DE10002895B4 - Coimmobilization of several chemical species - Google Patents

Abstract

defined Coimmobilization of two or more chemical or biochemical Species of carriers, the surface is passivated and the nonspecific adsorption largely is completely suppressed and at least one of the chemical and biochemical species as Blocker (passivating agent) acts and at least one of the chemical or biochemical species is the analyte or a derivative thereof, characterized in that the blocker is selected from the group of lipophilic, Enzymes, enzyme complexes, haptens, hormones, agonists, antagonists, Antibodies, antibody fragments, Antigens, oligonucleotides, peptide nucleic acids or derivatives thereof, Polysaccharides, lipids, enzyme substrates, enzyme inhibitors, markers, Labels selected is.

Description

In affinity chromatography, In biochemical analysis and in biosensorics surfaces are equally needed high affinity on the one hand to a substance in question and on the other hand preferably few, ideally no, bind further substances. This problem will often by adding possible Interferents, eg. As proteins, tries to minimize. denatured Proteins, bovine serum albumin or casein or nucleic acids without specific sequence for the Research questions are used as blocking reagents before or during the Purification or added to the assay. These substances are but little specific and can in many cases not be used, for. As in the purification or analysis of haptens limited or not used at all.

The The object of the invention relates to the simultaneous, covalent coupling two or more different chemical or biochemical species on a carrier, Called immobilization. According to the invention, the surface becomes partial passivated, d. H. Unspecific adsorption is largely completely suppressed. application areas the invention are purification and defection in biotechnology, Bioanalytics, biomedicine, clinical chemistry and diagnostics as well the sensor.

At least one of the surface coupled substances is a binding partner of a chemical or biochemical recognition reaction. Is the surface, on which has been coupled, part of a detection system, at more than two simultaneously coupled components, are multiple analyzes at one or in different places possible.

The The invention relates to the simultaneous, covalent coupling of several different chemical species on a support (immobilization), the is designed so that the surface is passivated, d. H. unspecific adsorption is optionally suppressed. Areas of application of the invention are biotechnology, bioanalytics, Biomedicine, clinical chemistry and diagnostics as well as sensors

is the surface a sensor, one of the coupled substances can be a binding partner and Z. B. be used in the form of a competitive (immune) assay. If more than two components are coupled simultaneously, there are several Analyzes in one place (sequential) or different places the surface (simultaneously) possible.

The Difficulty of coupling biochemical units to carriers now that these Coupling should be defined, reproducible and stable and for different Substance groups must be suitable.

The Problem with the purification z. B. by affinity chromatography or Detection by means of a solid phase consists in the unspecific Adsorption to the respective carrier surface, the competes with the specific coupling. This will be usual way Masking agent is used, which wets the surface with a "repellent" layer. So z. B. in immunoassays frequently denatured proteins for suppression unspecific bonds used.

JP-04019561 A discloses an immunoassay in which in addition to the analyte polyvinyl alcohol (PVA) adsorbed to a support surface to serve as a preservative and a non-specific To prevent adsorption of antisera.

The Problem with the sensitive defection of different substances after coupling to the surface is that conventional Masking agents in addition to the unspecific often the specific Cover binding sites with and thus affects the measurand. That's why it was here described Koimmobilisierung introduced.

The Treatment according to the invention the surface passivated defines it in a defined way, thereby enabling masking the analyte-like substances from the measuring solutions, So that the Sample pretreatment is minimized and interference by non-specific binding still be open-minded.

The The invention has the aim of the detection of individual substances by means of one (upper) surface-sensitive To make the process sensitive.

The Co-mobilization can be controlled by the choice of co-immobilization they will for targeted surface treatments according to the respective task can be used.

The Invention will be explained in more detail below using an exemplary embodiment.

embodiment

The task was to detect a lipophilic substance in the blood by means of a surface-sensitive method. The sensitivity of the procedure made a competitive assay tion of antibodies is required. A derivative of the analyte was covalently coupled to the surface and the binding of the antibody to the surface was monitored. For this purpose, it was necessary to passivate the surface between the specific binding sites - here the immobilized analyte (derivative) molecules.

