Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
2001-02-21
2004-06-22
Snay, Jeffrey R. (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C356S445000
Reexamination Certificate
active
06752963
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to providing a SPR sensor array capable of simultaneously sensing a plurality of samples, to methods for its production, measurement assemblies as well as to adjustment and measurement methods for parallel readout of the sensor system and to use thereof in the search for active substances and in high-throughput screening.
BACKGROUND OF THE INVENTION
One modern approach in the search for active substances involves generating a large number of diverse chemical compounds by means of automated synthesizers, this plurality of diverse structures then being tested for binding to interaction partners often represented by biomacromolecules such as proteins. One automated method which assays a large number of samples in this way is also termed high-throughput screening.
Due to the biological dispersion of the results in studying the bindings it is particularly important to achieve exactly the same conditions for all compounds in the binding test. This is why the test ideally should be implemented for all samples simultaneously, where possible, and with the same solution of the interaction partner under test to eliminate the effects of ageing and temperature drift as well as differences in the binding times for the compounds. Due to complexities involved in purifying biomacromolecules the quantities needed for the test should be kept to a minimum.
One particularly effective method of implementing the binding test is surface plasmon resonance (SPR) spectroscopy. Unlike fluorescence and chemiluminescence methods no dye-marked samples and also no antibodies are needed in SPR for the protein to be tested. In SPR an interaction partner (e.g. ligand) is immobilized on a metal surface and its binding to another interaction partner (e.g. receptor) demonstrated. For this purpose an optical substrate (usually a prism) is coated with gold and the drop in internal reflectivity in the prism detected as a function of the set angle or as a function of the wavelength (Kretschmann configuration). What is demonstrated ultimately is a change in the refractive index of the medium at the side opposite the gold film which occurs when molecules bind to the surface.
FIG. 1
a
shows diagrammatically the so-called Kretschmann geometry which is often used to measure the SPR effect. In this case a thin gold film
125
applied to a prism
20
is brought into wetting contact with the solution
160
to be assayed. What is usually measured is the internal reflectivity at glass/gold/fluid interfaces either as a function of the angle of incidence ∂ or as a function of the wavelength &lgr;. At a suitable resonance condition the reflectivity is greatly reduced, the energy of the light then being converted into electron charge density waves (plasmons) along the gold/fluid interface. The condition for resonance approximates (from Chapter 4, “Surface Plasmon Resonance” in G. Ramsay, Commercial Biosensors, John Wiley & Sons (1998) ) to:
2
π
λ
n
prism
sin
ϑ
≈
2
π
λ
n
metal
2
(
λ
)
n
sample
2
n
metal
2
(
λ
)
+
n
sample
2
where n
prism
is the refractive index of the prism, n
metal
the complex refractive index of the metal coating and n
sample
that of the sample. ∂ and &lgr; stand for the angle of incidence and wavelength of the incident light, respectively. The wavelength spectra (
FIG. 1
b
) respectively the angle spectra (
FIG. 1
c
) exhibit a reduction in reflectivity in the wavelength range or in the angle range respectively in which the resonance condition as cited above is satisfied. Changing the refractive index in the solution n
sample
alters the resonance condition, as a result of which the resonance curves are shifted by a value which for small changes in the refractive index is linear to this change (a calibration being made, where necessary, for larger changes). Since the reflected light penetrates into the fluid only by a few 100 nm the change in the refractive index is measured locally in this range. When the target molecules (e.g. proteins)
162
in the solution bind to suitable interaction partners
161
immobilized on the surface (i.e. association and dissociation forming an equilibrium) the local concentration of the target molecule at the surface increases which can then be demonstrated as a change in the refractive index.
WO 99/60382 describes an SPR sensor capable of simultaneously sensing a plurality of samples. A measurement assembly for reading out such a SPR sensor system in parallel is disclosed in WO 00/31515. This proposes an apparatus for implementing SPR measurements on a plurality of samples in parallel which is based on the principle of wavelength measurement, but does not use a prism, but an array of sensor fingers capable of carrying another substance on each sensor finger. This array can be coated in a microtiter plate (MTP) and measured, i.e. each sensor finger is able to measure another solution. The contrast between the sensor fields and the intermediate regions is dictated by the geometry of the waveguides. In this case light passes through the array only at the regions at which a sensor field is applied, resulting in a high contrast. The disadvantage is the expense in producing the sensor fingers and their sensitiveness to physical contact as well as the relatively high sample consumption in coating.
WO 98/34098 shows sample fields on an SPR-compatible gold film applied to a prism. The contrast is determined by setting suitable resonance conditions. The disadvantage here is that the surfaces need to be very homogenous since it is only the region of the sensor surface area that shows a contrast in imaging under SPR conditions that exhibits the same layer thicknesses.
Another SPR imaging system is described in B. P. Nelson et al., Anal. Chemical 1999, 71, pages 3928-3934. In this case a uniform gold surface applied to a non-structured glass plate is patterned with an array of 500×500 &mgr;m large squares covered with DNA, the DNA squares being separated by squares covered with alkanethiol intended to prevent adsorption of the protein outside of the DNA squares. The DNA squares are then brought into contact with a protein sample and an image of the gold surface produced at the SPR angle on an CCD chip before and after contact is made. Here, distinguishing the DNA squares from the other regions depends on the molecular weight of the immobilized chemical or biological molecules, the contrast sinking with a reduction in the molecular weight. Also a disadvantage in this system is the relatively large pixel region to which a DNA square needs to be assigned on the CCD camera to ensure adequate contrast. These requirements conflict with the need for a miniaturized SPR sensor array for universal application.
Described in WO 90/05305 is a replaceable sensor unit for use in an optical biosensor system (WO 90/05295) in which the geometry and arrangement of the sample fields on the non-structured sensor unit is not dictated by the latter. Assigning the sample fields on the sensor unit is done by bringing it into contact with a block unit for handling the fluids, e.g. the throughflow system as disclosed in WO 90/05295, the throughflow system defining the arrangement of the sensor surfaces one-dimensionally (one-dimensional array). The disadvantage in this case is that making use of a throughflow system makes it difficult to use and miniaturize a two-dimensional sample array (two-dimensional array).
OBJECT OF THE PRESENT INVENTION
The present invention is based on the object of providing an improved SPR sensor array.
SUMMARY OF THE PRESENT INVENTION
This object is achieved by the characterizing features of the claim
1
and the subject matter of parallel claims respectively. Advantageous aspects are the subject matter of the dependent claims.
In accordance with the invention separating means or separators are provided for structuring the SPR sensor array so that a two-dimensional sample array is made possible. A
Burkert Klaus
Dickopf Stefan
Schmidt Kristina
Vetter Dirk
Berkowitz Marvin C.
Graffinity Pharmaceutical Design GmbH
Nath Gary M.
Nath & Associates PLLC
Snay Jeffrey R.
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