Optics: measuring and testing – Of light reflection
Reexamination Certificate
2002-08-19
2004-09-21
Stafira, Michael P. (Department: 2877)
Optics: measuring and testing
Of light reflection
Reexamination Certificate
active
06795192
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to providing SPR sensors capable of assaying a plurality of samples simultaneously, methods for their production, measuring assemblies for scanning the sensors in accordance with the invention in parallel as well as to their use in the search for active agents and in high-throughput screening (HTS).
BACKGROUND OF THE INVENTION
Continuing progress in automating the search for active agents has resulted in the question of miniaturization and parallelization gaining ever-increasing interest. Miniaturizing sample containers and the apparatus used in synthesis causes a plurality of substances to be assayed in ever-decreasing volume. This is why it is necessary in achieving novel detector and sensor systems to configure them so that several measurements can be run simultaneously in parallel, or a large number of samples assayed in sequence in shortest time whilst minimizing the volume of the substance needed therefor (high-throughput screening). Salient to this is enhancing the degree of automation.
There is furthermore a need to provide also the sensors used for analysis in a parallel and miniaturized format so that assaying a plurality of samples in shortest time and with minimum volume and consumption is achievable in thereby boosting the throughput of the substances to be characterized.
One highly sensitive measurement method for boundary layer characterization is known which is termed surface plasmon resonance (SPR) spectroscopy in pertinent literature. This method is based on optical excitation of surface plasmons along the boundary layer of films of metal.
It is usual in this arrangement to detect the light reflected by a thin gold film. When the condition for resonance is suitable (angle of incidence and wavelength of the light and film thickness of the gold film) the intensity of the reflected light decreases. In absorption of the light, excitation of the charge density waves of the electron gas occurs at the gold surface. These charge density waves are called plasma oscillations, their quantized excitation states plasmons.
To observe the resonance there are two methodic approaches. Either use is made of monochromatic light and the intensity of the reflected light is recorded as a function of the angle of incidence, or the angle of incidence is maintained constant and the wavelength of the light is varied. In both cases there is a shift in the location of resonance when the refractive index of the medium changes on the side of the gold film facing away from the incident light.
These methods are described in prior art as detailed, among others, by Striebel, Ch; Brecht, A; Gauglitz, G in Biosensors & Bioelectronics 9 (1994), 139-146. The resonance conditions for surface plasmon excitation greatly depend on the optical properties of the dielectric surrounding the metal film. Determining the refractive index or film thickness of thin dielectric films is basically possible with high accuracy by known prior art.
SPR spectroscopy is finding increasing application e.g. in biochemical analysis by making it possible to assay the interaction between partners (for example in the biomolecular reactions of antibodies and antigens) directly and without marking. For this purpose, an interaction partner (e.g. ligand) is immobilized on the surface of the metal, the other interaction partner (e.g. analyt) directed in solution over the surface. The interaction can be directly verified as the increase in film thickness via the change in the refractive index.
One task often involved in the miniaturization and parallel measurement of many samples is bringing the sensor sites into contact with fluid without, for example, cross-contamination occurring.
In the search for active agents conventional SPR sensors employ a prism coated with a thin film of metal. The sample to be assayed is brought into contact with the metal or the modified metal surface, and the SPR reflection spectrum of the sample is measured by coupling light into the prism and measuring the intensity of the reflected light as a function of the angle of incidence (cf. Chapter 8, “Small Molecule Drug Screening based on Surface Plasmon Resonance” in
Advances in Drug Discovery Techniques
, John Wiley & Sons Ltd, London 1998).
A parallel approach to analyzing a sample array is SPR microscopy (SPM) (see e.g. EP 388 874 A2 or M. Zizlsperger, W. Knoll, Progr. Colloid Polym. Sci. 1998, 109, pp. 244-253) involving coating various sites on various samples of a gold surface applied to a prism and obtaining an image of the gold surface at the SPR angle on a CCD chip. During the measurement the angle is altered by a mechanical scanner. This method is, however, restricted to objects of small diameter.
A more recent SPR method is disclosed in WO 94/16312 A1 in which detecting the binding of small amounts of substance is achieved by optical fibers partially coated with a gold film. However, here too, the problem still exists in designing a sensor array required to assay a plurality of samples in parallel in accordance with this principle. Such an array of gold-coated fibers is, on the one hand, expensive and highly sensitive to mechanical stress, and, on the other, producing the array in parallel as proposed therein is difficult to achieve technically.
Optical fibers are also employed as it reads from WO 98/32002 A1. To protect them from being damaged physically the fiber cable is housed in a pipette. To achieve an array it is proposed to use a series arrangement of such pipettes. However, miniaturizing such an arrangement is difficult to achieve, especially for parallel measurement of many different samples.
WO 97/15819 A1 describes a dual-channel sensor comprising an objective mount as used in microscopy. Supplying fluid to the gold sensor site is achieved with the aid of a flow-through cuvette. Such a configuration is complicated especially in miniaturized HTS.
Known from U.S. Pat. No. 5,485,277 is a SPR sensor array using a waveguide configured so that multiple reflection occurs therein in thus simplifying signal analysis. Several spatial channels, e.g. a sensor channel and a reference channel, are employed in the measurement. There is no mention, however, of how the separate channels are generated.
Known from DE-196 15 366 A1 is a method and a means for simultaneously assaying a plurality of samples e.g. in a matrix arrangement. Separating the samples is done by applying the samples spatially separated.
WO 99/41594 describes a SPR system in which for a better time resolution the material properties of sites bordering a full-size SPR-sensitizing coating are modified to permit determining the intensity of the radiation reflected by the surface resolved in time or space.
WO 90/05295 describes a SPR system comprising a plurality of sensor surfaces. The SPR sensor system consists of a plate of glass coated with a film of metal, covered in turn by a dielectric film. The ligands making measurement possible are applied to the dielectric film. Separate sensor sites are generated by the separate application of the ligands.
A further possibility of simultaneously assaying a plurality of samples is described in WO 99/60382 A1 from which the preamble of the present claim
1
is known. In the arrangement as described several strip-type lightguides are arranged at a defined spacing on a planar backing and provided with a thin film of metal to permit excitation of such plasmons, whereby means are provided which separate the sensitized sites of the individual thin metal films by interruption thereof such that each lightguide can be assigned one sample only.
The lightguide described in WO 99/60382 A1 is fixed to a backing plate. This necessitates very high precision in production. The light-guiding film is deposited in a separate coating step. There is a risk of inhomogeneities materializing from one sensor to the other. Should solvent gain access to the interlayer between backing and light-guiding film problems may be encountered in the bonding of the deposited film.
OBJECTIVE OF THE INVENTION
The present i
Dickopf Stefan
Schmidt Kristina
Vetter Dirk
Berkowitz Marvin C.
Graffinity Pharmaceutical Design GmbH
Nath & Associates PLLC
Richmond Derek
Stafira Michael P.
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