Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
2000-06-15
2003-04-15
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C436S518000, C436S526000, C436S525000, C422S082010, C422S098000, C422S186010, C422S186040, C422S051000, C204S403060, C435S007100, C435S285200
Reexamination Certificate
active
06548311
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a process for detection of analytes and a device for carrying out the process.
Such devices and processes, which are also designated below with the term of assay describing both, serve for qualitative and quantitative recording of specific bonds between at least two molecules. This includes, for example the recording of receptor-ligand interactions, antibody-antigen interaction, recognition of nucleic acids, interaction between oligonucleotides and DNA as well as other molecular interactions. Processes and devices of this type can be used, for example in chemistry, clinical analysis, pharmaceutical development, environmental analysis and in routine molecular biology work including sequencing of nucleic acids.
It is known that immunoassays having different detection methods are carried out. This includes radioactive, fluorescence-assisted and chemoluminescence-assisted and enzymatic processes (C. P. Prince, D. J. Newman: Principles and Practice of Immunoassays, Macmillan Publishers Ltd., 1991 U.K.).
In a particular form of immunoassay there is an agglutination of latex particles due to antibody-antigen bonds, which may be detected, for example visually (J. M. Singer, C. M. Plotz: The Latex Fixation Test, American Journal of Medicine, December 1965, pp. 888-892). 10
5
molecules may be detected using microspheres of 10 &mgr;m diameter, 10
8
molecules using microspheres having a diameter of 1 &mgr;m, and 10
13
molecules using microspheres having a diameter of 0.1 &mgr;m with such agglutination tests. Using the example of IgG (MW=100,000) theoretical sensitivities of 10 fM, 10 pM or 10 nM are given. The highest sensitivity is thus seen for relatively large microspheres, the use of which is however limited by their sedimentation behaviour,
Furthermore, recently nucleic acids, for example oligonucleotides, RNA and DNA may be detected via such interactions by means of DNA microchip technology (Nature, Genetics, Volume 14, No 4, December 96, and D. Noble: DNA Sequencing on a chip, Anal Chemistry, Volume 67, No. 5. Mar. 1, 1995). However, the chip technology is not used here as an electrical measuring process, but it serves as a novel synthesis process and for producing microstructures. The actual detection mechanism is of visual type. The combination of electrical processes for synthesis of a ligand and the visual marking and detection is however very expensive.
The disadvantage of the state of the art is that detection processes based on radioactivity are burdened with problems of radiation protection and disposal of the radioactive waste thus produced. In enzymatic detection methods which facilitate electrochemical detection of the analytes, a chemical reaction with a substance as the chemical reaction substrate must take place as an additional working step.
In all detection processes and assays known in the state of the art, a final washing step is necessary to remove excess reactants before detection of the analyte to minimise nonspecific signals as far as possible.
The object of the invention is to provide a device and a process which facilitates detection of analytes in rapid, simple and precise manner and in which a washing step may be omitted.
2. Description of the Related Art
World application 9005300 relates to a process for detection of binding reaction between specific substances, in which electrodes, between which a gap exists, are applied to a support. A layer of, for example antigens, is applied to the support in the gap. A measuring solution containing marker particles with antibodies and antigens is passed over the support, wherein marker particles with antibodies bind to the antigen layer of the support. After a preset reaction time, the non-bound marker particles are removed by washing and after drying the resistance is measured between the two electrodes which is a measure for binding which has taken place. Depending on the design of the support (conducting or non-conducting), a signal is measured when the gap is not completely bridged by conductive particles or only when the gap is filled.
European application 0 745 843 discloses an electrochemical biosensor which has electrodes and a matrix with different reagents. The reagents include conjugates of analytes and cytolytic reagent, receptors and liposomes containing electroactive materials. The liposomes release the electroactive material under the action of the analyte.
In a biosensor disclosed in European application 0 402 917, an electrically conducting polymer layer, to which a binding partner is coupled, is arranged between the electrodes. When binding the particular binding partner, the electrical properties of the polymer layer are changed.
European application 0 495 519 describes a process for measuring a specific substance in a sample, wherein an alternating current is applied to electrodes shaped like ridges to accelerate the aggregration of charged particles. Aggregation is then measured visually.
This object is achieved according to the invention by the features of the main and sub-claim.
If marker particles having electrical and/or electrochemical properties, which differ from those of their surroundings, the measuring solution, are introduced into an electric field, the electric field is disturbed by this. These disturbances of an electric field produced in the measuring solution may be determined simply, rapidly and very precisely.
SUMMARY OF THE INVENTION
The process of the invention is thus based on a detection method which may be regarded as essentially purely electrical. Therefore it has the very high sensitivity or precision possible for the determination of electrical or electrochemical properties and consequently a very low detection limit at high sensitivity.
With suitable dimensions and/or positioning of the electrodes which produce the electric field, the electric field is virtually exclusively influenced by marker particles having different material composition, different specific resistance of electric surface charge or different dielectric constant, which have undergone specific binding, for example with the analyte or with a substrate, that is bodies or materials suitable as substrate. Excess non-bound marker particles do not lead to a signal, so that a washing step for removing non-bound marker particles from the measuring solution may be omitted.
The measuring range of the bioassay according to the invention (process and/or device) may be adjusted by fixing the electrode or substrate surfaces and by selecting the size of the marker particles.
The process of the invention and the device of the invention may be used for detection and for concentration determination of any analytes which can be recorded via molecular interactions. These include, for example the interactions of receptor-ligand, antibody-antigen, antibody-hapten, antibody fragment-antigen, aptamers, proteins, nucleic acids, oligonucleotides, DNA and all molecular interactions, in which at least one of the molecular partners may be marked with a marker particle. This includes the interaction of materials with the surfaces of whole cells. It is possible in principle to realise all known immunoassay formats according to the state of the art.
The advantages achieved using the invention consequently exist particularly in the fact that a purely electrical detection method is used having all its advantages with reference to precision, rapidity and sensitivity, and that also when using very small electrodes and marker particles of comparable size, individual bond detection is possible.
Advantageous further developments of the process of the invention and the device of the invention are given in the dependent claims.
Particularly in the near-field of an electrode producing an electric field, even the presence of a single marker particle may lead to adequately high changes in the electric field. Non-specifically bound marker particles further removed from the electrode lead to fewer impairments of the electric field, so that with suitable arran
Counts Gary
Le Long V.
Marshall & Melhorn LLC
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