detector array needs to be quite substantial, thereby increasing the overall size of the system. For luminescence detection in extremely small measurement fields and volumes, confocal laser fluorescence microscopy is a very sensitive method. The detection of individual molecules has been demonstrated with excitation areas as small as the diffraction-limited focus of the laser excitation light, i.e. of the order of one wavelength. However, the excitation and detection of a large number of measurement cells in an array requires a lateral translation of the sample with respect to the measuring arrangement to allow sequential measurement of each measurement cell in the array. Accordingly, the time required to receive the signals of a substantial array of measurement fields is prolonged and the relative cost of this type of system itself is expensive due to the size and complexity of the instrument.
According to a first aspect of the present invention, there is provided an optical sensing unit which comprises at least one sample measurement cell, at least one excitation light source acting upon the or each measurement cell to provide one or more sensor fields defining an array of measurement fields, a photoelectric detector array for detecting the intensity of light emitted from the or each measurement cell in response to excitation light, and an array of waveguides or channels for directing light emitted from each measurement field to a respective portion of the photoelectric detector array, characterized in that the array wave-guides or channels have separate beam guiding of the excitation light and emission light for each waveguide or channel.
The present invention addresses the need to improve the ratio of a detected luminescence signal to background “noise” in a luminescence based measurement method. It achieves this by providing a form of beam guiding for light emitted from an array of relatively small measurement fields associated with a number of sensing fields to eliminate optical cross-talk usually associated with conventional macroscopic imaging of adjacent measurement fields. In the preferred examples, an array of waveguides or channels are used with separate beam guiding of the excitation light and emission light for each waveguide or channel.
In one preferred example of the present invention, the one or more sensor fields are provided by the use of a number of planar evanescent field transducers. Preferably, the sensor fields comprise a number of spaced apart optical waveguides, which are preferably arranged in parallel segments. The excitation light is coupled into the array of optical waveguides to establish a number of spatially separated evanescent sensor fields.
Preferably, the or each waveguide is a single-mode metal oxide planar transducer. Where an array of waveguides is provided, they may be integrated on a single substrate. In a preferred embodiment, the spatially
Bruno-Raimondi Alfredo Emilio
Dändliker René
Duveneck Gert Ludwig
Effenhauser Carlo Stefan
Herzig Hans-Peter
Gabor Otilia
Wenderoth , Lind & Ponack, L.L.P.
Zeptosens AG
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