The usual in immunoassays Method with casein or serum albumin or denatured proteins to mask, could not be applied here because the immobilized Derivatives would also be covered. Therefore, became a second Substance, which is also lipophilic, co-immobilized in high density. The entire surface thus becomes largely hydrophobic and the adsorption more soluble Proteins from the blood plasma effectively suppressed.

1 : Co-immobilization by activation and covalent attachment of two different substrates (according to claims 1-12), which function as analyte or blocker depending on the task. The density of the blocker or analyte derivatives can be adjusted so as to optimize the loading capacity or measuring range of a sensor.

2 : Example of a competitive immune hormone assay in whole blood on a surface-sensitive biosensor (optical grating coupler). Antibody to the assay was added to the samples and competed by the presence of free assay. Data 1 shows the assay with passivated surface, data 2 with simply in the mobilized analyte derivative. The signal / noise ratio improved by a factor of 5, the lower detection limit could thus be reduced by 2 orders of magnitude in this example.

Claims (12)

Defined co-immobilization of two or more chemical or biochemical species on carriers, the surface being passivated and the unspecific adsorption being largely completely suppressed and at least one of the chemical and biochemical species acting as a blocker (passivant) and at least one of the chemical or biochemical species An analyte or a derivative thereof, characterized in that the blocker is selected from the group of lipophiles, enzymes, enzyme complexes, haptens, hormones, agonists, antagonists, antibodies, antibody fragments, antigens, oligonucleotides, peptide nucleic acids or derivatives thereof, polysaccharides, lipids, Enzyme substrates, enzyme inhibitors, markers, labels is selected. Defined Koimmobilisierung according to claim 1, characterized that the Analyte or the analyte derivative from the group of lipophiles, enzymes, Enzyme complexes, haptens, hormones, agonists, antagonists, antibodies, antibody fragments, Antigens, oligonucleotides, peptide nucleic acids or derivatives thereof, Polysaccharides, lipids, enzyme substrates, enzyme inhibitors, markers, labels selected is. Defined Koimmobilisierung according to claim 1 or 2, characterized characterized in that the markers or labels are fluorochromes, redox labels or spin label. Defined Koimmobilisierung according to claim 1 or 2, characterized in that the blocker and / or the analyte or the analyte derivative as a mixture of any of the specified in claim 1 or 2 substances is present. Defined Koimmobilisierung according to any one of claims 1-4, characterized that the carrier surface through a solid or soluble inorganic or organic (polymeric) carrier is. Defined Koimmobilisierung according to any one of claims 1-4, characterized that the carrier surface through a silicon wafer, a glass surface or sol / gel materials given is. Defined Koimmobilisierung according to any one of claims 1-4, characterized that the carrier surface through Plastics in the form of titer plates, reaction vessels (Eppendorf vessels), flow reactors, contained therein carrier material or hoses given is. Defined Koimmobilisierung according to any one of claims 1-4, characterized that the carrier surface off a precious metal. Defined Koimmobilisierung according to any one of claims 1-4, characterized that the carrier surface the Forming a scanning probe microscopy tip, in particular AFM tip (Atomic Force Microscope, a SNOM tip (optical Near-field microscope), a SEM tip (scanning electrochemical microscope) or another Scanning Probe Microscopy tip. Defined Koimmobilisierung according to any one of claims 1-4, characterized that the carrier surface off Latex or derivatives consists of or optionally coated or activated magnetic particles, in particular for chromatographic or cleansing purposes. A defined co-immobilization according to any one of claims 1-4, characterized in that the surface has the shape of a transducer; these are electrodes of all kinds or optical sensors, Optical waveguides or coated waveguides (simple or interferometric structure). Defined Koimmobilisierung according to any one of claims 1-4, characterized that the analyte photometrically, fluorimetrically or by means of chemiluminescence is detected.