Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1998-04-13
2001-02-27
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C356S318000, C356S445000, C422S082050, C422S082080, C422S082110, C435S287100, C435S287200, C435S288700, C435S808000, C436S172000, C436S524000, C436S525000, C436S527000, C436S805000
Reexamination Certificate
active
06194223
ABSTRACT:
The invention concerns a method for the detection of an analyte wherein a combination of plasmon resonance and fluorescence measurement is carried out for the detection and it also concerns a device which is suitable for carrying out this method.
Surface plasmon spectroscopy is used to determine optical layer thicknesses of adsorbed substances on solid phases. No label is required for this detection method and it is possible to carry out so-called real time measurements which enable the determination of the kinetics of reactions of biomolecular interactions and adsorption processes. The disadvantages of surface plasmon resonance are the low sensitivity with a detection limit of about 10
−10
mol/l as well as difficulties and limitations in measuring relatively small molecules.
Fluorescence detection using fluorescent labels is used for example in biochemistry and also in diagnostics. The excitation of fluorescence on surfaces can for example be achieved by an external light source and also by so-called evanescent light. Such excitation of fluorescence by evanescent light takes place for example in the e-wave concept in which the light conducted in a light conductor excites the labels immobilized on the surface of this light conductor to fluoresce (W. F. Love et al., Biosensors and Bioelectronics 7 (1992), 38-48). It is possible to use plasmon light as evanescent light to excite fluorescent labels bound on the surface. The intensity of the evanescent light is several-fold higher than the intensity of the irradiated light so that a considerable increase in the sensitivity of the fluorescence detection can be achieved. Hence the field amplifying effect of surface plasmon resonance is used to increase the fluorescence signal as described for example by Kitson et al., Journal of Modern Optics, vol. 43 (3), (1996), 573-582 and EP-0 353 937. The methods described in these publications are sensitive variants of fluorescence detection which, however, have the disadvantage that kinetic measurements are not possible.
Therefore one object of the present invention was to provide a method which enables an analyte to be detected with high sensitivity and simultaneously allows the determination of reaction kinetics.
This object is achieved according to the invention by a method for the detection of an analyte which is characterized in that the binding of the analyte to a solid phase is determined by independent analysis of the signals from a plasmon resonance and a fluorescence measurement. Surprisingly the method according to the invention enables real time measurements to be carried out while concomitantly achieving a higher sensitivity. According to the invention two independent measurement signals are obtained by the combination of the two methods plasmon resonance detection and fluorescence detection.
An important advantage of the method according to the invention is that plasmon resonance in which the layer thickness is determined also enables the detection of unspecific interactions of the sample containing the analyte with the solid phase whereas fluorescence detection only detects the specific interaction of the analyte with the solid phase. This enables the amount of specific and unspecific binding in a sample to be determined and enables differentiation between both types of binding. Moreover kinetic measurements are additionally possible with plasmon resonance e.g. adsorption can be observed over time whereas end point measurements can be carried out with the fluorescence detection. In this connection a sensitivity of 10
−13
to 10
−14
mol/l is achieved with the fluorescence detection whereas a sensitivity of about 10
−10
mol/l is achieved with the plasmon resonance.
Field amplification by resonant coupling in of the incident light at the reflectivity minimum of the plasmon resonance enables an additional improvement of the sensitivity of the fluorescence detection. This field amplification can be used to excite adsorbed dye molecules as a result of which a surface sensitive detection method is obtained since the evanescent character of the field strength causes a dominant excitation of fluorophores adsorbed to the surface. Since this excitation occurs specifically only for dye molecules bound to the surface it is not necessary to separate dye that is not bound to the surface from bound dye.
The method according to the invention enables the examination of for example biomolecular interactions, reaction kinetics and adsorption processes and also enables analytes to be determined qualitatively or/and quantitatively.
The method according to the invention can be carried out utilizing the intrinsic fluorescence of a reaction partner participating in the detection reaction of an analyte. However, at least one fluorescently labelled reagent is used for the detection.
An optically transparent support which is coated with a metal or metal/metal oxide layer is preferably used as the solid phase for the method according to the invention. The optically transparent support, for example made of glass or quartz glass, can for this purpose be for example vapour-coated with a relatively thin metal or metal/metal oxide layer. The metal/metal oxide layer is in this case preferably composed of a metal layer which is firstly vapour-deposited on the optically transparent support and a metal oxide layer which is subsequently applied onto the metal layer. The thickness of the coating is preferably between 10 nm and 1 &mgr;m particularly preferably between 30 nm and 100 nm. If the support is vapour-coated with a metal layer one preferably uses noble metals and particularly preferably gold or silver. Preferred metal oxide layers include SiO
2
, TiO
2
, Al
2
O
3
and Ag
2
O.
A solid phase binding matrix via which the analyte is linked to the solid phase is preferably located on the metal or metal oxide layer. A solid phase binding matrix comprises a solid phase reactant which can specifically interact with a binding partner e.g. an analyte to be determined. The solid phase reactant can be covalently or adsorptively linked to the surface via anchor groups, optionally via spacer molecules. The linkage is particularly preferably achieved by means of a self-assembled monolayer (SAM) in which thiol or disulfide groups are adsorptively bound to a metal surface. Such self-assembled monolayers are described for example in DE 40 39 677 the contents of which become part of this application by reference. A dilute and essentially laterally homogeneous binding layer which has particularly advantageous binding properties is preferably formed on the surface of the support material by addition of diluent molecules or by treating a surface with solid phase reactants present in a high dilution. It is, however, also possible to use conventional Langmuir layers as a solid phase binding matrix (Blankenburg et al., Biochemistry 28 (1989) 8214; Ahlers et al., Thin Solid Films 180 (1989) 93-99). Especially suitable silane compounds for application onto metal oxide layers are described in DE 4401450.
A laser which provides monochromatic light is preferably used as a light source for the plasmon resonance and for the fluorescence detection. It is, however, also possible to use other light sources.
The distance between the solid phase surface and the fluorophore used for the fluorescence detection is preferably ≧5 nm, particularly preferably ≧20 nm in order to avoid the metal surface having a disadvantageous influence on the radiated fluorescence. The spacing can for example be achieved by using suitable spacer molecules. The plasmon resonance can be detected in a known manner e.g. by means of a photodiode. The fluorescence can also be detected in any known manner e.g. with a photomultiplier. By using appropriate devices such as for example microscope optics to broaden and parallelize the laser ray and detection via a scanner or CCD camera, it is possible to carry out the method as a combination of plasmon resonance microscopy (A detailed description of plasmon resonance microscopy is given for exampl
Herrmann Rupert
Knoll Wolfgang
Liebermann Thorstein
Sluka Peter
Arent Fox Kintner & Plotkin & Kahn, PLLC
Chin Christopher L.
Roche Diagnostics GmbH
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